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Hosseini N, Kazeminejad E, Oladnabi M, Khosravi A. Isolation and characterization of a new SHED cell line as a standard source for stem cell research and clinical translation. Tissue Cell 2025; 93:102649. [PMID: 39637488 DOI: 10.1016/j.tice.2024.102649] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/11/2024] [Revised: 11/28/2024] [Accepted: 11/28/2024] [Indexed: 12/07/2024]
Abstract
BACKGROUND AND AIMS Stem cells from human exfoliated deciduous teeth (SHED) are multi-potent mesenchymal stem/stromal cells (MSCs) and are inspected a favorable, non-invasive source beneficial to stem cell-mediated regeneration of damaged tissues. Our aim was to establish and characterize a non-immortalized SHED cell line as an accessible resource and novel platform for stem cell research and tissue regeneration studies. METHODS A Healthy exfoliated deciduous molar was extracted from a 12-year-old girl and shipped to an animal cell culture laboratory. Outgrowing primary cells from explanted small pulp tissues were monitored daily and characterized after passage 3 both morphologically and functionally. The SHED cell line was characterized by calculation of doubling time, cytogenetic analyses, STR analysis, adherence to cell culture flasks under standard cell culture media, and immunophenotypic analysis of specific MSC markers (CD90+, CD73+, CD34- and CD45-) using flow cytometry method. Differentiation potential to osteoblast, adipocyte, and chondrocyte was evaluated under standard differentiation media Expression of OCT-4 and NANOG genes was also assessed using RT-PCR method. RESULTS After the third day, SHED cells were visible. SHED cells were subcultured when they reached 90 % confluence after approximately 17 days. The doubling time of SHED cells was forty seven hours. SHED immunophenotyping showed the high expression level of CD90 (99.2 %) and CD73 (45.9 %), and approximately no expression of CD34 (0.079 %) and CD45 (0.19 %). The human origin, female gender and chromosomal normality of SHED cells was confirmed by cytogenetic analysis. The STR matching analysis showed that SHED cells are well-identified and authentic. No genetic instability and cross-contamination were observed in SHED cells. CONCLUSIONS This study provides a new SHED cell line with a normal karyotype and all the characteristics of MSCs, which can be used as a favorable model cell line in biomedical research and a promising source for clinical translation.
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Affiliation(s)
- Niloufar Hosseini
- Department of Medical Biotechnology, Faculty of Advanced Medical Technologies, Golestan University of Medical Sciences, Gorgan, Iran
| | - Ezatolah Kazeminejad
- Stem Cell Research Center, Golestan University of Medical Sciences, Gorgan, Iran; Dental Research Center, Golestan University of Medical Sciences, Gorgan, Iran
| | - Morteza Oladnabi
- Stem Cell Research Center, Golestan University of Medical Sciences, Gorgan, Iran; Gorgan Congenital Malformations Research Center, Golestan University of Medical Sciences, Gorgan, Iran
| | - Ayyoob Khosravi
- Stem Cell Research Center, Golestan University of Medical Sciences, Gorgan, Iran; Department of Molecular Medicine, Faculty of Advanced Medical Technologies, Golestan University of Medical Sciences, Gorgan, Iran.
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Moos HK, Patel R, Flaherty SK, Loverde SM, Nikolova EN. H2A.Z facilitates Sox2-nucleosome interaction by promoting DNA and histone H3 tail mobility. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2025:2025.03.06.641691. [PMID: 40093108 PMCID: PMC11908261 DOI: 10.1101/2025.03.06.641691] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 03/19/2025]
Abstract
Epigenetic regulation of eukaryotic chromatin structure and function can be modulated by histone variants and post-translational modifications. The conserved variant H2A.Z has been functionally linked to pioneer factors Sox2 and Oct4 that open chromatin and initiate cell fate-specific expression programs. However, the molecular basis for their interaction remains unknown. Using biochemistry, nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics (MD) simulations, we examine the role of H2A.Z nucleosome dynamics in pioneer factor binding. We find that H2A.Z facilitates Sox2 and Oct4 binding at distinct locations in 601 nucleosomes. We further link this to increased DNA accessibility and perturbed dynamics of the H3 N-terminal tail, which we show competes with Sox2 for DNA binding. Our simulations validate a coupling between H2A.Z-mediated DNA unwrapping and altered H3 N-tail conformations with fewer contacts to DNA and the H2A.Z C- terminal tail. This destabilizing effect of H2A.Z is DNA sequence dependent and enhanced with the less stable Lin28B nucleosome. Collectively, our findings suggest that H2A.Z promotes pioneer factor binding by increasing access to DNA and reducing competition with H3 tails. This could have broader implications for how epigenetic marks or oncogenic mutations tune pioneer factor engagement with chromatin and thus affect its structure and recognition.
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Ugalde-Morales E, Wilf R, Pluta J, Ploner A, Fan M, Damra M, Aben KK, Anson-Cartwright L, Chen C, Cortessis VK, Daneshmand S, Ferlin A, Gamulin M, Gietema JA, Gonzalez-Niera A, Grotmol T, Hamilton RJ, Harland M, Haugen TB, Hauser R, Hildebrandt MAT, Karlsson R, Kiemeney LA, Kim J, Lessel D, Lothe RA, Loveday C, Chanock SJ, McGlynn KA, Meijer C, Nead KT, Nsengimana J, Popovic M, Rafnar T, Richiardi L, Rocca MS, Schwartz SM, Skotheim RI, Stefansson K, Stewart DR, Turnbull C, Vaughn DJ, Winge SB, Zheng T, Monteiro AN, Almstrup K, Kanetsky PA, Nathanson KL, Wiklund F. Identification of genes associated with testicular germ cell tumor susceptibility through a transcriptome-wide association study. Am J Hum Genet 2025; 112:630-643. [PMID: 39999848 PMCID: PMC11947167 DOI: 10.1016/j.ajhg.2025.01.022] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/05/2024] [Revised: 01/20/2025] [Accepted: 01/28/2025] [Indexed: 02/27/2025] Open
Abstract
Transcriptome-wide association studies (TWASs) have the potential to identify susceptibility genes associated with testicular germ cell tumors (TGCTs). We conducted a comprehensive TGCT TWAS by integrating genome-wide association study (GWAS) summary data with predicted expression models from normal testis, TGCT tissues, and a cross-tissue panel that encompasses shared regulatory features across 22 normal tissues, including the testis. Gene associations were evaluated while accounting for variant-level effects from GWASs, followed by fine-mapping analyses in regions exhibiting multiple TWAS signals, and finally supplemented by colocalization analysis. Expression and protein patterns of identified TWAS genes were further examined in relevant tissues. Our analysis tested 19,805 gene-disease links, revealing 165 TGCT-associated genes with a false discovery rate of less than 0.01. We prioritized 46 candidate genes by considering GWAS-inflated signals, correlations between neighboring genes, and evidence of colocalization. Among these, 23 genes overlap with 22 GWAS loci, with 7 being associations not previously implicated in TGCT risk. Additionally, 23 genes located within 21 loci are at least 1 Mb away from published GWAS index variants. The 46 prioritized genes display expression levels consistent with expected expression levels in human gonadal cell types and precursor tumor cells and significant enrichment in TGCTs. Additionally, immunohistochemistry revealed protein-level accumulation of two candidate genes, ARID3B and GINM1, in both precursor and tumor cells. These findings enhance our understanding of the genetic predisposition to TGCTs and underscore the importance of further functional investigations into these candidate genes.
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Affiliation(s)
- Emilio Ugalde-Morales
- Department of Medical Epidemiology and Biostatistics, Karolinska Institute, Stockholm, Sweden
| | - Rona Wilf
- Division of Translational Medicine and Human Genetics, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA
| | - John Pluta
- Division of Translational Medicine and Human Genetics, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA
| | - Alexander Ploner
- Department of Medical Epidemiology and Biostatistics, Karolinska Institute, Stockholm, Sweden
| | - Mengyao Fan
- Division of Translational Medicine and Human Genetics, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA
| | - Mohammad Damra
- Division of Translational Medicine and Human Genetics, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA
| | - Katja K Aben
- Netherlands Comprehensive Cancer Organization, Radboud University Medical Center, Utrecht, the Netherlands; Radboud University Medical Center, Nijmegen, the Netherlands
| | - Lynn Anson-Cartwright
- Department of Surgery (Urology), University of Toronto and The Princess Margaret Cancer Centre, Toronto, ON, Canada
| | - Chu Chen
- Epidemiology Program, Fred Hutchinson Cancer Center, Seattle, WA, USA; Department of Epidemiology, University of Washington, Seattle, WA, USA
| | - Victoria K Cortessis
- Departments of Preventive Medicine and Obstetrics and Gynecology, Keck School of Medicine at the University of Southern California, Los Angeles, CA, USA
| | - Siamak Daneshmand
- Departments of Urology, Keck School of Medicine at the University of Southern California, Los Angeles, CA, USA
| | - Alberto Ferlin
- Department of Medicine, University of Padova, Padua, Italy
| | - Marija Gamulin
- Department of Oncology, University Hospital Center Zagreb, University of Zagreb School of Medicine, Zagreb, Croatia
| | - Jourik A Gietema
- Department of Medical Oncology, University Medical Center Groningen, University of Groningen, Groningen, the Netherlands
| | - Anna Gonzalez-Niera
- Human Genotyping Core Unit, Spanish National Cancer Centre (CNIO), Madrid, Spain
| | - Tom Grotmol
- Cancer Registry of Norway, Oslo Metropolitan University, Oslo, Norway
| | | | - Mark Harland
- Department of Surgery (Urology), University of Toronto and The Princess Margaret Cancer Centre, Toronto, ON, Canada
| | - Trine B Haugen
- Department of Life Sciences and Health, Oslo Metropolitan University, Oslo, Norway
| | - Russ Hauser
- Department of Environmental Health, Harvard T.H. Chan School of Public Health, Boston, MA, USA
| | - Michelle A T Hildebrandt
- Department of Lymphoma and Myeloma, University of Texas, MD Anderson Cancer Center, Houston, TX, USA
| | - Robert Karlsson
- Department of Medical Epidemiology and Biostatistics, Karolinska Institute, Stockholm, Sweden
| | | | - Jung Kim
- Division of Cancer Epidemiology and Genetics, National Cancer Institute, Bethesda, MD, USA
| | - Davor Lessel
- Institute of Human Genetics, University of Regensburg, Regensburg, Germany; Institute of Clinical Human Genetics, University Hospital Regensburg, Regensburg, Germany
| | - Ragnhild A Lothe
- Department of Molecular Oncology, Institute for Cancer Research, Oslo University Hospital-Radiumhospitalet, Oslo, Norway
| | - Chey Loveday
- Division of Genetics & Epidemiology, The Institute of Cancer Research, London, UK; William Harvey Research Institute, Queen Mary University, London, UK
| | - Stephen J Chanock
- Division of Cancer Epidemiology and Genetics, National Cancer Institute, Bethesda, MD, USA
| | - Katherine A McGlynn
- Division of Cancer Epidemiology and Genetics, National Cancer Institute, Bethesda, MD, USA
| | - Coby Meijer
- Department of Medical Oncology, University Medical Center Groningen, University of Groningen, Groningen, the Netherlands
| | - Kevin T Nead
- Department of Lymphoma and Myeloma, University of Texas, MD Anderson Cancer Center, Houston, TX, USA
| | - Jeremie Nsengimana
- Biostatistics Research Group, Population Health Sciences Institute, Faculty of Medical Sciences, Newcastle University, Newcastle, UK
| | - Maja Popovic
- Cancer Epidemiology Unit, Department of Medical Sciences, University of Turin and CPO-Piemonte, Turin, Italy
| | | | - Lorenzo Richiardi
- Cancer Epidemiology Unit, Department of Medical Sciences, University of Turin and CPO-Piemonte, Turin, Italy
| | - Maria S Rocca
- Department of Medicine, University of Padova, Padua, Italy
| | - Stephen M Schwartz
- Epidemiology Program, Fred Hutchinson Cancer Center, Seattle, WA, USA; Department of Epidemiology, University of Washington, Seattle, WA, USA
| | - Rolf I Skotheim
- Department of Molecular Oncology, Institute for Cancer Research, Oslo University Hospital-Radiumhospitalet, Oslo, Norway
| | | | - Douglas R Stewart
- Division of Cancer Epidemiology and Genetics, National Cancer Institute, Bethesda, MD, USA
| | - Clare Turnbull
- Division of Genetics & Epidemiology, The Institute of Cancer Research, London, UK
| | - David J Vaughn
- Division of Hematology/Oncology, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA
| | - Sofia B Winge
- Department of Growth and Reproduction, Copenhagen University Hospital - Rigshospitalet, Copenhagen, Denmark
| | - Tongzhang Zheng
- Department of Epidemiology, Brown School of Public Health, Brown University, Providence, RI, USA
| | - Alvaro N Monteiro
- Department of Cancer Epidemiology, Moffitt Cancer Center and Research Institute, Tampa, FL, USA
| | - Kristian Almstrup
- Department of Growth and Reproduction, Copenhagen University Hospital - Rigshospitalet, Copenhagen, Denmark; Department of Cellular and Molecular Medicine, University of Copenhagen, Copenhagen, Denmark
| | - Peter A Kanetsky
- Department of Cancer Epidemiology, Moffitt Cancer Center and Research Institute, Tampa, FL, USA
| | - Katherine L Nathanson
- Division of Translational Medicine and Human Genetics, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA; Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA
| | - Fredrik Wiklund
- Department of Medical Epidemiology and Biostatistics, Karolinska Institute, Stockholm, Sweden.
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da Silva Pescador G, Baia Amaral D, Varberg JM, Zhang Y, Hao Y, Florens L, Bazzini AA. Protein profiling of zebrafish embryos unmasks regulatory layers during early embryogenesis. Cell Rep 2024; 43:114769. [PMID: 39302832 PMCID: PMC11544563 DOI: 10.1016/j.celrep.2024.114769] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/12/2024] [Revised: 07/22/2024] [Accepted: 08/30/2024] [Indexed: 09/22/2024] Open
Abstract
The maternal-to-zygotic transition is crucial in embryonic development, marked by the degradation of maternally provided mRNAs and initiation of zygotic gene expression. However, the changes occurring at the protein level during this transition remain unclear. Here, we conducted protein profiling throughout zebrafish embryogenesis using quantitative mass spectrometry, integrating transcriptomics and translatomics datasets. Our data show that, unlike RNA changes, protein changes are less dynamic. Further, increases in protein levels correlate with mRNA translation, whereas declines in protein levels do not, suggesting active protein degradation processes. Interestingly, proteins from pure zygotic genes are present at fertilization, challenging existing mRNA-based gene classifications. As a proof of concept, we utilized CRISPR-Cas13d to target znf281b mRNA, a gene whose protein significantly accumulates within the first 2 h post-fertilization, demonstrating its crucial role in development. Consequently, our protein profiling, coupled with CRISPR-Cas13d, offers a complementary approach to unraveling maternal factor function during embryonic development.
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Affiliation(s)
| | | | - Joseph M Varberg
- Stowers Institute for Medical Research, Kansas City, MO 64110, USA
| | - Ying Zhang
- Stowers Institute for Medical Research, Kansas City, MO 64110, USA
| | - Yan Hao
- Stowers Institute for Medical Research, Kansas City, MO 64110, USA
| | - Laurence Florens
- Stowers Institute for Medical Research, Kansas City, MO 64110, USA
| | - Ariel A Bazzini
- Stowers Institute for Medical Research, Kansas City, MO 64110, USA; Department of Molecular and Integrative Physiology, University of Kansas School of Medicine, Kansas City, KS 66160, USA.
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Choi WH, Cho Y, Cha JH, Lee DH, Jeong JG, Jung SH, Song JJ, Lee JH, Lee SY. Functional pathogenicity of ESRRB variant of uncertain significance contributes to hearing loss (DFNB35). Sci Rep 2024; 14:21215. [PMID: 39261511 PMCID: PMC11390957 DOI: 10.1038/s41598-024-70795-8] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/24/2024] [Accepted: 08/21/2024] [Indexed: 09/13/2024] Open
Abstract
Advances in next-generation sequencing technologies have led to elucidation of sensorineural hearing loss genetics and associated clinical impacts. However, studies on the functional pathogenicity of variants of uncertain significance (VUS), despite their close association with clinical phenotypes, are lacking. Here we identified compound heterozygous variants in ESRRB transcription factor gene linked to DFNB35, specifically a novel splicing variant (NM_004452.4(ESRRB): c.397 + 2T>G) in trans with a missense variant (NM_004452.4(ESRRB): c.1144C>T p.(Arg382Cys)) whose pathogenicity remains unclear. The splicing variant (c.397 + 2T>G) caused exon 4 skipping, leading to premature stop codon formation and nonsense-mediated decay. The p.(Arg382Cys) variant was classified as a VUS due to its particularly higher allele frequency among East Asian population despite disease-causing in-silico predictions. However, functional assays showed that p.(Arg382Cys) variant disrupted key intramolecular interactions, leading to protein instability. This variant also reduced transcriptional activity and altered expression of downstream target genes essential for inner ear function, suggesting genetic contribution to disease phenotype. This study expanded the phenotypic and genotypic spectrum of ESRRB in DFNB35 and revealed molecular mechanisms underlying ESRRB-associated DFNB35. These findings suggest that variants with high allele frequencies can also possess functional pathogenicity, providing a breakthrough for cases where VUS, previously unexplored, could be reinterpreted by elucidating their functional roles and disease-causing characteristics.
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Affiliation(s)
- Won Hoon Choi
- Department of Otorhinolaryngology-Head and Neck Surgery, Seoul National University Hospital, Seoul National University College of Medicine, Seoul, Republic of Korea
| | - Yeijean Cho
- Seoul National University College of Medicine, Seoul, South Korea
| | - Ju Hyuen Cha
- Department of Otorhinolaryngology-Head and Neck Surgery, Seoul National University Hospital, Seoul National University College of Medicine, Seoul, Republic of Korea
| | - Dae Hee Lee
- CTCELLS, Inc., 21, Yuseong-Daero, 1205 Beon-Gil, Yuseong-Gu, Daejeon, Republic of Korea
| | - Jong Gwan Jeong
- CTCELLS, Inc., 21, Yuseong-Daero, 1205 Beon-Gil, Yuseong-Gu, Daejeon, Republic of Korea
| | - Sung Ho Jung
- Department of Otorhinolaryngology-Head and Neck Surgery, Seoul National University Hospital, Seoul National University College of Medicine, Seoul, Republic of Korea
| | - Jae-Jin Song
- Department of Otorhinolaryngology-Head and Neck Surgery, Seoul National University Hospital, Seoul National University College of Medicine, Seoul, Republic of Korea
| | - Jun Ho Lee
- Department of Otorhinolaryngology-Head and Neck Surgery, Seoul National University Hospital, Seoul National University College of Medicine, Seoul, Republic of Korea
| | - Sang-Yeon Lee
- Department of Otorhinolaryngology-Head and Neck Surgery, Seoul National University Hospital, Seoul National University College of Medicine, Seoul, Republic of Korea.
- Sensory Organ Research Institute, Seoul National University Medical Research Center, Seoul, Republic of Korea.
- Department of Genomic Medicine, Seoul National University Hospital, Seoul National University College of Medicine, Seoul, Republic of Korea.
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6
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Ngule C, Shi R, Ren X, Jia H, Oyelami F, Li D, Park Y, Kim J, Hemati H, Zhang Y, Xiong X, Shinkle A, Vanderford NL, Bachert S, Zhou BP, Wang J, Song J, Liu X, Yang JM. NAC1 promotes stemness and regulates myeloid-derived cell status in triple-negative breast cancer. Mol Cancer 2024; 23:188. [PMID: 39243032 PMCID: PMC11378519 DOI: 10.1186/s12943-024-02102-y] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/02/2024] [Accepted: 08/27/2024] [Indexed: 09/09/2024] Open
Abstract
Triple negative breast cancer (TNBC) is a particularly lethal breast cancer (BC) subtype driven by cancer stem cells (CSCs) and an immunosuppressive microenvironment. Our study reveals that nucleus accumbens associated protein 1 (NAC1), a member of the BTB/POZ gene family, plays a crucial role in TNBC by maintaining tumor stemness and influencing myeloid-derived suppressor cells (MDSCs). High NAC1 expression correlates with worse TNBC prognosis. NAC1 knockdown reduced CSC markers and tumor cell proliferation, migration, and invasion. Additionally, NAC1 affects oncogenic pathways such as the CD44-JAK1-STAT3 axis and immunosuppressive signals (TGFβ, IL-6). Intriguingly, the impact of NAC1 on tumor growth varies with the host immune status, showing diminished tumorigenicity in natural killer (NK) cell-competent mice but increased tumorigenicity in NK cell-deficient ones. This highlights the important role of the host immune system in TNBC progression. In addition, high NAC1 level in MDSCs also supports TNBC stemness. Together, this study implies NAC1 as a promising therapeutic target able to simultaneously eradicate CSCs and mitigate immune evasion.
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Affiliation(s)
- Chrispus Ngule
- Department of Toxicology and Cancer Biology, Department of Pharmacology and Nutritional Science, and Markey Cancer Center, University of Kentucky College of Medicine, Lexington, KY, 40536, USA
| | - Ruyi Shi
- Department of Toxicology and Cancer Biology, Department of Pharmacology and Nutritional Science, and Markey Cancer Center, University of Kentucky College of Medicine, Lexington, KY, 40536, USA
- Present Address: Department of Cell Biology and Genetics, Shanxi Medical University, Taiyuan, Shanxi, China
| | - Xingcong Ren
- Department of Toxicology and Cancer Biology, Department of Pharmacology and Nutritional Science, and Markey Cancer Center, University of Kentucky College of Medicine, Lexington, KY, 40536, USA
| | - Hongyan Jia
- Department of Toxicology and Cancer Biology, Department of Pharmacology and Nutritional Science, and Markey Cancer Center, University of Kentucky College of Medicine, Lexington, KY, 40536, USA
- Present Address: Department of Breast Surgery, First Hospital of Shanxi Medical University, Taiyuan, Shanxi, China
| | - Felix Oyelami
- Department of Toxicology and Cancer Biology, Department of Pharmacology and Nutritional Science, and Markey Cancer Center, University of Kentucky College of Medicine, Lexington, KY, 40536, USA
| | - Dong Li
- Department of Toxicology and Cancer Biology, Department of Pharmacology and Nutritional Science, and Markey Cancer Center, University of Kentucky College of Medicine, Lexington, KY, 40536, USA
| | - Younhee Park
- Department of Toxicology and Cancer Biology, Department of Pharmacology and Nutritional Science, and Markey Cancer Center, University of Kentucky College of Medicine, Lexington, KY, 40536, USA
| | - Jinhwan Kim
- Department of Biochemistry, and Markey Cancer Center, University of Kentucky College of Medicine, Lexington, KY, 40536, USA
| | - Hami Hemati
- Department of Toxicology and Cancer Biology, Department of Pharmacology and Nutritional Science, and Markey Cancer Center, University of Kentucky College of Medicine, Lexington, KY, 40536, USA
| | - Yi Zhang
- Department of Toxicology and Cancer Biology, Department of Pharmacology and Nutritional Science, and Markey Cancer Center, University of Kentucky College of Medicine, Lexington, KY, 40536, USA
- Present Address: Department of Pharmacology, College of Pharmaceutical Sciences, Soochow University, Suzhou, Jiangsu, China
| | - Xiaofang Xiong
- Department of Microbial Pathogenesis and Immunology, Texas A&M University Health Science Center, Bryan, TX, 77807, USA
| | - Andrew Shinkle
- Department of Toxicology and Cancer Biology, Department of Pharmacology and Nutritional Science, and Markey Cancer Center, University of Kentucky College of Medicine, Lexington, KY, 40536, USA
| | - Nathan L Vanderford
- Department of Toxicology and Cancer Biology, Department of Pharmacology and Nutritional Science, and Markey Cancer Center, University of Kentucky College of Medicine, Lexington, KY, 40536, USA
| | - Sara Bachert
- Department of Pathology and Laboratory Medicine, University of Kentucky, Lexington, KY, 40536, USA
| | - Binhua P Zhou
- Department of Biochemistry, and Markey Cancer Center, University of Kentucky College of Medicine, Lexington, KY, 40536, USA
| | - Jianlong Wang
- Department of Medicine, Columbia Center for Human Development and Stem Cell Therapies, Columbia University Irving Medical Center, New York, NY, 10032, USA
| | - Jianxun Song
- Department of Microbial Pathogenesis and Immunology, Texas A&M University Health Science Center, Bryan, TX, 77807, USA.
| | - Xia Liu
- Department of Toxicology and Cancer Biology, Department of Pharmacology and Nutritional Science, and Markey Cancer Center, University of Kentucky College of Medicine, Lexington, KY, 40536, USA.
| | - Jin-Ming Yang
- Department of Toxicology and Cancer Biology, Department of Pharmacology and Nutritional Science, and Markey Cancer Center, University of Kentucky College of Medicine, Lexington, KY, 40536, USA.
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Gayer FA, Klaus L, Reichardt SD, Fichtner A, Reichardt HM. Phenotype and gene signature of testicular tumors in 129.MOLF-Chr19 mice resemble human teratomas. Andrology 2024. [PMID: 39074032 DOI: 10.1111/andr.13717] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/27/2024] [Revised: 07/02/2024] [Accepted: 07/18/2024] [Indexed: 07/31/2024]
Abstract
BACKGROUND Testicular germ cell tumor (TGCT) is the most common type of tumor in young men. Type II germ cell tumors including postpubertal-type teratomas are derived from the germ cell neoplasia in situ (GCNIS), whereas prepubertal-type teratomas arise independently of the GCNIS. The consomic mouse strain 129.MOLF-Chr19 (M19) is a suitable murine model of such tumors, but its characterization remains incomplete. OBJECTIVE Here, we interrogated the suitability of testicular tumors in M19 mice as a model of human TGCT by analyzing their histological features and gene expression signature. MATERIAL AND METHODS Testes collected from M19 mice of different ages were categorized by macroscopic appearance based on size and the degree of suspected tumorigenesis. Histological sections from selected tumors were stained with Hematoxylin and Eosin, and expression of genes associated with tumorigenesis was determined in frozen tissue samples from a large range of tumors of different subclasses using RT-qPCR and Fluidigm Dynamic Arrays. RESULTS Macroscopically, testicular specimens appeared very heterogeneous concerning size and signs indicating the presence of a tumor. Histological analysis confirmed the development of teratomas with areas of cells corresponding to all three germ cell layers. Gene expression analyses indicated upregulation of markers related to proliferation, vascular invasive potential and pluripotency, and revealed a strong correlation of gene expression with tumor size and a significant intercorrelation of individual genes. DISCUSSION AND CONCLUSION TGCT in M19 mice is reminiscent of human testicular teratomas presenting with areas of cells derived from all germ layers and showing a typical gene signature. We thus confirm that these mice can serve as a suitable murine model of pure teratomas for preclinical research.
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Affiliation(s)
- Fabian A Gayer
- Institute for Cellular and Molecular Immunology, University Medical Center Göttingen, Göttingen, Germany
- Clinic of Urology, University Medical Center Göttingen, Göttingen, Germany
| | - Lucas Klaus
- Institute for Cellular and Molecular Immunology, University Medical Center Göttingen, Göttingen, Germany
| | - Sybille D Reichardt
- Institute for Cellular and Molecular Immunology, University Medical Center Göttingen, Göttingen, Germany
| | - Alexander Fichtner
- Institute of Pathology, University Medical Center Göttingen, Göttingen, Germany
| | - Holger M Reichardt
- Institute for Cellular and Molecular Immunology, University Medical Center Göttingen, Göttingen, Germany
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Kulkarni S, Alampally H, Guddattu V, Rodrigues G, Carnelio S. Expression of Fascin and SALL4 in odontogenic cysts and tumors: an immunohistochemical appraisal. F1000Res 2024; 11:1578. [PMID: 38895097 PMCID: PMC11184278 DOI: 10.12688/f1000research.126091.3] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Accepted: 06/25/2024] [Indexed: 06/21/2024] Open
Abstract
Background Various stemness markers (SOX2, OCT4, and NANOG) have been studied in odontogenic cysts and tumors. However, studies on SALL4 having similar properties of stemness has not been documented. Additionally, insight into fascin as a migratory molecule is less explored. In this study, the expression of SALL4 and fascin were evaluated in ameloblastoma, adenomatoid odontogenic tumor (AOT), odontogenic keratocyst (OKC), dentigerous cyst (DC), radicular cyst (RC), and calcifying odontogenic cyst (COC). Methods Semi-quantitative analysis of fascin and SALL4 immuno-positive cells was done in a total of 40 cases of ameloblastoma (11 plexiform, 12 follicular, 12 unicystic, and 5 desmoplastic) variants, 6 cases of AOT, 15 each of OKC, DC, RC and 5 of COC. Chi-square test was applied to evaluate the association between SALL4 and fascin expression in odontogenic cysts and tumors. Results Fascin immunopositivity was observed in peripheral ameloblast-like cells, and the expression was weak or absent in stellate reticulum-like cells. A moderate to weak immune-reactivity to SALL4 was observed in the cytoplasm of ameloblastoma, epithelial cells of dentigerous and radicular cysts, having a marked inflammatory infiltrate, which was an interesting observation. COC and AOT had negative to weak expressions. No recurrence has been reported. Conclusions Expression of fascin in ameloblastomas elucidate their role in motility and localized invasion. Its expression in less aggressive lesions like DC, COC, AOT will incite to explore the other functional properties of fascin. SALL4 expression in the cytoplasm of odontogenic cysts and tumors may represent inactive or mutant forms which requires further validation.
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Affiliation(s)
- Spoorti Kulkarni
- Oral Pathology and Microbiology, Manipal College of Dental Sciences, Manipal Academy of Higher Education (MAHE), Manipal, Karnataka, 576104, India
| | - Harishanker Alampally
- Oral Pathology and Microbiology, Manipal College of Dental Sciences, Manipal Academy of Higher Education (MAHE), Manipal, Karnataka, 576104, India
| | - Vasudev Guddattu
- Department of Data Science, Prasanna School of Public Health, Manipal Academy of Higher Education, Manipal, Karnataka, 576104, India
| | - Gabriel Rodrigues
- Department of General Surgery, Kasturba Medical College, Manipal Academy of Higher Education, Manipal, Karnataka, 576104, India
| | - Sunitha Carnelio
- Oral Pathology and Microbiology, Manipal College of Dental Sciences, Manipal Academy of Higher Education (MAHE), Manipal, Karnataka, 576104, India
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9
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Noor Z, Guo S, Zhao Z, Qin Y, Shi G, Ma H, Zhang Y, Li J, Yu Z. Identification and involvement of DAX1 gene in spermatogenesis of boring giant clam Tridacna crocea. Gene 2024; 911:148338. [PMID: 38438056 DOI: 10.1016/j.gene.2024.148338] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/02/2023] [Revised: 02/20/2024] [Accepted: 03/01/2024] [Indexed: 03/06/2024]
Abstract
DAX1 (dosage-sensitive sex reversal, adrenal hypoplasia congenital critical region on X chromosome gene 1), a key sex determinant in various species, plays a vital role in gonad differentiation and development and controls spermatogenesis. However, the identity and function of DAX1 are still unclear in bivalves. In the present study, we identified a DAX1 (designed as Tc-DAX1) gene from the boring giant clam Tridacna crocea, a tropical marine bivalve. The full length of Tc-DAX1 was 1877 bp, encoding 462 amino acids, with a Molecular weight of 51.81 kDa and a theoretical Isoelectric point of 5.87 (pI). Multiple sequence alignments and phylogenetic analysis indicated a putative ligand binding domain (LBD) conserved regions clustered with molluscans DAX1 homologs. The tissue distributions in different reproductive stages revealed a dimorphic pattern, with the highest expression trend in the male reproductive stage, indicating its role in spermatogenesis. The DAX1 expression data from embryonic stages shows its highest expression profile (P < 0.05) in the zygote stage, followed by decreasing trends in the larvae stages (P > 0.05). The localization of DAX1 transcripts has also been confirmed by whole mount in situ hybridization, showing high positive signals in the fertilized egg, 2, and 4-cell stage, and gastrula. Moreover, RNAi knockdown of the Tc-DAX1 transcripts shows a significantly lower expression profile in the ds-DAX1 group compared to the ds-EGFP group. Subsequent histological analysis of gonads revealed that spermatogenesis was affected in a ds-DAX1 group compared to the ds-EGFP group. All these results indicate that Tc-DAX1 is involved in the spermatogenesis and early embryonic development of T. crocea, providing valuable information for the breeding and aquaculture of giant clams.
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Affiliation(s)
- Zohaib Noor
- Key Laboratory of Tropical Marine Bio-resources and Ecology, Guangdong Provincial Key Laboratory of Applied Marine Biology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, China; University of Chinese Academy of Sciences, Beijing 100049, China; Hainan Key Laboratory of Tropical Marine Biotechnology, Hainan Sanya Marine Ecosystem National Observation and Research Station, Sanya 572024, China
| | - Shuming Guo
- Key Laboratory of Tropical Marine Bio-resources and Ecology, Guangdong Provincial Key Laboratory of Applied Marine Biology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, China; University of Chinese Academy of Sciences, Beijing 100049, China; Hainan Key Laboratory of Tropical Marine Biotechnology, Hainan Sanya Marine Ecosystem National Observation and Research Station, Sanya 572024, China
| | - Zhen Zhao
- Key Laboratory of Tropical Marine Bio-resources and Ecology, Guangdong Provincial Key Laboratory of Applied Marine Biology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, China; Hainan Key Laboratory of Tropical Marine Biotechnology, Hainan Sanya Marine Ecosystem National Observation and Research Station, Sanya 572024, China
| | - Yanpin Qin
- Key Laboratory of Tropical Marine Bio-resources and Ecology, Guangdong Provincial Key Laboratory of Applied Marine Biology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, China; Hainan Key Laboratory of Tropical Marine Biotechnology, Hainan Sanya Marine Ecosystem National Observation and Research Station, Sanya 572024, China
| | - Gongpengyang Shi
- Key Laboratory of Tropical Marine Bio-resources and Ecology, Guangdong Provincial Key Laboratory of Applied Marine Biology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, China; Hainan Key Laboratory of Tropical Marine Biotechnology, Hainan Sanya Marine Ecosystem National Observation and Research Station, Sanya 572024, China
| | - Haitao Ma
- Key Laboratory of Tropical Marine Bio-resources and Ecology, Guangdong Provincial Key Laboratory of Applied Marine Biology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, China; Hainan Key Laboratory of Tropical Marine Biotechnology, Hainan Sanya Marine Ecosystem National Observation and Research Station, Sanya 572024, China
| | - Yuehuan Zhang
- Key Laboratory of Tropical Marine Bio-resources and Ecology, Guangdong Provincial Key Laboratory of Applied Marine Biology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, China; University of Chinese Academy of Sciences, Beijing 100049, China; Hainan Key Laboratory of Tropical Marine Biotechnology, Hainan Sanya Marine Ecosystem National Observation and Research Station, Sanya 572024, China
| | - Jun Li
- Key Laboratory of Tropical Marine Bio-resources and Ecology, Guangdong Provincial Key Laboratory of Applied Marine Biology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, China; Hainan Key Laboratory of Tropical Marine Biotechnology, Hainan Sanya Marine Ecosystem National Observation and Research Station, Sanya 572024, China.
| | - Ziniu Yu
- Key Laboratory of Tropical Marine Bio-resources and Ecology, Guangdong Provincial Key Laboratory of Applied Marine Biology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, China; Hainan Key Laboratory of Tropical Marine Biotechnology, Hainan Sanya Marine Ecosystem National Observation and Research Station, Sanya 572024, China.
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10
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Li-Bao L, Díaz-Díaz C, Raiola M, Sierra R, Temiño S, Moya FJ, Rodriguez-Perales S, Santos E, Giovinazzo G, Bleckwehl T, Rada-Iglesias Á, Spitz F, Torres M. Regulation of Myc transcription by an enhancer cluster dedicated to pluripotency and early embryonic expression. Nat Commun 2024; 15:3931. [PMID: 38729993 PMCID: PMC11087473 DOI: 10.1038/s41467-024-48258-5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/12/2022] [Accepted: 04/23/2024] [Indexed: 05/12/2024] Open
Abstract
MYC plays various roles in pluripotent stem cells, including the promotion of somatic cell reprogramming to pluripotency, the regulation of cell competition and the control of embryonic diapause. However, how Myc expression is regulated in this context remains unknown. The Myc gene lies within a ~ 3-megabase gene desert with multiple cis-regulatory elements. Here we use genomic rearrangements, transgenesis and targeted mutation to analyse Myc regulation in early mouse embryos and pluripotent stem cells. We identify a topologically-associated region that homes enhancers dedicated to Myc transcriptional regulation in stem cells of the pre-implantation and early post-implantation embryo. Within this region, we identify elements exclusively dedicated to Myc regulation in pluripotent cells, with distinct enhancers that sequentially activate during naive and formative pluripotency. Deletion of pluripotency-specific enhancers dampens embryonic stem cell competitive ability. These results identify a topologically defined enhancer cluster dedicated to early embryonic expression and uncover a modular mechanism for the regulation of Myc expression in different states of pluripotency.
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Affiliation(s)
- Lin Li-Bao
- Cardiovascular Regeneration Program, Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain
- Centro Andaluz de Biología del Desarrollo (CABD), Sevilla, Spain
| | - Covadonga Díaz-Díaz
- Cardiovascular Regeneration Program, Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain
- Centro de Investigación Biomédica en Red de Enfermedades Cardiovasculares (CIBERCV), Madrid, Spain
| | - Morena Raiola
- Cardiovascular Regeneration Program, Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain
| | - Rocío Sierra
- Cardiovascular Regeneration Program, Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain
- Centro de Investigación Biomédica en Red de Enfermedades Cardiovasculares (CIBERCV), Madrid, Spain
| | - Susana Temiño
- Cardiovascular Regeneration Program, Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain
- Centro de Investigación Biomédica en Red de Enfermedades Cardiovasculares (CIBERCV), Madrid, Spain
| | - Francisco J Moya
- Molecular Cytogenetics and Genome Editing Unit, Human Cancer Genetics Program, Centro Nacional de Investigaciones Oncológicas (CNIO), Madrid, Spain
| | - Sandra Rodriguez-Perales
- Molecular Cytogenetics and Genome Editing Unit, Human Cancer Genetics Program, Centro Nacional de Investigaciones Oncológicas (CNIO), Madrid, Spain
| | - Elisa Santos
- Pluripotent Cell Technology Unit, Centro Nacional de Investigaciones Cardiovasculares, CNIC, Madrid, Spain
| | - Giovanna Giovinazzo
- Centro de Investigación Biomédica en Red de Enfermedades Cardiovasculares (CIBERCV), Madrid, Spain
- Pluripotent Cell Technology Unit, Centro Nacional de Investigaciones Cardiovasculares, CNIC, Madrid, Spain
| | - Tore Bleckwehl
- Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany
- Institute of Experimental Medicine and Systems Biology, RWTH Aachen University, Aachen, Germany
| | - Álvaro Rada-Iglesias
- Institute of Biomedicine and Biotechnology of Cantabria (IBBTEC), CSIC/University of Cantabria, Santander, Spain
| | - Francois Spitz
- Department of Human Genetics, The University of Chicago, Chicago, IL, USA
- Developmental Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany
| | - Miguel Torres
- Cardiovascular Regeneration Program, Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain.
- Centro de Investigación Biomédica en Red de Enfermedades Cardiovasculares (CIBERCV), Madrid, Spain.
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11
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Wang F, Chander A, Yoon Y, Welton JM, Wallingford MC, Espejo-Serrano C, Bustos F, Findlay GM, Mager J, Bach I. Roles of the Rlim-Rex1 axis during X chromosome inactivation in mice. Proc Natl Acad Sci U S A 2023; 120:e2313200120. [PMID: 38113263 PMCID: PMC10756295 DOI: 10.1073/pnas.2313200120] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/01/2023] [Accepted: 11/17/2023] [Indexed: 12/21/2023] Open
Abstract
In female mice, the gene dosage from X chromosomes is adjusted by a process called X chromosome inactivation (XCI) that occurs in two steps. An imprinted form of XCI (iXCI) that silences the paternally inherited X chromosome (Xp) is initiated at the 2- to 4-cell stages. As extraembryonic cells including trophoblasts keep the Xp silenced, epiblast cells that give rise to the embryo proper reactivate the Xp and undergo a random form of XCI (rXCI) around implantation. Both iXCI and rXCI require the lncRNA Xist, which is expressed from the X to be inactivated. The X-linked E3 ubiquitin ligase Rlim (Rnf12) in conjunction with its target protein Rex1 (Zfp42), a critical repressor of Xist, have emerged as major regulators of iXCI. However, their roles in rXCI remain controversial. Investigating early mouse development, we show that the Rlim-Rex1 axis is active in pre-implantation embryos. Upon implantation Rex1 levels are downregulated independently of Rlim specifically in epiblast cells. These results provide a conceptual framework of how the functional dynamics between Rlim and Rex1 ensures regulation of iXCI but not rXCI in female mice.
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Affiliation(s)
- Feng Wang
- Department of Molecular, Cell and Cancer Biology, University of Massachusetts Chan Medical School, Worcester, MA01605
| | - Ashmita Chander
- Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, MA01003
| | - Yeonsoo Yoon
- Division of Genes and Development, Department of Pediatrics, University of Massachusetts Chan Medical School, Worcester, MA01605
| | - Janelle M. Welton
- Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, MA01003
| | - Mary C. Wallingford
- Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, MA01003
| | - Carmen Espejo-Serrano
- Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, DundeeDD1 5EH, United Kingdom
| | - Francisco Bustos
- Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, DundeeDD1 5EH, United Kingdom
| | - Greg M. Findlay
- Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, DundeeDD1 5EH, United Kingdom
| | - Jesse Mager
- Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, MA01003
| | - Ingolf Bach
- Department of Molecular, Cell and Cancer Biology, University of Massachusetts Chan Medical School, Worcester, MA01605
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12
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Zheng C, Wei Y, Zhang P, Lin K, He D, Teng H, Manyam G, Zhang Z, Liu W, Lee HRL, Tang X, He W, Islam N, Jain A, Chiu Y, Cao S, Diao Y, Meyer-Gauen S, Höök M, Malovannaya A, Li W, Hu M, Wang W, Xu H, Kopetz S, Chen Y. CRISPR-Cas9-based functional interrogation of unconventional translatome reveals human cancer dependency on cryptic non-canonical open reading frames. Nat Struct Mol Biol 2023; 30:1878-1892. [PMID: 37932451 PMCID: PMC10716047 DOI: 10.1038/s41594-023-01117-1] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/16/2021] [Accepted: 09/06/2023] [Indexed: 11/08/2023]
Abstract
Emerging evidence suggests that cryptic translation beyond the annotated translatome produces proteins with developmental or physiological functions. However, functions of cryptic non-canonical open reading frames (ORFs) in cancer remain largely unknown. To fill this gap and systematically identify colorectal cancer (CRC) dependency on non-canonical ORFs, we apply an integrative multiomic strategy, combining ribosome profiling and a CRISPR-Cas9 knockout screen with large-scale analysis of molecular and clinical data. Many such ORFs are upregulated in CRC compared to normal tissues and are associated with clinically relevant molecular subtypes. We confirm the in vivo tumor-promoting function of the microprotein SMIMP, encoded by a primate-specific, long noncoding RNA, the expression of which is associated with poor prognosis in CRC, is low in normal tissues and is specifically elevated in CRC and several other cancer types. Mechanistically, SMIMP interacts with the ATPase-forming domains of SMC1A, the core subunit of the cohesin complex, and facilitates SMC1A binding to cis-regulatory elements to promote epigenetic repression of the tumor-suppressive cell cycle regulators encoded by CDKN1A and CDKN2B. Thus, our study reveals a cryptic microprotein as an important component of cohesin-mediated gene regulation and suggests that the 'dark' proteome, encoded by cryptic non-canonical ORFs, may contain potential therapeutic or diagnostic targets.
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Affiliation(s)
- Caishang Zheng
- Department of Bioinformatics and Computational Biology, the University of Texas MD Anderson Cancer Center, Houston, TX, USA
| | - Yanjun Wei
- Department of Bioinformatics and Computational Biology, the University of Texas MD Anderson Cancer Center, Houston, TX, USA
| | - Peng Zhang
- Department of Bioinformatics and Computational Biology, the University of Texas MD Anderson Cancer Center, Houston, TX, USA
- Key Laboratory of RNA Biology, Center for Big Data Research in Health, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China
| | - Kangyu Lin
- Department of Bioinformatics and Computational Biology, the University of Texas MD Anderson Cancer Center, Houston, TX, USA
- Department of Gastrointestinal Medical Oncology, University of Texas MD Anderson Cancer Center, Houston, TX, USA
| | - Dandan He
- Department of Bioinformatics and Computational Biology, the University of Texas MD Anderson Cancer Center, Houston, TX, USA
- Sema4, Inc., Stamford, CT, USA
| | - Hongqi Teng
- Department of Experimental Radiation Oncology, the University of Texas MD Anderson Cancer Center, Houston, TX, USA
| | - Ganiraju Manyam
- Department of Bioinformatics and Computational Biology, the University of Texas MD Anderson Cancer Center, Houston, TX, USA
| | - Zhao Zhang
- Department of Biochemistry and Molecular Biology, McGovern Medical School, the University of Texas Health Science Center at Houston, Houston, TX, USA
- MOE Key Laboratory of Metabolism and Molecular Medicine, Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Fudan University, Shanghai, China
| | - Wen Liu
- Center for Infectious and Inflammatory Diseases, Texas A&M Health Science Center, Institute of Biosciences of Technology, Houston, TX, USA
| | - Hye Rin Lindsay Lee
- Department of Quantitative Health Sciences, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH, USA
| | - Ximing Tang
- Department of Translational Molecular Pathology, the University of Texas MD Anderson Cancer Center, Houston, TX, USA
| | - Wei He
- Department of Epigenetics and Molecular Carcinogenesis, the University of Texas MD Anderson Cancer Center, Houston, TX, USA
| | - Nelufa Islam
- Mass Spectrometry Proteomics Core, Baylor College of Medicine, Houston, TX, USA
| | - Antrix Jain
- Mass Spectrometry Proteomics Core, Baylor College of Medicine, Houston, TX, USA
| | - Yulun Chiu
- Department of Melanoma Medical Oncology, the University of Texas MD Anderson Cancer Center, Houston, TX, USA
| | - Shaolong Cao
- Department of Bioinformatics and Computational Biology, the University of Texas MD Anderson Cancer Center, Houston, TX, USA
| | - Yarui Diao
- Department of Cell Biology, Duke University Medical Center, Durham, NC, USA
- Duke Regeneration Center, Duke University Medical Center, Durham, NC, USA
- Department of Orthopedic Surgery, Duke University Medical Center, Durham, NC, USA
| | - Sherita Meyer-Gauen
- Department of Translational Molecular Pathology, the University of Texas MD Anderson Cancer Center, Houston, TX, USA
| | - Magnus Höök
- Center for Infectious and Inflammatory Diseases, Texas A&M Health Science Center, Institute of Biosciences of Technology, Houston, TX, USA
| | - Anna Malovannaya
- Mass Spectrometry Proteomics Core, Baylor College of Medicine, Houston, TX, USA
- Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX, USA
- Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX, USA
- Dan L. Duncan Cancer Center, Baylor College of Medicine, Houston, TX, USA
| | - Wenbo Li
- Department of Biochemistry and Molecular Biology, McGovern Medical School, the University of Texas Health Science Center at Houston, Houston, TX, USA
- Graduate School of Biomedical Sciences, University of Texas MD Anderson Cancer Center and UTHealth, Houston, TX, USA
| | - Ming Hu
- Department of Quantitative Health Sciences, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH, USA
| | - Wenyi Wang
- Department of Bioinformatics and Computational Biology, the University of Texas MD Anderson Cancer Center, Houston, TX, USA
- Quantitative Sciences Program, MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences, Houston, TX, USA
| | - Han Xu
- Department of Bioinformatics and Computational Biology, the University of Texas MD Anderson Cancer Center, Houston, TX, USA
- Department of Epigenetics and Molecular Carcinogenesis, the University of Texas MD Anderson Cancer Center, Houston, TX, USA
- Quantitative Sciences Program, MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences, Houston, TX, USA
- Genetics and Epigenetics Program, MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences, Houston, TX, USA
| | - Scott Kopetz
- Department of Gastrointestinal Medical Oncology, University of Texas MD Anderson Cancer Center, Houston, TX, USA
| | - Yiwen Chen
- Department of Bioinformatics and Computational Biology, the University of Texas MD Anderson Cancer Center, Houston, TX, USA.
- Quantitative Sciences Program, MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences, Houston, TX, USA.
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13
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Hernández-Quiles M, Martinez Campesino L, Morris I, Ilyas Z, Reynolds S, Soon Tan N, Sobrevals Alcaraz P, Stigter ECA, Varga Á, Varga J, van Es R, Vos H, Wilson HL, Kiss-Toth E, Kalkhoven E. The pseudokinase TRIB3 controls adipocyte lipid homeostasis and proliferation in vitro and in vivo. Mol Metab 2023; 78:101829. [PMID: 38445671 PMCID: PMC10663684 DOI: 10.1016/j.molmet.2023.101829] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/17/2023] [Revised: 10/11/2023] [Accepted: 10/19/2023] [Indexed: 03/07/2024] Open
Abstract
OBJECTIVE In vivo studies in humans and mice have implicated the pseudokinase Tribbles 3 (TRIB3) in various aspects of energy metabolism. Whilst cell-based studies indicate a role for TRIB3 in adipocyte differentiation and function, it is unclear if and how these cellular functions may contribute to overall metabolic health. METHODS We investigated the metabolic phenotype of whole-body Trib3 knockout (Trib3KO) mice, focusing on adipocyte and adipose tissue functions. In addition, we combined lipidomics, transcriptomics, interactomics and phosphoproteomics analyses to elucidate cell-intrinsic functions of TRIB3 in pre- and mature adipocytes. RESULTS Trib3KO mice display increased adiposity, but their insulin sensitivity remains unaltered. Trib3KO adipocytes are smaller and display higher Proliferating Cell Nuclear Antigen (PCNA) levels, indicating potential alterations in either i) proliferation-differentiation balance, ii) impaired expansion after cell division, or iii) an altered balance between lipid storage and release, or a combination thereof. Lipidome analyses suggest TRIB3 involvement in the latter two processes, as triglyceride storage is reduced and membrane composition, which can restrain cellular expansion, is altered. Integrated interactome, phosphoproteome and transcriptome analyses support a role for TRIB3 in all three cellular processes through multiple cellular pathways, including Mitogen Activated Protein Kinase- (MAPK/ERK), Protein Kinase A (PKA)-mediated signaling and Transcription Factor 7 like 2 (TCF7L2) and Beta Catenin-mediated gene expression. CONCLUSIONS Our findings support TRIB3 playing multiple distinct regulatory roles in the cytoplasm, nucleus and mitochondria, ultimately controlling adipose tissue homeostasis, rather than affecting a single cellular pathway.
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Affiliation(s)
- Miguel Hernández-Quiles
- Center for Molecular Medicine, University Medical Center Utrecht, Utrecht University, 3C584 CG Utrecht, The Netherlands
| | - Laura Martinez Campesino
- Division of Clinical Medicine, School of Medicine and Population Health, University of Sheffield, Sheffield S10 2TN, UK
| | - Imogen Morris
- Center for Molecular Medicine, University Medical Center Utrecht, Utrecht University, 3C584 CG Utrecht, The Netherlands
| | - Zabran Ilyas
- Division of Clinical Medicine, School of Medicine and Population Health, University of Sheffield, Sheffield S10 2TN, UK
| | - Steve Reynolds
- Division of Clinical Medicine, School of Medicine and Population Health, University of Sheffield, Sheffield S10 2TN, UK
| | - Nguan Soon Tan
- Lee Kong Chian School of Medicine, Nanyang Technological University Singapore, Clinical Sciences Building, 11 Mandalay Road, 308232 Singapore, Singapore; School of Biological Sciences, Nanyang Technological University Singapore, 60 Nanyang Drive, 637551 Singapore, Singapore
| | - Paula Sobrevals Alcaraz
- Oncode Institute and Molecular Cancer Research, Center for Molecular Medicine, University Medical Center Utrecht, Utrecht University, 3C584 CG Utrecht, The Netherlands
| | - Edwin C A Stigter
- Center for Molecular Medicine, University Medical Center Utrecht, Utrecht University, 3C584 CG Utrecht, The Netherlands
| | - Ákos Varga
- Department of Dermatology and Allergology, University of Szeged, H-6720 Szeged, Hungary
| | - János Varga
- Department of Dermatology and Allergology, University of Szeged, H-6720 Szeged, Hungary
| | - Robert van Es
- Oncode Institute and Molecular Cancer Research, Center for Molecular Medicine, University Medical Center Utrecht, Utrecht University, 3C584 CG Utrecht, The Netherlands
| | - Harmjan Vos
- Oncode Institute and Molecular Cancer Research, Center for Molecular Medicine, University Medical Center Utrecht, Utrecht University, 3C584 CG Utrecht, The Netherlands
| | - Heather L Wilson
- Division of Clinical Medicine, School of Medicine and Population Health, University of Sheffield, Sheffield S10 2TN, UK
| | - Endre Kiss-Toth
- Division of Clinical Medicine, School of Medicine and Population Health, University of Sheffield, Sheffield S10 2TN, UK
| | - Eric Kalkhoven
- Center for Molecular Medicine, University Medical Center Utrecht, Utrecht University, 3C584 CG Utrecht, The Netherlands.
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Wiegreffe C, Ehricke S, Schmid L, Andratschke J, Britsch S. Using i-GONAD for Cell-Type-Specific and Systematic Analysis of Developmental Transcription Factors In Vivo. BIOLOGY 2023; 12:1236. [PMID: 37759634 PMCID: PMC10526018 DOI: 10.3390/biology12091236] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/12/2023] [Revised: 09/08/2023] [Accepted: 09/12/2023] [Indexed: 09/29/2023]
Abstract
Transcription factors (TFs) regulate gene expression via direct DNA binding together with cofactors and in chromatin remodeling complexes. Their function is thus regulated in a spatiotemporal and cell-type-specific manner. To analyze the functions of TFs in a cell-type-specific context, genome-wide DNA binding, as well as the identification of interacting proteins, is required. We used i-GONAD (improved genome editing via oviductal nucleic acids delivery) in mice to genetically modify TFs by adding fluorescent reporter and affinity tags that can be exploited for the imaging and enrichment of target cells as well as chromatin immunoprecipitation and pull-down assays. As proof-of-principle, we showed the functional genetic modification of the closely related developmental TFs, Bcl11a and Bcl11b, in defined cell types of newborn mice. i-GONAD is a highly efficient procedure for modifying TF-encoding genes via the integration of small insertions, such as reporter and affinity tags. The novel Bcl11a and Bcl11b mouse lines, described in this study, will be used to improve our understanding of the Bcl11 family's function in neurodevelopment and associated disease.
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Affiliation(s)
- Christoph Wiegreffe
- Medical Faculty, Institute of Molecular and Cellular Anatomy, Ulm University, Albert-Einstein-Allee 11, 89081 Ulm, Germany
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15
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Zisis V, Andreadis D, Anastasiadou PA, Akrivou M, Vizirianakis IS, Anagnostou L, Malamos D, Paraskevopoulos K, Poulopoulos A. Expression of the Embryonic Cancer Stem Cells' Biomarkers SOX2 and OCT3/4 in Oral Leukoplakias and Squamous Cell Carcinomas: A Preliminary Study. Cureus 2023; 15:e45482. [PMID: 37859926 PMCID: PMC10584277 DOI: 10.7759/cureus.45482] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 09/18/2023] [Indexed: 10/21/2023] Open
Abstract
INTRODUCTION Cancer stem cells (CSCs) are incriminated for initiating the process of carcinogenesis either de novo or through the transformation of oral potentially malignant disorders (OPMDs) to oral squamous cell carcinoma (OSCC). The aim of this study was to detect the expression of embryonic-type CSC markers OCT3/4 and SOX2 in OSCCs and oral leukoplakias (OLs), the most common of OPMDs. MATERIALS AND METHODS The study type is experimental, and the study design is characterized as semiquantitative research, which belongs to the branch of experimental research. The experiment was conducted in the Department of Oral Medicine/Pathology, School of Dentistry, Aristotle University of Thessaloniki, Greece. This study focuses on the semiquantitative immunohistochemical (IHC) pattern of expression of CSCs protein-biomarkers SOX2 and OCT3/4, in paraffin embedded samples of 21 OSCCs of different grades of differentiation and 30 cases of OLs with different grades of dysplasia, compared to five cases of normal oral mucosa in both terms of cells' stain positivity and intensity. Statistical analysis was performed through SPSS 2017 Pearson Chi-square and the significance level was set at 0.05 (p=0.05). The expression of the respective genes of SOX2 and OCT3/4 was studied through quantitative polymerase chain reaction (qPCR), in paraffin-embedded samples of 12 cases of OLs with mild/non dysplasia and 19 cases moderately/poorly differentiated OSCCs(n=19) and five normal mucosa using the Independent Paired T-test. RESULTS The genes SOX2 and Oct3/4 were expressed in all examined cases although no statistically significant correlations among normal, OL and OSCC, were established. A nuclear/membrane staining of OCT3/4 was noticed only in three out of 21 OSCCs but in none of OLs or normal cases (without statistical significance). A characteristic nuclear staining of SOX2 was noticed in the majority of the samples, mostly in the basal and parabasal layers of the epithelium. SOX2 was significantly detected in the OSCCs group (strong positivity in 17/21) than in the OL group (30 cases, mostly mildly stained) (p-value=0.007), and the normal oral epithelium (mild stained, p=0.065). Furthermore, SOX2 was overexpressed in well differentiated OSCCs group (5/OSCCs, strongly stained) rather than in mildly dysplastic and non-dysplastic OLs samples (14/OLs, mildly stained) (p-value =0.035). CONCLUSION The characteristic expression of SOX2 but not of OCT3/4 in OLs' and OSCCs' lesions suggests the presence of neoplastic cells with certain CSC characteristics whose implication in the early stages of oral tumorigenesis could be further evaluated. The clinical use of SOX2, as prognostic factor, requires further experimental evaluation in larger number of samples.
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Affiliation(s)
- Vasileios Zisis
- Oral Medicine/Pathology, School of Dentistry, Aristotle University of Thessaloniki, Thessaloniki, GRC
| | - Dimitrios Andreadis
- Oral Medicine/Pathology, School of Dentistry, Aristotle University of Thessaloniki, Thessaloniki, GRC
| | - Pinelopi A Anastasiadou
- Oral Medicine/Pathology, School of Dentistry, Aristotle University of Thessaloniki, Thessaloniki, GRC
| | - Meni Akrivou
- Pharmacology, Aristotle University of Thessaloniki, Thessaloniki, GRC
| | - Ioannis S Vizirianakis
- Health Sciences, University of Nicosia, Nicosia, CYP
- Pharmacy, Aristotle University of Thessaloniki, Thessaloniki, GRC
| | - Lefteris Anagnostou
- Oral Medicine/Pathology, School of Dentistry, Aristotle University of Thessaloniki, Thessaloniki, GRC
| | - Dimitrios Malamos
- Oral Medicine, National and Kapodistrian University of Athens, Athens, GRC
| | | | - Athanasios Poulopoulos
- Oral Medicine/Pathology, School of Dentistry, Aristotle University of Thessaloniki, Thessaloniki, GRC
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16
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Wang J, Huang Y, Zhang C, Ruan Y, Tian Y, Wang F, Xu Y, Yu M, Wang J, Cheng Y, Liu L, Yang R, Wang J, Yang Y, Xiong J, Hu Y, Jian R, Ni B, Wu W, Zhang J. Identification and Functional Evaluation of Alternative Splice Variants of Dax1 in Mouse Embryonic Stem Cells. Stem Cells Dev 2023; 32:554-564. [PMID: 37261981 DOI: 10.1089/scd.2023.0037] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 06/03/2023] Open
Abstract
Dax1 (Nr0b1; Dosage-sensitive sex reversal-adrenal hypoplasia congenital on the X-chromosome gene-1) is an important component of the transcription factor network that governs pluripotency in mouse embryonic stem cells (ESCs). Functional evaluation of alternative splice variants of pluripotent transcription factors has shed additional insight on the maintenance of ESC pluripotency and self-renewal. Dax1 splice variants have not been identified and characterized in mouse ESCs. We identified 18 new transcripts of Dax1 with putative protein-coding properties and compared their protein structures with known Dax1 protein (Dax1-472). The expression pattern analysis showed that the novel isoforms were cotranscribed with Dax1-472 in mouse ESCs, but they had transcriptional heterogeneity among single cells and the subcellular localization of the encoded proteins differed. Cell function experiments indicated that Dax1-404 repressed Gata6 transcription and functionally replaced Dax1-472, while Dax1-38 and Dax1-225 partially antagonized Dax1-472 transcriptional repression. This study provided a comprehensive characterization of the Dax1 splice variants in mouse ESCs and suggested complex effects of Dax1 variants in a self-renewal regulatory network.
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Affiliation(s)
- Jiaqi Wang
- Laboratory of Stem Cell and Developmental Biology, Department of Histology and Embryology, College of Basic Medical Science, Army Medical University, Chongqing, China
- Department of Pathophysiology, College of High-Altitude Military Medicine, Army Medical University, Chongqing, China
| | - Yi Huang
- Biomedical Analysis Center, Army Medical University, Chongqing, China
| | - Chen Zhang
- Laboratory of Stem Cell and Developmental Biology, Department of Histology and Embryology, College of Basic Medical Science, Army Medical University, Chongqing, China
| | - Yan Ruan
- Laboratory of Stem Cell and Developmental Biology, Department of Histology and Embryology, College of Basic Medical Science, Army Medical University, Chongqing, China
| | - Yanping Tian
- Laboratory of Stem Cell and Developmental Biology, Department of Histology and Embryology, College of Basic Medical Science, Army Medical University, Chongqing, China
| | - Fengsheng Wang
- Laboratory of Stem Cell and Developmental Biology, Department of Histology and Embryology, College of Basic Medical Science, Army Medical University, Chongqing, China
- State Key Laboratory of NBC Protection for Civilian, Beijing, China
| | - Yixiao Xu
- Laboratory of Stem Cell and Developmental Biology, Department of Histology and Embryology, College of Basic Medical Science, Army Medical University, Chongqing, China
| | - Meng Yu
- Laboratory of Stem Cell and Developmental Biology, Department of Histology and Embryology, College of Basic Medical Science, Army Medical University, Chongqing, China
- Department of Joint Surgery, Southwest Hospital, The First Hospital Affiliated to Army Medical University, Chongqing, China
| | - Jiangjun Wang
- Laboratory of Stem Cell and Developmental Biology, Department of Histology and Embryology, College of Basic Medical Science, Army Medical University, Chongqing, China
- Department of Cell Biology, College of Basic Medical Science, Army Medical University, Chongqing, China
| | - Yuda Cheng
- Laboratory of Stem Cell and Developmental Biology, Department of Histology and Embryology, College of Basic Medical Science, Army Medical University, Chongqing, China
| | - Lianlian Liu
- Laboratory of Stem Cell and Developmental Biology, Department of Histology and Embryology, College of Basic Medical Science, Army Medical University, Chongqing, China
| | - Ran Yang
- Laboratory of Stem Cell and Developmental Biology, Department of Histology and Embryology, College of Basic Medical Science, Army Medical University, Chongqing, China
- Department of Pathophysiology, College of High-Altitude Military Medicine, Army Medical University, Chongqing, China
| | - Jiali Wang
- Laboratory of Stem Cell and Developmental Biology, Department of Histology and Embryology, College of Basic Medical Science, Army Medical University, Chongqing, China
| | - Yi Yang
- Experimental Center of Basic Medicine, College of Basic Medical Science, Army Medical University, Chongqing, China
| | - Jiaxiang Xiong
- Experimental Center of Basic Medicine, College of Basic Medical Science, Army Medical University, Chongqing, China
| | - Yan Hu
- Department of Military Basic Training and Army Management, Army Health Service Training Base, Army Medical University, Chongqing, China
| | - Rui Jian
- Laboratory of Stem Cell and Developmental Biology, Department of Histology and Embryology, College of Basic Medical Science, Army Medical University, Chongqing, China
| | - Bing Ni
- Department of Pathophysiology, College of High-Altitude Military Medicine, Army Medical University, Chongqing, China
| | - Wei Wu
- Department of Department of Thoracic Surgery, Southwest Hospital, The First Hospital Affiliated to Army Medical University, Chongqing, China
| | - Junlei Zhang
- Laboratory of Stem Cell and Developmental Biology, Department of Histology and Embryology, College of Basic Medical Science, Army Medical University, Chongqing, China
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17
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Kabir M, Stuart HM, Lopes FM, Fotiou E, Keavney B, Doig AJ, Woolf AS, Hentges KE. Predicting congenital renal tract malformation genes using machine learning. Sci Rep 2023; 13:13204. [PMID: 37580336 PMCID: PMC10425350 DOI: 10.1038/s41598-023-38110-z] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/03/2023] [Accepted: 07/03/2023] [Indexed: 08/16/2023] Open
Abstract
Congenital renal tract malformations (RTMs) are the major cause of severe kidney failure in children. Studies to date have identified defined genetic causes for only a minority of human RTMs. While some RTMs may be caused by poorly defined environmental perturbations affecting organogenesis, it is likely that numerous causative genetic variants have yet to be identified. Unfortunately, the speed of discovering further genetic causes for RTMs is limited by challenges in prioritising candidate genes harbouring sequence variants. Here, we exploited the computer-based artificial intelligence methodology of supervised machine learning to identify genes with a high probability of being involved in renal development. These genes, when mutated, are promising candidates for causing RTMs. With this methodology, the machine learning classifier determines which attributes are common to renal development genes and identifies genes possessing these attributes. Here we report the validation of an RTM gene classifier and provide predictions of the RTM association status for all protein-coding genes in the mouse genome. Overall, our predictions, whilst not definitive, can inform the prioritisation of genes when evaluating patient sequence data for genetic diagnosis. This knowledge of renal developmental genes will accelerate the processes of reaching a genetic diagnosis for patients born with RTMs.
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Affiliation(s)
- Mitra Kabir
- CentreDivision of Evolution, Infection and Genomics, Faculty of Biology, Medicine and Health, Manchester Academic Health Science Centre, The University of Manchester, Oxford Road, Manchester, M13 9PT, UK
| | - Helen M Stuart
- CentreDivision of Evolution, Infection and Genomics, Faculty of Biology, Medicine and Health, Manchester Academic Health Science Centre, The University of Manchester, Oxford Road, Manchester, M13 9PT, UK
- Manchester Centre for Genomic Medicine, St. Mary's Hospital, Health Innovation Manchester, Manchester University Foundation NHS Trust, Manchester, M13 9WL, UK
| | - Filipa M Lopes
- Division of Cell Matrix Biology and Regenerative Medicine, School of Biological Sciences, Faculty of Biology, Medicine and Health, The University of Manchester, Manchester, M13 9PL, UK
| | - Elisavet Fotiou
- Division of Cardiovascular Sciences, School of Medical Sciences, Faculty of Biology, Medicine, and Health, The University of Manchester, Manchester, M13 9PL, UK
- C.B.B Lifeline Biotech Ltd, 5 Propontidos Street, Strovolos, 2033, Nicosia, Cyprus
| | - Bernard Keavney
- Division of Cardiovascular Sciences, School of Medical Sciences, Faculty of Biology, Medicine, and Health, The University of Manchester, Manchester, M13 9PL, UK
- Manchester Heart Institute, Manchester University NHS Foundation Trust, Manchester Academic Health Science Centre, Manchester, M13 9WL, UK
| | - Andrew J Doig
- Division of Neuroscience, School of Biological Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Stopford Building, Manchester, M13 9BL, UK
| | - Adrian S Woolf
- Division of Cell Matrix Biology and Regenerative Medicine, School of Biological Sciences, Faculty of Biology, Medicine and Health, The University of Manchester, Manchester, M13 9PL, UK
- Department of Nephrology, Royal Manchester Children's Hospital, Manchester Academic Health Science Centre, Manchester, M13 9WL, UK
| | - Kathryn E Hentges
- CentreDivision of Evolution, Infection and Genomics, Faculty of Biology, Medicine and Health, Manchester Academic Health Science Centre, The University of Manchester, Oxford Road, Manchester, M13 9PT, UK.
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18
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Zhang Y, Li X, Gao S, Liao Y, Luo Y, Liu M, Bian Y, Xiong H, Yue Y, He A. Genetic reporter for live tracing fluid flow forces during cell fate segregation in mouse blastocyst development. Cell Stem Cell 2023; 30:1110-1123.e9. [PMID: 37541214 DOI: 10.1016/j.stem.2023.07.003] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/28/2023] [Revised: 06/02/2023] [Accepted: 07/10/2023] [Indexed: 08/06/2023]
Abstract
Mechanical forces are known to be important in mammalian blastocyst formation; however, due to limited tools, specific force inputs and how they relay to first cell fate control of inner cell mass (ICM) and/or trophectoderm (TE) remain elusive. Combining in toto live imaging and various perturbation experiments, we demonstrate and measure fluid flow forces existing in the mouse blastocyst cavity and identify Klf2(Krüppel-like factor 2) as a fluid force reporter with force-responsive enhancers. Long-term live imaging and lineage reconstructions reveal that blastomeres subject to higher fluid flow forces adopt ICM cell fates. These are reinforced by internal ferrofluid-induced flow force assays. We also utilize ex vivo fluid flow force mimicking and pharmacological perturbations to confirm mechanosensing specificity. Together, we report a genetically encoded reporter for continuously monitoring fluid flow forces and cell fate decisions and provide a live imaging framework to infer force information enriched lineage landscape during development. VIDEO ABSTRACT.
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Affiliation(s)
- Youdong Zhang
- Institute of Molecular Medicine, National Biomedical Imaging Center, College of Future Technology, Peking-Tsinghua Center for Life Sciences, Peking University, Beijing 100871, China
| | - Xin Li
- Institute of Molecular Medicine, National Biomedical Imaging Center, College of Future Technology, Peking-Tsinghua Center for Life Sciences, Peking University, Beijing 100871, China
| | - Shu Gao
- Institute of Molecular Medicine, National Biomedical Imaging Center, College of Future Technology, Peking-Tsinghua Center for Life Sciences, Peking University, Beijing 100871, China
| | - Yuanhui Liao
- School of Software and Microelectronics, Peking University, Beijing 100871, China
| | - Yingjie Luo
- Institute of Molecular Medicine, National Biomedical Imaging Center, College of Future Technology, Peking-Tsinghua Center for Life Sciences, Peking University, Beijing 100871, China
| | - Min Liu
- Institute of Molecular Medicine, National Biomedical Imaging Center, College of Future Technology, Peking-Tsinghua Center for Life Sciences, Peking University, Beijing 100871, China
| | - Yunkun Bian
- Institute of Molecular Medicine, National Biomedical Imaging Center, College of Future Technology, Peking-Tsinghua Center for Life Sciences, Peking University, Beijing 100871, China
| | - Haiqing Xiong
- Institute of Molecular Medicine, National Biomedical Imaging Center, College of Future Technology, Peking-Tsinghua Center for Life Sciences, Peking University, Beijing 100871, China
| | - Yanzhu Yue
- Institute of Molecular Medicine, National Biomedical Imaging Center, College of Future Technology, Peking-Tsinghua Center for Life Sciences, Peking University, Beijing 100871, China; Department of Cell Fate and Diseases, Jilin Provincial Key Laboratory of Women's Reproductive Health, the First Hospital of Jilin University, Changchun, Jilin 130061, China.
| | - Aibin He
- Institute of Molecular Medicine, National Biomedical Imaging Center, College of Future Technology, Peking-Tsinghua Center for Life Sciences, Peking University, Beijing 100871, China.
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19
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Varzideh F, Gambardella J, Kansakar U, Jankauskas SS, Santulli G. Molecular Mechanisms Underlying Pluripotency and Self-Renewal of Embryonic Stem Cells. Int J Mol Sci 2023; 24:8386. [PMID: 37176093 PMCID: PMC10179698 DOI: 10.3390/ijms24098386] [Citation(s) in RCA: 16] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/01/2023] [Revised: 04/29/2023] [Accepted: 05/02/2023] [Indexed: 05/15/2023] Open
Abstract
Embryonic stem cells (ESCs) are derived from the inner cell mass (ICM) of the blastocyst. ESCs have two distinctive properties: ability to proliferate indefinitely, a feature referred as "self-renewal", and to differentiate into different cell types, a peculiar characteristic known as "pluripotency". Self-renewal and pluripotency of ESCs are finely orchestrated by precise external and internal networks including epigenetic modifications, transcription factors, signaling pathways, and histone modifications. In this systematic review, we examine the main molecular mechanisms that sustain self-renewal and pluripotency in both murine and human ESCs. Moreover, we discuss the latest literature on human naïve pluripotency.
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Affiliation(s)
- Fahimeh Varzideh
- Department of Medicine (Division of Cardiology), Wilf Family Cardiovascular Research Institute, Einstein Institute for Aging Research, Institute for Neuroimmunology and Inflammation (INI), Albert Einstein College of Medicine, New York, NY 10461, USA
| | - Jessica Gambardella
- Department of Molecular Pharmacology, Einstein-Mount Sinai Diabetes Research Center (ES-DRC), Fleischer Institute for Diabetes and Metabolism (FIDAM), Albert Einstein College of Medicine, New York, NY 10461, USA
| | - Urna Kansakar
- Department of Medicine (Division of Cardiology), Wilf Family Cardiovascular Research Institute, Einstein Institute for Aging Research, Institute for Neuroimmunology and Inflammation (INI), Albert Einstein College of Medicine, New York, NY 10461, USA
| | - Stanislovas S. Jankauskas
- Department of Medicine (Division of Cardiology), Wilf Family Cardiovascular Research Institute, Einstein Institute for Aging Research, Institute for Neuroimmunology and Inflammation (INI), Albert Einstein College of Medicine, New York, NY 10461, USA
| | - Gaetano Santulli
- Department of Medicine (Division of Cardiology), Wilf Family Cardiovascular Research Institute, Einstein Institute for Aging Research, Institute for Neuroimmunology and Inflammation (INI), Albert Einstein College of Medicine, New York, NY 10461, USA
- Department of Molecular Pharmacology, Einstein-Mount Sinai Diabetes Research Center (ES-DRC), Fleischer Institute for Diabetes and Metabolism (FIDAM), Albert Einstein College of Medicine, New York, NY 10461, USA
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20
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Uribe-Etxebarria V, Pineda JR, García-Gallastegi P, Agliano A, Unda F, Ibarretxe G. Notch and Wnt Signaling Modulation to Enhance DPSC Stemness and Therapeutic Potential. Int J Mol Sci 2023; 24:ijms24087389. [PMID: 37108549 PMCID: PMC10138690 DOI: 10.3390/ijms24087389] [Citation(s) in RCA: 6] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/27/2023] [Revised: 04/16/2023] [Accepted: 04/16/2023] [Indexed: 04/29/2023] Open
Abstract
The Dental Pulp of permanent human teeth is home to stem cells with remarkable multilineage differentiation ability: human Dental Pulp Stem Cells (DPSCs). These cells display a very notorious expression of pluripotency core factors, and the ability to give rise to mature cell lineages belonging to the three embryonic layers. For these reasons, several researchers in the field have long considered human DPSCs as pluripotent-like cells. Notably, some signaling pathways such as Notch and Wnt contribute to maintaining the stemness of these cells through a complex network involving metabolic and epigenetic regulatory mechanisms. The use of recombinant proteins and selective pharmacological modulators of Notch and Wnt pathways, together with serum-free media and appropriate scaffolds that allow the maintenance of the non-differentiated state of hDPSC cultures could be an interesting approach to optimize the potency of these stem cells, without a need for genetic modification. In this review, we describe and integrate findings that shed light on the mechanisms responsible for stemness maintenance of hDPSCs, and how these are regulated by Notch/Wnt activation, drawing some interesting parallelisms with pluripotent stem cells. We summarize previous work on the stem cell field that includes interactions between epigenetics, metabolic regulations, and pluripotency core factor expression in hDPSCs and other stem cell types.
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Affiliation(s)
| | - Jose Ramon Pineda
- Cell Biology and Histology Department, University of the Basque Country (UPV/EHU), Barrio Sarriena s/n, 48940 Leioa, Spain
- Achucarro Basque Center for Neuroscience Fundazioa Leioa, Sede Building, 3rd Floor, 48940 Leioa, Spain
| | - Patricia García-Gallastegi
- Physiology Department, University of the Basque Country (UPV/EHU), Barrio Sarriena s/n, 48940 Leioa, Spain
| | - Alice Agliano
- Division of Radiotherapy and Imaging, Cancer Research UK Cancer Imaging Centre, The Institute of Cancer Research and The Royal Marsden NHS Foundation Trust, London SW7 3RP, UK
- Department of Materials and Department of Bioengineering, Institute of Biomedical Engineering, Imperial College London, Exhibition Road, London SW7 2AZ, UK
| | - Fernando Unda
- Cell Biology and Histology Department, University of the Basque Country (UPV/EHU), Barrio Sarriena s/n, 48940 Leioa, Spain
| | - Gaskon Ibarretxe
- Cell Biology and Histology Department, University of the Basque Country (UPV/EHU), Barrio Sarriena s/n, 48940 Leioa, Spain
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21
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Liu C, Yu P, Ren Z, Yao F, Wang L, Hu G, Li P, Zhao Q. Rif1 Regulates Self-Renewal and Impedes Mesendodermal Differentiation of Mouse Embryonic Stem Cells. Stem Cell Rev Rep 2023:10.1007/s12015-023-10525-1. [PMID: 36971904 PMCID: PMC10366267 DOI: 10.1007/s12015-023-10525-1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 03/02/2023] [Indexed: 03/29/2023]
Abstract
Abstract
Background
RAP1 interacting factor 1 (Rif1) is highly expressed in mice embryos and mouse embryonic stem cells (mESCs). It plays critical roles in telomere length homeostasis, DNA damage, DNA replication timing and ERV silencing. However, whether Rif1 regulates early differentiation of mESC is still unclear.
Methods
In this study, we generated a Rif1 conditional knockout mouse embryonic stem (ES) cell line based on Cre-loxP system. Western blot, flow cytometry, quantitative real-time polymerase chain reaction (qRT-PCR), RNA high-throughput sequencing (RNA-Seq), chromatin immunoprecipitation followed high-throughput sequencing (ChIP-Seq), chromatin immunoprecipitation quantitative PCR (ChIP-qPCR), immunofluorescence, and immunoprecipitation were employed for phenotype and molecular mechanism assessment.
Results
Rif1 plays important roles in self-renewal and pluripotency of mESCs and loss of Rif1 promotes mESC differentiation toward the mesendodermal germ layers. We further show that Rif1 interacts with histone H3K27 methyltransferase EZH2, a subunit of PRC2, and regulates the expression of developmental genes by directly binding to their promoters. Rif1 deficiency reduces the occupancy of EZH2 and H3K27me3 on mesendodermal gene promoters and activates ERK1/2 activities.
Conclusion
Rif1 is a key factor in regulating the pluripotency, self-renewal, and lineage specification of mESCs. Our research provides new insights into the key roles of Rif1 in connecting epigenetic regulations and signaling pathways for cell fate determination and lineage specification of mESCs.
Graphical abstract
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Markman S, Zada M, David E, Giladi A, Amit I, Zelzer E. A single-cell census of mouse limb development identifies complex spatiotemporal dynamics of skeleton formation. Dev Cell 2023; 58:565-581.e4. [PMID: 36931270 DOI: 10.1016/j.devcel.2023.02.013] [Citation(s) in RCA: 9] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/17/2022] [Revised: 10/20/2022] [Accepted: 02/20/2023] [Indexed: 03/18/2023]
Abstract
Limb development has long served as a model system for coordinated spatial patterning of progenitor cells. Here, we identify a population of naive limb progenitors and show that they differentiate progressively to form the skeleton in a complex, non-consecutive, three-dimensional pattern. Single-cell RNA sequencing of the developing mouse forelimb identified three progenitor states: naive, proximal, and autopodial, as well as Msx1 as a marker for the naive progenitors. In vivo lineage tracing confirmed this role and localized the naive progenitors to the outer margin of the limb, along the anterior-posterior axis. Sequential pulse-chase experiments showed that the progressive transition of Msx1+ naive progenitors into proximal and autopodial progenitors coincides with their differentiation to Sox9+ chondroprogenitors, which occurs along all the forming skeletal segments. Indeed, tracking the spatiotemporal sequence of differentiation showed that the skeleton forms progressively in a complex pattern. These findings suggest an alternative model for limb skeleton development.
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Affiliation(s)
- Svetlana Markman
- Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 7610001, Israel
| | - Mor Zada
- Department of Immunology, Weizmann Institute of Science, Rehovot 7610001, Israel
| | - Eyal David
- Department of Immunology, Weizmann Institute of Science, Rehovot 7610001, Israel
| | - Amir Giladi
- Department of Immunology, Weizmann Institute of Science, Rehovot 7610001, Israel
| | - Ido Amit
- Department of Immunology, Weizmann Institute of Science, Rehovot 7610001, Israel.
| | - Elazar Zelzer
- Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 7610001, Israel.
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23
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A Simplified and Effective Approach for the Isolation of Small Pluripotent Stem Cells Derived from Human Peripheral Blood. Biomedicines 2023; 11:biomedicines11030787. [PMID: 36979766 PMCID: PMC10045871 DOI: 10.3390/biomedicines11030787] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/25/2023] [Revised: 02/24/2023] [Accepted: 03/03/2023] [Indexed: 03/08/2023] Open
Abstract
Pluripotent stem cells are key players in regenerative medicine. Embryonic pluripotent stem cells, despite their significant advantages, are associated with limitations such as their inadequate availability and the ethical dilemmas in their isolation and clinical use. The discovery of very small embryonic-like (VSEL) stem cells addressed the aforementioned limitations, but their isolation technique remains a challenge due to their small cell size and their efficiency in isolation. Here, we report a simplified and effective approach for the isolation of small pluripotent stem cells derived from human peripheral blood. Our approach results in a high yield of small blood stem cell (SBSC) population, which expresses pluripotent embryonic markers (e.g., Nanog, SSEA-3) and the Yamanaka factors. Further, a fraction of SBSCs also co-express hematopoietic markers (e.g., CD45 and CD90) and/or mesenchymal markers (e.g., CD29, CD105 and PTH1R), suggesting a mixed stem cell population. Finally, quantitative proteomic profiling reveals that SBSCs contain various stem cell markers (CD9, ITGA6, MAPK1, MTHFD1, STAT3, HSPB1, HSPA4), and Transcription reg complex factors (e.g., STAT5B, PDLIM1, ANXA2, ATF6, CAMK1). In conclusion, we present a novel, simplified and effective isolating process that yields an abundant population of small-sized cells with characteristics of pluripotency from human peripheral blood.
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Zheng C, Wei Y, Zhang P, Xu L, Zhang Z, Lin K, Hou J, Lv X, Ding Y, Chiu Y, Jain A, Islam N, Malovannaya A, Wu Y, Ding F, Xu H, Sun M, Chen X, Chen Y. CRISPR/Cas9 screen uncovers functional translation of cryptic lncRNA-encoded open reading frames in human cancer. J Clin Invest 2023; 133:e159940. [PMID: 36856111 PMCID: PMC9974104 DOI: 10.1172/jci159940] [Citation(s) in RCA: 20] [Impact Index Per Article: 10.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/16/2022] [Accepted: 01/19/2023] [Indexed: 03/02/2023] Open
Abstract
Emerging evidence suggests that cryptic translation within long noncoding RNAs (lncRNAs) may produce novel proteins with important developmental/physiological functions. However, the role of this cryptic translation in complex diseases (e.g., cancer) remains elusive. Here, we applied an integrative strategy combining ribosome profiling and CRISPR/Cas9 screening with large-scale analysis of molecular/clinical data for breast cancer (BC) and identified estrogen receptor α-positive (ER+) BC dependency on the cryptic ORFs encoded by lncRNA genes that were upregulated in luminal tumors. We confirmed the in vivo tumor-promoting function of an unannotated protein, GATA3-interacting cryptic protein (GT3-INCP) encoded by LINC00992, the expression of which was associated with poor prognosis in luminal tumors. GTE-INCP was upregulated by estrogen/ER and regulated estrogen-dependent cell growth. Mechanistically, GT3-INCP interacted with GATA3, a master transcription factor key to mammary gland development/BC cell proliferation, and coregulated a gene expression program that involved many BC susceptibility/risk genes and impacted estrogen response/cell proliferation. GT3-INCP/GATA3 bound to common cis regulatory elements and upregulated the expression of the tumor-promoting and estrogen-regulated BC susceptibility/risk genes MYB and PDZK1. Our study indicates that cryptic lncRNA-encoded proteins can be an important integrated component of the master transcriptional regulatory network driving aberrant transcription in cancer, and suggests that the "hidden" lncRNA-encoded proteome might be a new space for therapeutic target discovery.
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Affiliation(s)
- Caishang Zheng
- Department of Bioinformatics and Computational Biology, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA
| | - Yanjun Wei
- Department of Bioinformatics and Computational Biology, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA
| | - Peng Zhang
- Department of Bioinformatics and Computational Biology, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA
| | - Longyong Xu
- Department of Molecular and Cellular Biology
- Lester and Sue Smith Breast Center, and
- Dan L. Duncan Comprehensive Cancer Center, Baylor College of Medicine, Houston, Texas, USA
| | - Zhenzhen Zhang
- Department of Physics and Astronomy, Clemson University, Clemson, South Carolina, USA
| | - Kangyu Lin
- Department of Bioinformatics and Computational Biology, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA
| | - Jiakai Hou
- Department of Bioinformatics and Computational Biology, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA
| | - Xiangdong Lv
- Department of Molecular and Cellular Biology
- Lester and Sue Smith Breast Center, and
- Dan L. Duncan Comprehensive Cancer Center, Baylor College of Medicine, Houston, Texas, USA
| | - Yao Ding
- Department of Molecular and Cellular Biology
- Lester and Sue Smith Breast Center, and
- Dan L. Duncan Comprehensive Cancer Center, Baylor College of Medicine, Houston, Texas, USA
| | - Yulun Chiu
- Department of Melanoma Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA
| | | | | | - Anna Malovannaya
- Dan L. Duncan Comprehensive Cancer Center, Baylor College of Medicine, Houston, Texas, USA
- Mass Spectrometry Proteomics Core and
- Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas, USA
| | - Yun Wu
- Department of Pathology, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA
| | - Feng Ding
- Department of Physics and Astronomy, Clemson University, Clemson, South Carolina, USA
| | - Han Xu
- Department of Bioinformatics and Computational Biology, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA
- Department of Epigenetics and Molecular Carcinogenesis, The University of Texas MD Anderson Cancer Center
- Genetics and Epigenetics Program, and
- Quantitative Sciences Program, MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences, Houston, Texas, USA
| | - Ming Sun
- Department of Bioinformatics and Computational Biology, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA
| | - Xi Chen
- Department of Molecular and Cellular Biology
- Lester and Sue Smith Breast Center, and
- Dan L. Duncan Comprehensive Cancer Center, Baylor College of Medicine, Houston, Texas, USA
| | - Yiwen Chen
- Department of Bioinformatics and Computational Biology, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA
- Quantitative Sciences Program, MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences, Houston, Texas, USA
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25
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Zhang X, Fang B, Huang YF. Transcription factor binding sites are frequently under accelerated evolution in primates. Nat Commun 2023; 14:783. [PMID: 36774380 PMCID: PMC9922303 DOI: 10.1038/s41467-023-36421-3] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/30/2022] [Accepted: 01/31/2023] [Indexed: 02/13/2023] Open
Abstract
Recent comparative genomic studies have identified many human accelerated elements (HARs) with elevated substitution rates in the human lineage. However, it remains unknown to what extent transcription factor binding sites (TFBSs) are under accelerated evolution in humans and other primates. Here, we introduce two pooling-based phylogenetic methods with dramatically enhanced sensitivity to examine accelerated evolution in TFBSs. Using these new methods, we show that more than 6000 TFBSs annotated in the human genome have experienced accelerated evolution in Hominini, apes, and Old World monkeys. Although these TFBSs individually show relatively weak signals of accelerated evolution, they collectively are more abundant than HARs. Also, we show that accelerated evolution in Pol III binding sites may be driven by lineage-specific positive selection, whereas accelerated evolution in other TFBSs might be driven by nonadaptive evolutionary forces. Finally, the accelerated TFBSs are enriched around developmental genes, suggesting that accelerated evolution in TFBSs may drive the divergence of developmental processes between primates.
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Affiliation(s)
- Xinru Zhang
- Department of Biology, Pennsylvania State University, University Park, PA, 16802, USA. .,Huck Institutes of the Life Sciences, Pennsylvania State University, University Park, PA, 16802, USA. .,Bioinformatics and Genomics Graduate Program, Pennsylvania State University, University Park, PA, 16802, USA.
| | - Bohao Fang
- Department of Organismic and Evolutionary Biology and the Museum of Comparative Zoology, Harvard University, Boston, MA, 02135, USA
| | - Yi-Fei Huang
- Department of Biology, Pennsylvania State University, University Park, PA, 16802, USA. .,Huck Institutes of the Life Sciences, Pennsylvania State University, University Park, PA, 16802, USA.
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26
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Malaga Gadea FC, Nikolova EN. Structural Plasticity of Pioneer Factor Sox2 and DNA Bendability Modulate Nucleosome Engagement and Sox2-Oct4 Synergism. J Mol Biol 2023; 435:167916. [PMID: 36495920 PMCID: PMC10184184 DOI: 10.1016/j.jmb.2022.167916] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/01/2022] [Revised: 12/01/2022] [Accepted: 12/01/2022] [Indexed: 12/12/2022]
Abstract
Pioneer transcription factors (pTFs) can bind directly to silent chromatin and promote vital transcriptional programs. Here, by integrating high-resolution nuclear magnetic resonance (NMR) spectroscopy with biochemistry, we reveal new structural and mechanistic insights into the interaction of pluripotency pTFs and functional partners Sox2 and Oct4 with nucleosomes. We find that the affinity and conformation of Sox2 for solvent-exposed nucleosome sites depend strongly on their position and DNA sequence. Sox2, which is partially disordered but becomes structured upon DNA binding and bending, forms a super-stable nucleosome complex at superhelical location +5 (SHL+5) with similar affinity and conformation to that with naked DNA. However, at suboptimal internal and end-positioned sites where DNA may be harder to deform, Sox2 favors partially unfolded and more dynamic states that are encoded in its intrinsic flexibility. Importantly, Sox2 structure and DNA bending can be stabilized by synergistic Oct4 binding, but only on adjacent motifs near the nucleosome edge and with the full Oct4 DNA-binding domain. Further mutational studies reveal that strategically impaired Sox2 folding is coupled to reduced DNA bending and inhibits nucleosome binding and Sox2-Oct4 cooperation, while increased nucleosomal DNA flexibility enhances Sox2 association. Together, our findings fit a model where the site-specific DNA bending propensity and structural plasticity of Sox2 govern distinct modes of nucleosome engagement and modulate Sox2-Oct4 synergism. The principles outlined here can potentially guide pTF site selection in the genome and facilitate interaction with other chromatin factors or chromatin opening in vivo.
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Affiliation(s)
- Fabiana C Malaga Gadea
- T.C. Jenkins Department of Biophysics, Johns Hopkins University, Baltimore, MD 21218, USA
| | - Evgenia N Nikolova
- T.C. Jenkins Department of Biophysics, Johns Hopkins University, Baltimore, MD 21218, USA.
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27
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Wen D, Hu M, Guo W, Wu J, Wu Y. Multi-SUMOylation of NAC1 is essential for the growth of prostate cancer cells. Biochem Biophys Res Commun 2023; 641:148-154. [PMID: 36527749 DOI: 10.1016/j.bbrc.2022.12.015] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/13/2022] [Revised: 11/21/2022] [Accepted: 12/03/2022] [Indexed: 12/14/2022]
Abstract
Nucleus accumbens-associated 1 (NAC1) is a member of pox virus and zinc finger/bric-a-brac tramtrack broad complex (BTB/POZ) gene family. Overexpression of NAC1 is implicated in cancer development, recurrence and chemotherapy resistance. In our previous study, we found NAC1 was a potential small ubiquitin-like modifier (SUMO) substrate in prostate cancer cells. However, there was still lack of evidences to further support and validate the result. In this work, we found that NAC1 is a multi-SUMO-sites acceptor. The SUMO acceptor lysines were K167, K318, K368, K483 and K498. SUMOylation didn't alter the localization of NAC1, but facilitated the formation of NAC1 nuclear bodies. Compared with NAC1 wild type (NAC1 WT), the SUMO-sites mutant of NAC1 (NAC1 SM) suppressed cell proliferation and tumor growth in cellular and animal levels. This work uncovered the function of SUMOylation of NAC1 in prostate cancer cells.
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Affiliation(s)
- Donghua Wen
- Department of Laboratory Medicine, Shanghai East Hospital, Tongji University School of Medicine, Shanghai, 200123, China.
| | - Min Hu
- Department of Laboratory Medicine, Shanghai East Hospital, Tongji University School of Medicine, Shanghai, 200123, China
| | - Wenzheng Guo
- Department of Laboratory Medicine, Shanghai East Hospital, Tongji University School of Medicine, Shanghai, 200123, China
| | - Jingjing Wu
- Department of Laboratory Medicine, Shanghai East Hospital, Tongji University School of Medicine, Shanghai, 200123, China
| | - Yingli Wu
- Hongqiao International Institute of Medicine, Shanghai Tongren Hospital / Faculty of Basic Medicine, Key Laboratory of Cell Differentiation and Apoptosis of the Chinese Ministry of Education, Chinese Academy of Medical Sciences Research Unit 2019RU043, Shanghai Jiao Tong University School of Medicine (SJTU-SM), Shanghai, 200025, China.
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28
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Xiong H, Liu B, Liu XY, Xia ZK, Lu M, Hu CH, Liu P. circ_rac GTPase-Activating Protein 1 Facilitates Stemness and Metastasis of Non-Small Cell Lung Cancer via Polypyrimidine Tract-Binding Protein 1 Recruitment to Promote Sirtuin-3-Mediated Replication Timing Regulatory Factor 1 Deacetylation. J Transl Med 2023; 103:100010. [PMID: 36748197 DOI: 10.1016/j.labinv.2022.100010] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/07/2022] [Revised: 07/26/2022] [Accepted: 08/20/2022] [Indexed: 01/19/2023] Open
Abstract
Circular RNAs have been identified as diagnostic and therapeutic targets for various tumors. The expression of circ_rac GTPase-activating protein 1 (circRACGAP1) is reported to drive the development of non-small cell lung cancer (NSCLC). This study further explored the potential mechanism of circRACGAP1-mediated development of NSCLC. The circRACGAP1 level was detected by quantitative RT-PCR. Sphere formation, CD133-positive cell percentage, and expression of octamer-binding transcription factor 4, Sox2, Nanog, and CD133 were detected to evaluate stemness of NSCLC. Migration and invasion were determined using wound healing and transwell assays. Protein expression was measured using Western blotting. The molecular mechanism was evaluated using RNA pull-down, RNA immunoprecipitation, and coimmunoprecipitation assays. In vivo tumor growth and metastasis were determined in nude mice. circRACGAP1 was highly expressed in NSCLC and was associated with stemness marker Sox2 expression. The stemness, metastasis, and epithelial mesenchymal transformation were repressed in circRACGAP1-depleted NSCLC cells. Mechanistically, circRACGAP1 recruited RNA-binding protein polypyrimidine tract-binding protein 1 to enhance the stability and expression of sirtuin-3 (SIRT3), which subsequently led to replication timing regulatory factor 1 (RIF1) deacetylation and activation of the Wnt/β-catenin pathway. circRACGAP1 overexpression counteracted SIRT3 or RIF1 knockdown-mediated inhibition in stemness and metastasis of NSCLC cells. The in vivo tumor growth and metastasis were repressed by circRACGAP1 depletion. Patients with NSCLC with a higher serum exosomal circRACGAP1 level had a lower overall survival rate. In conclusion, circRACGAP1 facilitated stemness and metastasis of NSCLC cells through the recruitment of polypyrimidine tract-binding protein 1 to promote SIRT3-mediated RIF1 deacetylation. Our results uncover a novel regulatory mechanism of circRACGAP1 in NSCLC and identify circRACGAP1 as a promising therapeutic target.
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Affiliation(s)
- Hui Xiong
- Department of Oncology, The Second Xiangya Hospital of Central South University, Changsha, Hunan Province, China
| | - Bin Liu
- Department of Oncology, The Second Xiangya Hospital of Central South University, Changsha, Hunan Province, China
| | - Xiao-Yu Liu
- Department of Oncology, The Second Xiangya Hospital of Central South University, Changsha, Hunan Province, China
| | - Zhen-Kun Xia
- Department of Thoracic Surgery, The Second Xiangya Hospital of Central South University, Changsha, Hunan Province, China
| | - Min Lu
- Department of Oncology, The Second Xiangya Hospital of Central South University, Changsha, Hunan Province, China
| | - Chun-Hong Hu
- Department of Oncology, The Second Xiangya Hospital of Central South University, Changsha, Hunan Province, China
| | - Ping Liu
- Department of Oncology, The Second Xiangya Hospital of Central South University, Changsha, Hunan Province, China.
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Zhang X, Qiu X, Zhao W, Song L, Zhang X, Yang L, Tao M. Over-Expression of ARID3B Suppresses Tumor Progression and Predicts Better Prognosis in Patients With Gastric Cancer. Cancer Control 2023; 30:10732748231169403. [PMID: 37071790 PMCID: PMC10126794 DOI: 10.1177/10732748231169403] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 04/20/2023] Open
Abstract
BACKGROUND ARID3B (AT-rich interaction domain 3B) has been demonstrated to be associated with the progression and patient prognosis of several human tumors. We conducted the present study to investigate the biological behavior and clinical relevance of ARID3B in gastric cancer (GC). METHODS Detection of the expression level in GC tissues and cell lines were performed by Western blot and immunohistochemistry. We also retrospectively analyzed the correlation of ARID3B with clinicopathological characteristics and patient prognosis in gastric cancer. The biological functions of ARID3B in GC cells were further explored by transwell migration assays, wound healing assays and cell proliferation assay. RESULTS The present study suggested that the expression of ARID3B was significantly lower in GC tissues than in adjacent normal tissues. IHC staining in tissues of 406 GC patients from training and validation sets verified that ARID3B over-expression correlated with clinicopathological features, such as degree of differentiation and clinical stage. Meanwhile, ARID3B was proved to be an independent prognostic factor for GC prognosis. Furthermore, over-expression of ARID3B suppressed proliferation in GC cells according CCK8 assay. We found that over-expression of ARID3B inhibited GC cell migration by transwell assay and wound healing assay. Furthermore, EMT markers were detected in ARID3B over-expression GC cells, which showed that ARID3B may inhibit metastasis of GC cells. CONCLUSION Our results firstly revealed that the expression level of ARID3B was closely correlated with clinicopathological features and may serve as an independent prognostic factor for GC patients. More importantly, ARID3B could suppress GC progression, including cell proliferation, migration and metastasis.
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Affiliation(s)
- Xunlei Zhang
- Department of Oncology, The First Affiliated Hospital of Soochow University, Suzhou, Jiangsu, China
- Department of Oncology, Affiliated Tumor Hospital of Nantong University, Nantong, Jiangsu, China
| | - Xinyue Qiu
- Department of Oncology, Affiliated Tumor Hospital of Nantong University, Nantong, Jiangsu, China
| | - Wenjing Zhao
- Cancer Research Center, Affiliated Tumor Hospital of Nantong University, Nantong, Jiangsu, China
| | - Li Song
- Department of Oncology, Affiliated Tumor Hospital of Nantong University, Nantong, Jiangsu, China
| | - Xingsong Zhang
- Department of Pathology, Affiliated Tumor Hospital of Nantong University, Nantong Jiangsu, China
| | - Lei Yang
- Department of Oncology, Affiliated Tumor Hospital of Nantong University, Nantong, Jiangsu, China
| | - Min Tao
- Department of Oncology, The First Affiliated Hospital of Soochow University, Suzhou, Jiangsu, China
- Department of Oncology, Dushu Lake Hospital Affiliated to Soochow University, Suzhou, Jiangsu, China
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30
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Mechanisms of Drug Resistance in Ovarian Cancer and Associated Gene Targets. Cancers (Basel) 2022; 14:cancers14246246. [PMID: 36551731 PMCID: PMC9777152 DOI: 10.3390/cancers14246246] [Citation(s) in RCA: 37] [Impact Index Per Article: 12.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/29/2022] [Revised: 11/30/2022] [Accepted: 12/08/2022] [Indexed: 12/24/2022] Open
Abstract
In the United States, over 100,000 women are diagnosed with a gynecologic malignancy every year, with ovarian cancer being the most lethal. One of the hallmark characteristics of ovarian cancer is the development of resistance to chemotherapeutics. While the exact mechanisms of chemoresistance are poorly understood, it is known that changes at the cellular and molecular level make chemoresistance challenging to treat. Improved therapeutic options are needed to target these changes at the molecular level. Using a precision medicine approach, such as gene therapy, genes can be specifically exploited to resensitize tumors to therapeutics. This review highlights traditional and novel gene targets that can be used to develop new and improved targeted therapies, from drug efflux proteins to ovarian cancer stem cells. The review also addresses the clinical relevance and landscape of the discussed gene targets.
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31
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Hu S, Metcalf E, Mahat DB, Chan L, Sohal N, Chakraborty M, Hamilton M, Singh A, Singh A, Lees JA, Sharp PA, Garg S. Transcription factor antagonism regulates heterogeneity in embryonic stem cell states. Mol Cell 2022; 82:4410-4427.e12. [PMID: 36356583 PMCID: PMC9722640 DOI: 10.1016/j.molcel.2022.10.022] [Citation(s) in RCA: 10] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/15/2022] [Revised: 05/19/2022] [Accepted: 10/20/2022] [Indexed: 11/10/2022]
Abstract
Gene expression heterogeneity underlies cell states and contributes to developmental robustness. While heterogeneity can arise from stochastic transcriptional processes, the extent to which it is regulated is unclear. Here, we characterize the regulatory program underlying heterogeneity in murine embryonic stem cell (mESC) states. We identify differentially active and transcribed enhancers (DATEs) across states. DATEs regulate differentially expressed genes and are distinguished by co-binding of transcription factors Klf4 and Zfp281. In contrast to other factors that interact in a positive feedback network stabilizing mESC cell-type identity, Klf4 and Zfp281 drive opposing transcriptional and chromatin programs. Abrogation of factor binding to DATEs dampens variation in gene expression, and factor loss alters kinetics of switching between states. These results show antagonism between factors at enhancers results in gene expression heterogeneity and formation of cell states, with implications for the generation of diverse cell types during development.
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Affiliation(s)
- Sofia Hu
- Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Harvard-MIT Division of Health Sciences and Technology, Cambridge, MA 02139, USA
| | - Emily Metcalf
- Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
| | - Dig Bijay Mahat
- Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
| | - Lynette Chan
- Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
| | - Noor Sohal
- Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
| | - Meenakshi Chakraborty
- Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
| | - Maxwell Hamilton
- Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
| | - Arundeep Singh
- Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
| | - Abhyudai Singh
- Department of Electrical and Computer Engineering, University of Delaware, Newark, DE 19716, USA
| | - Jacqueline A Lees
- Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
| | - Phillip A Sharp
- Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
| | - Salil Garg
- Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Harvard-MIT Division of Health Sciences and Technology, Cambridge, MA 02139, USA; Department of Pathology, Massachusetts General Hospital, Boston, MA 02114, USA; Department of Laboratory Medicine, Yale Stem Cell Center and Center for RNA Science and Medicine, Yale School of Medicine, New Haven, CT 06510, USA.
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Chen Z, Wu H, Jiang S, Liu X, Luo M, Yuan Y. Serum SALL4 as a predictive biomarker for the prognosis of patients with hepatocellular carcinoma who underwent nonsurgical treatment. Medicine (Baltimore) 2022; 101:e31200. [PMID: 36316931 PMCID: PMC9622602 DOI: 10.1097/md.0000000000031200] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/05/2023] Open
Abstract
To investigate the role of serum spalt like transcription factor 4 (SALL4) in the hepatocellular carcinoma (HCC) patients with nonsurgical treatment. Serum samples were collected from 101 patients with HCC without surgical treatment, then the SALL4 was detected by enzyme linked immunosorbent assay. According to subsequent treatment, patients were divided into 2 groups, best supportive care (BSC) (58 cases) and nonsurgical anticancer treatment (NSAT) (48 cases). Kaplan-Meier survival analysis and Cox multivariate regression analysis were applied to evaluate the relationship between SALL4 and survival time of 2 groups. In BSC group, there was no significant difference of the survival time between 2 groups (SALL4 < 800 ng/mL or SALL4 ≥ 800 ng/mL) (P = .339). In NSAT group, the survival time of patients with low SALL4 concentration was significantly longer than patients with high SALL4 concentration (P = .005). SALL4 have no predictive effect in BSC patients with HCC. But for patients received NSAT, low SALL4 concentration in serum may indicate longer survival.
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Affiliation(s)
- Zhixian Chen
- Department of Oncology, Shunde Hospital, Southern Medical University, Foshan, Guangdong Province, China
| | - Huifeng Wu
- Department of Nursing, Shunde Hospital, Southern Medical University, Foshan, Guangdong Province, China
- Department of Urology, Shunde Hospital, Southern Medical University, Foshan, Guangdong province, China
| | - Simin Jiang
- Department of Radiation Oncology, Shunde Hospital, Southern Medical University, Foshan, Guangdong Province, China
| | - Xingli Liu
- Department of Oncology, Shunde Hospital, Southern Medical University, Foshan, Guangdong Province, China
| | - Meihua Luo
- Department of Oncology, Shunde Hospital, Southern Medical University, Foshan, Guangdong Province, China
| | - Yawei Yuan
- Department of Radiation Oncology, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong Province, China
- Department of Radiation Oncology, Affiliated Cancer Hospital & Institute of Guangzhou Medical University, Guangzhou, Guangdong Province, China
- * Correspondence: Yawei Yuan, Department of Radiation Oncology, Nanfang Hospital, Southern Medical University, 1838 Guangzhou Avenue North, Guangzhou, Guangdong Province, 510515, China (e-mail: )
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Li J, Cheng C, Xu J, Zhang T, Tokat B, Dolios G, Ramakrishnan A, Shen L, Wang R, Xu PX. The transcriptional coactivator Eya1 exerts transcriptional repressive activity by interacting with REST corepressors and REST-binding sequences to maintain nephron progenitor identity. Nucleic Acids Res 2022; 50:10343-10359. [PMID: 36130284 PMCID: PMC9561260 DOI: 10.1093/nar/gkac760] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/19/2022] [Revised: 08/18/2022] [Accepted: 08/27/2022] [Indexed: 11/15/2022] Open
Abstract
Eya1 is critical for establishing and maintaining nephron progenitor cells (NPCs). It belongs to a family of proteins called phosphatase-transcriptional activators but without intrinsic DNA-binding activity. However, the spectrum of the Eya1-centered networks is underexplored. Here, we combined transcriptomic, genomic and proteomic approaches to characterize gene regulation by Eya1 in the NPCs. We identified Eya1 target genes, associated cis-regulatory elements and partner proteins. Eya1 preferentially occupies promoter sequences and interacts with general transcription factors (TFs), RNA polymerases, different types of TFs, chromatin-remodeling factors with ATPase or helicase activity, and DNA replication/repair proteins. Intriguingly, we identified REST-binding motifs in 76% of Eya1-occupied sites without H3K27ac-deposition, which were present in many Eya1 target genes upregulated in Eya1-deficient NPCs. Eya1 copurified REST-interacting chromatin-remodeling factors, histone deacetylase/lysine demethylase, and corepressors. Coimmunoprecipitation validated physical interaction between Eya1 and Rest/Hdac1/Cdyl/Hltf in the kidneys. Collectively, our results suggest that through interactions with chromatin-remodeling factors and specialized DNA-binding proteins, Eya1 may modify chromatin structure to facilitate the assembly of regulatory complexes that regulate transcription positively or negatively. These findings provide a mechanistic basis for how Eya1 exerts its activity by forming unique multiprotein complexes in various biological processes to maintain the cellular state of NPCs.
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Affiliation(s)
- Jun Li
- Department of Genetics and Genomic Sciences, New York, NY 10029, USA
| | - Chunming Cheng
- Department of Genetics and Genomic Sciences, New York, NY 10029, USA
| | - Jinshu Xu
- Department of Genetics and Genomic Sciences, New York, NY 10029, USA
| | - Ting Zhang
- Department of Genetics and Genomic Sciences, New York, NY 10029, USA
| | - Bengu Tokat
- Department of Genetics and Genomic Sciences, New York, NY 10029, USA
| | - Georgia Dolios
- Department of Genetics and Genomic Sciences, New York, NY 10029, USA
| | | | - Li Shen
- Department of Neurosciences, New York, NY 10029, USA
| | - Rong Wang
- Department of Genetics and Genomic Sciences, New York, NY 10029, USA
| | - Pin-Xian Xu
- Department of Genetics and Genomic Sciences, New York, NY 10029, USA.,Department of Cell, Developmental and Regenerative Biology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA
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Machida K. HCV and tumor-initiating stem-like cells. Front Physiol 2022; 13:903302. [PMID: 36187761 PMCID: PMC9520593 DOI: 10.3389/fphys.2022.903302] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/24/2022] [Accepted: 07/11/2022] [Indexed: 12/24/2022] Open
Abstract
Neoplasms contain tumor-initiating stem-like cells (TICs) that are characterized by increased drug resistance. The incidence of many cancer types have trended downward except for few cancer types, including hepatocellular carcinoma (HCC). Therefore mechanism of HCC development and therapy resistance needs to be understood. These multiple hits by hepatitis C virus (HCV) eventually promotes transformation and TIC genesis, leading to HCC development. This review article describes links between HCV-associated HCC and TICs. This review discusses 1) how HCV promotes genesis of TICs and HCC development; 2) how this process avails itself as a novel therapeutic target for HCC treatment; and 3) ten hall marks of TIC oncogenesis and HCC development as targets for novel therapeutic modalities.
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The Divergent and Conserved Expression Profile of Turtle Nanog Gene Comparing with Fish and Mammals. BIOLOGY 2022; 11:biology11091342. [PMID: 36138820 PMCID: PMC9495436 DOI: 10.3390/biology11091342] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 07/27/2022] [Revised: 08/19/2022] [Accepted: 09/05/2022] [Indexed: 11/17/2022]
Abstract
Nanog is a homeodomain-containing transcription factor, and it plays a vital role in maintaining the pluripotency of embryonic stem cells. Nanog’s function has been well studied in many species. However, there is lack of reporting on the Nanog gene in reptile. Here, we identified a 1032 bp cDNA sequence of a Nanog gene in Pelidiscus sinensis, known as PsNanog. PsNanog has a highly conserved HD domain and shares a high identity with that of Chelonia mydas and the lowest identity with Oryzias latipes. Similarly, PsNanog presented a tight cluster with C. mydas Nanog, but was far from those of teleosts. Additionally, we cloned a length of 1870 bp PsNanog promoter. Dual luciferase assay showed that the DNA fragment of −1560 to +1 exhibited a high promoter activity. The RT-PCR and RT-qPCR results showed that PsNanog was predominantly expressed in ovary, and then in testis. The in situ hybridization and immunohistochemical analysis showed that PsNanog was expressed in the early primary oocytes and the cytoplasm of the cortical region of stage VIII oocytes in ovary, and distributed in most stages of germ cells in testis. Collectively, the results imply that PsNanog probably has the conserved function in regulating germ cell development across phyla and is also a pluripotent cell gene and expressed in germ cells, which is similar to that in teleosts and mammals.
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Jimenez E, Slevin CC, Song W, Chen Z, Frederickson SC, Gildea D, Wu W, Elkahloun AG, Ovcharenko I, Burgess SM. A regulatory network of Sox and Six transcription factors initiate a cell fate transformation during hearing regeneration in adult zebrafish. CELL GENOMICS 2022; 2. [PMID: 36212030 PMCID: PMC9540346 DOI: 10.1016/j.xgen.2022.100170] [Citation(s) in RCA: 12] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Indexed: 12/24/2022]
Abstract
Using adult zebrafish inner ears as a model for sensorineural regeneration, we ablated the mechanosensory receptors and characterized the single-cell epigenome and transcriptome at consecutive time points during hair cell regeneration. We utilized deep learning on the regeneration-induced open chromatin sequences and identified cell-specific transcription factor (TF) motif patterns. Enhancer activity correlated with gene expression and identified potential gene regulatory networks. A pattern of overlapping Sox- and Six-family TF gene expression and binding motifs was detected, suggesting a combinatorial program of TFs driving regeneration and cell identity. Pseudotime analysis of single-cell transcriptomic data suggested that support cells within the sensory epithelium changed cell identity to a “progenitor” cell population that could differentiate into hair cells. We identified a 2.6 kb DNA enhancer upstream of the sox2 promoter that, when deleted, showed a dominant phenotype that resulted in a hair-cell-regeneration-specific deficit in both the lateral line and adult inner ear. Jimenez et al. interrogate the epigenomic and transcriptomic landscape of regenerating adult zebrafish inner-ear sensory epithelia. They show that the support-cell population transitions to an intermediate “progenitor” cell state that becomes new hair cells, and they demonstrate that the cell fate decisions may be driven by the coordinate regulation and spatial co-binding of Sox and Six transcription factors. By functionally validating a predicted regeneration-responsive enhancer upstream of sox2, they show that precise timing of sox2 expression is critical for hearing regeneration in zebrafish.
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Affiliation(s)
- Erin Jimenez
- Translational and Functional Genomics Branch, National Human Genome Research Institute, Bethesda, MD, USA
| | - Claire C. Slevin
- Translational and Functional Genomics Branch, National Human Genome Research Institute, Bethesda, MD, USA
| | - Wei Song
- Computational Biology Branch, National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD 20894, USA
| | - Zelin Chen
- CAS Key Laboratory of Tropical Marine Bio-Resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, China
- Southern Marine Science and Engineering Guangdong Laboratory, Guangzhou, China
| | - Stephen C. Frederickson
- Translational and Functional Genomics Branch, National Human Genome Research Institute, Bethesda, MD, USA
| | - Derek Gildea
- Translational and Functional Genomics Branch, National Human Genome Research Institute, Bethesda, MD, USA
| | - Weiwei Wu
- Vaccine Immunology Program, Vaccine Research Center, National Institute of Allergy and Infectious Diseases, Bethesda, MD, USA
| | - Abdel G. Elkahloun
- Cancer Genetics and Comparative Genomics Branch, National Human Genome Research Institute, Bethesda, MD, USA
| | - Ivan Ovcharenko
- Computational Biology Branch, National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD 20894, USA
| | - Shawn M. Burgess
- Translational and Functional Genomics Branch, National Human Genome Research Institute, Bethesda, MD, USA
- Corresponding author
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Nel S, Durandt C, Murdoch C, Pepper MS. Determinants of Dental Pulp Stem Cell Heterogeneity. J Endod 2022; 48:1232-1240. [PMID: 35809811 DOI: 10.1016/j.joen.2022.06.013] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/25/2022] [Revised: 06/27/2022] [Accepted: 06/28/2022] [Indexed: 11/17/2022]
Abstract
INTRODUCTION The aim of this review is to provide a narrative review on the determinants of dental pulp stem cell (DPSC) heterogeneity that may affect the regenerative properties of these cells. METHODS PubMed, Scopus and Medline (Ovid) literature searches were done on human dental pulp stem cell (hDPSC) heterogeneity. The focus was on human dental pulp stem cells (hDPSCs) with a primary focus on DPSC heterogeneity. RESULTS DPSCs display significant heterogeneity as illustrated by the various subpopulations reported, including differences in proliferation and differentiation capabilities and the impact of various intrinsic and extrinsic factors. CONCLUSIONS The lack of consistent and reliable results in the clinical setting may be due to the heterogeneous nature of DPSC populations. Standardization in isolation techniques and in criteria to characterize DPSCs should lead to less variability in results reported and improve comparison of findings between studies. Single-cell RNA sequencing holds promise in elucidating DPSC heterogeneity and may contribute to the establishment of standardized techniques.
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Affiliation(s)
- Sulette Nel
- Department of Oral Pathology and Oral Biology, School of Dentistry, Faculty of Health Sciences, University of Pretoria, South Africa.
| | - Chrisna Durandt
- Institute for Cellular and Molecular Medicine (ICMM), Department of Immunology, and SAMRC Extramural Unit for Stem Cell Research and Therapy, Faculty of Health Sciences, University of Pretoria, South Africa
| | - Candice Murdoch
- Institute for Cellular and Molecular Medicine (ICMM), Department of Immunology, and SAMRC Extramural Unit for Stem Cell Research and Therapy, Faculty of Health Sciences, University of Pretoria, South Africa
| | - Michael S Pepper
- Institute for Cellular and Molecular Medicine (ICMM), Department of Immunology, and SAMRC Extramural Unit for Stem Cell Research and Therapy, Faculty of Health Sciences, University of Pretoria, South Africa
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Yang JM, Ren Y, Kumar A, Xiong X, Das JK, Peng HY, Wang L, Ren X, Zhang Y, Ji C, Cheng Y, Zhang L, Alaniz RC, de Figueiredo P, Fang D, Zhou H, Liu X, Wang J, Song J. NAC1 modulates autoimmunity by suppressing regulatory T cell-mediated tolerance. SCIENCE ADVANCES 2022; 8:eabo0183. [PMID: 35767626 PMCID: PMC9242588 DOI: 10.1126/sciadv.abo0183] [Citation(s) in RCA: 18] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 01/07/2022] [Accepted: 05/12/2022] [Indexed: 05/28/2023]
Abstract
We report here that nucleus accumbens-associated protein-1 (NAC1), a nuclear factor of the Broad-complex, Tramtrack, Bric-a-brac/poxvirus and zinc finger (BTB/POZ) gene family, is a negative regulator of FoxP3 in regulatory T cells (Tregs) and a critical determinant of immune tolerance. Phenotypically, NAC1-/- mice showed substantial tolerance to the induction of autoimmunity and generated a larger amount of CD4+ Tregs that exhibit a higher metabolic profile and immune-suppressive activity, increased acetylation and expression of FoxP3, and slower turnover of this transcription factor. Treatment of Tregs with the proinflammatory cytokines interleukin-1β or tumor necrosis factor-α induced a robust up-regulation of NAC1 but evident down-regulation of FoxP3 as well as the acetylated FoxP3. These findings imply that NAC1 acts as a trigger of the immune response through destabilization of Tregs and suppression of tolerance induction, and targeting of NAC1 warrants further exploration as a potential tolerogenic strategy for treatment of autoimmune disorders.
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Affiliation(s)
- Jin-Ming Yang
- Department of Toxicology and Cancer Biology, Department of Pharmacology and Nutritional Science, and Markey Cancer Center, University of Kentucky College of Medicine, Lexington, KY 40536, USA
| | - Yijie Ren
- Department of Microbial Pathogenesis and Immunology, Texas A&M University Health Science Center, Bryan, TX 77807, USA
| | - Anil Kumar
- Department of Microbial Pathogenesis and Immunology, Texas A&M University Health Science Center, Bryan, TX 77807, USA
| | - Xiaofang Xiong
- Department of Microbial Pathogenesis and Immunology, Texas A&M University Health Science Center, Bryan, TX 77807, USA
| | - Jugal Kishore Das
- Department of Microbial Pathogenesis and Immunology, Texas A&M University Health Science Center, Bryan, TX 77807, USA
| | - Hao-Yun Peng
- Department of Microbial Pathogenesis and Immunology, Texas A&M University Health Science Center, Bryan, TX 77807, USA
| | - Liqing Wang
- Department of Microbial Pathogenesis and Immunology, Texas A&M University Health Science Center, Bryan, TX 77807, USA
| | - Xingcong Ren
- Department of Toxicology and Cancer Biology, Department of Pharmacology and Nutritional Science, and Markey Cancer Center, University of Kentucky College of Medicine, Lexington, KY 40536, USA
| | - Yi Zhang
- Department of Toxicology and Cancer Biology, Department of Pharmacology and Nutritional Science, and Markey Cancer Center, University of Kentucky College of Medicine, Lexington, KY 40536, USA
| | - Cheng Ji
- Department of Toxicology and Cancer Biology, Department of Pharmacology and Nutritional Science, and Markey Cancer Center, University of Kentucky College of Medicine, Lexington, KY 40536, USA
| | - Yan Cheng
- Department of Toxicology and Cancer Biology, Department of Pharmacology and Nutritional Science, and Markey Cancer Center, University of Kentucky College of Medicine, Lexington, KY 40536, USA
| | - Li Zhang
- Department of Toxicology and Cancer Biology, Department of Pharmacology and Nutritional Science, and Markey Cancer Center, University of Kentucky College of Medicine, Lexington, KY 40536, USA
| | - Robert C. Alaniz
- Department of Microbial Pathogenesis and Immunology, Texas A&M University Health Science Center, Bryan, TX 77807, USA
| | - Paul de Figueiredo
- Department of Microbial Pathogenesis and Immunology, Texas A&M University Health Science Center, Bryan, TX 77807, USA
- Department of Veterinary Pathobiology, Texas A&M University, College Station, TX 77845, USA
- Norman Borlaug Center, Texas A&M University, College Station, TX 77845, USA
| | - Deyu Fang
- Department of Pathology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA
| | - Hongwei Zhou
- Department of Medicine, Columbia Center for Human Development, Columbia University Irving Medical Center, New York, NY 10032, USA
| | - Xiaoqi Liu
- Department of Toxicology and Cancer Biology, Department of Pharmacology and Nutritional Science, and Markey Cancer Center, University of Kentucky College of Medicine, Lexington, KY 40536, USA
| | - Jianlong Wang
- Department of Medicine, Columbia Center for Human Development, Columbia University Irving Medical Center, New York, NY 10032, USA
| | - Jianxun Song
- Department of Microbial Pathogenesis and Immunology, Texas A&M University Health Science Center, Bryan, TX 77807, USA
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Zuo F, Jiang J, Fu H, Yan K, Liefke R, Zhang J, Hong Y, Chang Z, Liu N, Wang Z, Xi Q. A TRIM66/DAX1/Dux axis suppresses the totipotent 2-cell-like state in murine embryonic stem cells. Cell Stem Cell 2022; 29:948-961.e6. [DOI: 10.1016/j.stem.2022.05.004] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/12/2021] [Revised: 03/22/2022] [Accepted: 05/09/2022] [Indexed: 12/22/2022]
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Choi KJ, Quan MD, Qi C, Lee JH, Tsoi PS, Zahabiyon M, Bajic A, Hu L, Prasad BVV, Liao SCJ, Li W, Ferreon ACM, Ferreon JC. NANOG prion-like assembly mediates DNA bridging to facilitate chromatin reorganization and activation of pluripotency. Nat Cell Biol 2022; 24:737-747. [PMID: 35484250 PMCID: PMC9106587 DOI: 10.1038/s41556-022-00896-x] [Citation(s) in RCA: 14] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/16/2021] [Accepted: 03/16/2022] [Indexed: 12/12/2022]
Abstract
Human NANOG expression resets stem cells to ground-state pluripotency. Here we identify the unique features of human NANOG that relate to its dose-sensitive function as a master transcription factor. NANOG is largely disordered, with a C-terminal prion-like domain that phase-transitions to gel-like condensates. Full-length NANOG readily forms higher-order oligomers at low nanomolar concentrations, orders of magnitude lower than typical amyloids. Using single-molecule Förster resonance energy transfer and fluorescence cross-correlation techniques, we show that NANOG oligomerization is essential for bridging DNA elements in vitro. Using chromatin immunoprecipitation sequencing and Hi-C 3.0 in cells, we validate that NANOG prion-like domain assembly is essential for specific DNA recognition and distant chromatin interactions. Our results provide a physical basis for the indispensable role of NANOG in shaping the pluripotent genome. NANOG's unique ability to form prion-like assemblies could provide a cooperative and concerted DNA bridging mechanism that is essential for chromatin reorganization and dose-sensitive activation of ground-state pluripotency.
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Affiliation(s)
- Kyoung-Jae Choi
- Department of Pharmacology and Chemical Biology, Baylor College of Medicine, Houston, TX, USA
| | - My Diem Quan
- Department of Pharmacology and Chemical Biology, Baylor College of Medicine, Houston, TX, USA
| | - Chuangye Qi
- Department of Biochemistry and Molecular Biology, McGovern Medical School, University of Texas Health Science Center, Houston, TX, USA
| | - Joo-Hyung Lee
- Department of Biochemistry and Molecular Biology, McGovern Medical School, University of Texas Health Science Center, Houston, TX, USA
| | - Phoebe S Tsoi
- Department of Pharmacology and Chemical Biology, Baylor College of Medicine, Houston, TX, USA
| | - Mahla Zahabiyon
- Department of Molecular and Human Genetics, Baylor College of Medicine and Jan and Dan Duncan Neurological Research Institute at Texas Children's Hospital, Houston, TX, USA
| | - Aleksandar Bajic
- Department of Molecular and Human Genetics, Baylor College of Medicine and Jan and Dan Duncan Neurological Research Institute at Texas Children's Hospital, Houston, TX, USA
| | - Liya Hu
- Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX, USA
| | - B V Venkataram Prasad
- Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX, USA
| | | | - Wenbo Li
- Department of Biochemistry and Molecular Biology, McGovern Medical School, University of Texas Health Science Center, Houston, TX, USA. .,Graduate School of Biomedical Sciences, University of Texas MD Anderson Cancer Center and UTHealth, Houston, TX, USA.
| | - Allan Chris M Ferreon
- Department of Pharmacology and Chemical Biology, Baylor College of Medicine, Houston, TX, USA.
| | - Josephine C Ferreon
- Department of Pharmacology and Chemical Biology, Baylor College of Medicine, Houston, TX, USA.
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Rif1 and Hmgn3 regulate the conversion of murine trophoblast stem cells. Cell Rep 2022; 38:110570. [PMID: 35354046 DOI: 10.1016/j.celrep.2022.110570] [Citation(s) in RCA: 14] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/08/2021] [Revised: 01/24/2022] [Accepted: 03/03/2022] [Indexed: 11/22/2022] Open
Abstract
The appearance of trophectoderm (TE) is a hallmark event in preimplantation development during murine embryogenesis. However, little is known about the mechanisms underlying TE specification. We find that the depletion of Rif1 breaks down the barrier to the transition from embryonic stem cells (ESCs) to trophoblast stem cells (TSCs). Rif1-null-induced TSCs show typical TE properties and the potential to differentiate into terminal trophoblast lineages. Global transcriptome analysis reveal that Rif1 deletion activates 2-cell embryo (2C)-related genes and induces a totipotent-like state. Chimeric assays further confirm that Rif1-null ESCs contribute to the functional placenta in addition to the fetus on embryonic day 12.5. Furthermore, we show overexpression of Hmgn3, one of the key upregulated gene in Rif1-null ESCs, facilitates the induction of TSCs. Therefore, we report two key genes regulating the conversion of TSCs and provide insights for investigating TE specification.
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Kulyyassov A, Ramankulov Y, Ogryzko V. Generation of Peptides for Highly Efficient Proximity Utilizing Site-Specific Biotinylation in Cells. Life (Basel) 2022; 12:life12020300. [PMID: 35207587 PMCID: PMC8875956 DOI: 10.3390/life12020300] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/19/2022] [Revised: 02/10/2022] [Accepted: 02/11/2022] [Indexed: 11/16/2022] Open
Abstract
Protein tags are peptide sequences genetically embedded into a recombinant protein for various purposes, such as affinity purification, Western blotting, and immunofluorescence. Another recent application of peptide tags is in vivo labeling and analysis of protein–protein interactions (PPI) by proteomics methods. One of the common workflows involves site-specific in vivo biotinylation of an AviTag-fused protein in the presence of the biotin ligase BirA. However, due to the rapid kinetics of labeling, this tag is not ideal for analysis of PPI. Here we describe the rationale, design, and protocol for the new biotin acceptor peptides BAP1070 and BAP1108 using modular assembling of biotin acceptor fragments, DNA sequencing, transient expression of proteins in cells, and Western blotting methods. These tags were used in the Proximity Utilizing Biotinylation (PUB) method, which is based on coexpression of BAP-X and BirA-Y in mammalian cells, where X or Y are candidate interacting proteins of interest. By changing the sequence of these peptides, a low level of background biotinylation is achieved, which occurs due to random collisions of proteins in cells. Over 100 plasmid constructs, containing genes of transcription factors, histones, gene repressors, and other nuclear proteins were obtained during implementation of projects related to this method.
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Affiliation(s)
- Arman Kulyyassov
- Republican State Enterprise “National Center for Biotechnology” under the Science Committee of Ministry of Education and Science of the Republic of Kazakhstan, 13/5 Kurgalzhynskoye Road, Nur-Sultan 010000, Kazakhstan;
- Correspondence: ; Tel.: +7-7172-707534
| | - Yerlan Ramankulov
- Republican State Enterprise “National Center for Biotechnology” under the Science Committee of Ministry of Education and Science of the Republic of Kazakhstan, 13/5 Kurgalzhynskoye Road, Nur-Sultan 010000, Kazakhstan;
| | - Vasily Ogryzko
- UMR8126, Institut de Cancerologie Gustave Roussy, Universite Paris-Sud 11, CNRS, 94805 Villejuif, France;
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Zhang T, Zhou H, Wang K, Wang X, Wang M, Zhao W, Xi X, Li Y, Cai M, Zhao W, Xu Y, Shao R. Role, molecular mechanism and the potential target of breast cancer stem cells in breast cancer development. Biomed Pharmacother 2022; 147:112616. [PMID: 35008001 DOI: 10.1016/j.biopha.2022.112616] [Citation(s) in RCA: 20] [Impact Index Per Article: 6.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/24/2021] [Revised: 01/01/2022] [Accepted: 01/02/2022] [Indexed: 02/06/2023] Open
Abstract
Breast cancer (BC) is one of the most common malignant tumors in women globally, and its occurrence has surpassed lung cancer and become the biggest threat for women. At present, breast cancer treatment includes surgical resection or postoperative chemotherapy and radiotherapy. However, tumor relapse and metastasis usually lead to current therapy failure thanks to breast cancer stem cells (BCSCs)-mediated tumorigenicity and drug resistance. Drug resistance is mainly due to the long-term quiescent G0 phase, strong DNA repairability, and high expression of ABC transporter, and the tumorigenicity is reflected in the activation of various proliferation pathways related to BCSCs. Therefore, understanding the characteristics of BCSCs and their intracellular and extracellular molecular mechanisms is crucial for the development of targeted drugs for BCSCs. To this end, we discussed the latest developments in BCSCs research, focusing on the analysis of specific markers, critical signaling pathways that maintain the stemness of BCSCs，such as NOTCH, Wnt/β-catenin, STAT3, Hedgehog, and Hippo-YAP signaling, immunomicroenviroment and summarizes targeting therapy strategies for stemness maintenance and differentiation, which provides a theoretical basis for further exploration of treating breast cancer and preventing relapse derived from BCSCs.
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Affiliation(s)
- Tianshu Zhang
- Key Laboratory of Antibiotic Bioengineering, Ministry of Health, Laboratory of Oncology, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China
| | - Huimin Zhou
- Key Laboratory of Antibiotic Bioengineering, Ministry of Health, Laboratory of Oncology, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China
| | - Kexin Wang
- Key Laboratory of Antibiotic Bioengineering, Ministry of Health, Laboratory of Oncology, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China
| | - Xiaowei Wang
- Key Laboratory of Antibiotic Bioengineering, Ministry of Health, Laboratory of Oncology, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China
| | - Mengyan Wang
- Key Laboratory of Antibiotic Bioengineering, Ministry of Health, Laboratory of Oncology, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China
| | - Wenxia Zhao
- Key Laboratory of Antibiotic Bioengineering, Ministry of Health, Laboratory of Oncology, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China
| | - Xiaoming Xi
- Key Laboratory of Antibiotic Bioengineering, Ministry of Health, Laboratory of Oncology, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China
| | - Yang Li
- Key Laboratory of Antibiotic Bioengineering, Ministry of Health, Laboratory of Oncology, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China
| | - Meilian Cai
- Key Laboratory of Antibiotic Bioengineering, Ministry of Health, Laboratory of Oncology, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China
| | - Wuli Zhao
- Key Laboratory of Antibiotic Bioengineering, Ministry of Health, Laboratory of Oncology, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China.
| | - Yanni Xu
- Key Laboratory of Antibiotic Bioengineering, Ministry of Health, Laboratory of Oncology, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China.
| | - Rongguang Shao
- Key Laboratory of Antibiotic Bioengineering, Ministry of Health, Laboratory of Oncology, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China
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Ordureau A, Kraus F, Zhang J, An H, Park S, Ahfeldt T, Paulo JA, Harper JW. Temporal proteomics during neurogenesis reveals large-scale proteome and organelle remodeling via selective autophagy. Mol Cell 2021; 81:5082-5098.e11. [PMID: 34699746 PMCID: PMC8688335 DOI: 10.1016/j.molcel.2021.10.001] [Citation(s) in RCA: 64] [Impact Index Per Article: 16.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/20/2021] [Revised: 08/23/2021] [Accepted: 10/01/2021] [Indexed: 12/18/2022]
Abstract
Cell state changes are associated with proteome remodeling to serve newly emergent cell functions. Here, we show that NGN2-driven conversion of human embryonic stem cells to induced neurons (iNeurons) is associated with increased PINK1-independent mitophagic flux that is temporally correlated with metabolic reprogramming to support oxidative phosphorylation. Global multiplex proteomics during neurogenesis revealed large-scale remodeling of functional modules linked with pluripotency, mitochondrial metabolism, and proteostasis. Differentiation-dependent mitophagic flux required BNIP3L and its LC3-interacting region (LIR) motif, and BNIP3L also promoted mitophagy in dopaminergic neurons. Proteomic analysis of ATG12-/- iNeurons revealed accumulation of endoplasmic reticulum, Golgi, and mitochondria during differentiation, indicative of widespread organelle remodeling during neurogenesis. This work reveals broad organelle remodeling of membrane-bound organelles during NGN2-driven neurogenesis via autophagy, identifies BNIP3L's central role in programmed mitophagic flux, and provides a proteomic resource for elucidating how organelle remodeling and autophagy alter the proteome during changes in cell state.
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Affiliation(s)
- Alban Ordureau
- Department of Cell Biology, Blavatnik Institute, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA.
| | - Felix Kraus
- Department of Cell Biology, Blavatnik Institute, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA
| | - Jiuchun Zhang
- Department of Cell Biology, Blavatnik Institute, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA
| | - Heeseon An
- Department of Cell Biology, Blavatnik Institute, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA
| | - Sookhee Park
- Department of Cell Biology, Blavatnik Institute, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA
| | - Tim Ahfeldt
- Nash Family Department of Neuroscience at Mount Sinai, New York, NY 10029, USA; Department of Neurology at Mount Sinai, New York, NY 10029, USA; Department of Cell, Developmental and Regenerative Biology at Mount Sinai, New York, NY 10029, USA; Ronald M. Loeb Center for Alzheimer's Disease at Mount Sinai, New York, NY 10029, USA; Friedman Brain Institute at Mount Sinai, New York, NY 10029, USA; Black Family Stem Cell Institute at Mount Sinai, New York, NY 10029, USA
| | - Joao A Paulo
- Department of Cell Biology, Blavatnik Institute, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA
| | - J Wade Harper
- Department of Cell Biology, Blavatnik Institute, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA.
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Effect of NANOG overexpression on porcine embryonic development and pluripotent embryonic stem cell formation in vitro. ZYGOTE 2021; 30:324-329. [PMID: 34879895 DOI: 10.1017/s0967199421000678] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
The efficiency of establishing pig pluripotent embryonic stem cell clones from blastocysts is still low. The transcription factor Nanog plays an important role in maintaining the pluripotency of mouse and human embryonic stem cells. Adequate activation of Nanog has been reported to increase the efficiency of establishing mouse embryonic stem cells from 3.5 day embryos. In mouse, Nanog starts to be strongly expressed as early as the morula stage, whereas in porcine NANOG starts to be strongly expressed by the late blastocyst stage. Therefore, here we investigated both the effect of expressing NANOG on porcine embryos early from the morula stage and the efficiency of porcine pluripotent embryonic stem cell clone formation. Compared with intact porcine embryos, NANOG overexpression induced a lower blastocyst rate, and did not show any advantages for embryo development and pluripotent embryonic stem cell line formation. These results indicated that, although NANOG is important pluripotent factor, NANOG overexpression is unnecessary for the initial formation of porcine pluripotent embryonic stem cell clones in vitro.
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Yoshizawa-Sugata N, Yamazaki S, Mita-Yoshida K, Ono T, Nishito Y, Masai H. Loss of full-length DNA replication regulator Rif1 in two-cell embryos is associated with zygotic transcriptional activation. J Biol Chem 2021; 297:101367. [PMID: 34736895 PMCID: PMC8686075 DOI: 10.1016/j.jbc.2021.101367] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/05/2021] [Revised: 10/17/2021] [Accepted: 10/20/2021] [Indexed: 11/21/2022] Open
Abstract
Rif1 regulates DNA replication timing and double-strand break repair, and its depletion induces transcriptional bursting of two-cell (2C) zygote-specific genes in mouse ES cells. However, how Rif1 regulates zygotic transcription is unclear. We show here that Rif1 depletion promotes the formation of a unique Zscan4 enhancer structure harboring both histone H3 lysine 27 acetylation (H3K27ac) and moderate levels of silencing chromatin mark H3K9me3. Curiously, another enhancer mark H3K4me1 is missing, whereas DNA methylation is still maintained in the structure, which spreads across gene bodies and neighboring regions within the Zscan4 gene cluster. We also found by function analyses of Rif1 domains in ES cells that ectopic expression of Rif1 lacking N-terminal domain results in upregulation of 2C transcripts. This appears to be caused by dominant negative inhibition of endogenous Rif1 protein localization at the nuclear periphery through formation of hetero-oligomers between the N-terminally truncated and endogenous forms. Strikingly, in murine 2C embryos, most of Rif1-derived polypeptides are expressed as truncated forms in soluble nuclear or cytosolic fraction and are likely nonfunctional. Toward the morula stage, the full-length form of Rif1 gradually increased. Our results suggest that the absence of the functional full-length Rif1 due to its instability or alternative splicing and potential inactivation of Rif1 through dominant inhibition by N-terminally truncated Rif1 polypeptides may be involved in 2C-specific transcription program.
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Key Words
- 2c, two-cell (embryo)
- 4-oht, 4-hydroxytamoxifen
- dox, doxycycline
- erv, endogenous retrovirus
- es, embryonic stem
- hpf, hours post fertilization
- idr, intrinsic disordered region
- ivf, in vitro fertilization
- kd, knockdown
- ko, knockout
- rt, room temperature
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Affiliation(s)
| | - Satoshi Yamazaki
- Genome Dynamics Project, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan
| | - Kaoru Mita-Yoshida
- Center for Basic Technology Research, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan
| | - Tomio Ono
- Center for Basic Technology Research, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan
| | - Yasumasa Nishito
- Center for Basic Technology Research, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan
| | - Hisao Masai
- Genome Dynamics Project, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan.
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Kulyyassov A. Application of Skyline for Analysis of Protein-Protein Interactions In Vivo. Molecules 2021; 26:molecules26237170. [PMID: 34885753 PMCID: PMC8658920 DOI: 10.3390/molecules26237170] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/02/2021] [Revised: 11/22/2021] [Accepted: 11/23/2021] [Indexed: 11/19/2022] Open
Abstract
Quantitative and qualitative analyses of cell protein composition using liquid chromatography/tandem mass spectrometry are now standard techniques in biological and clinical research. However, the quantitative analysis of protein–protein interactions (PPIs) in cells is also important since these interactions are the bases of many processes, such as the cell cycle and signaling pathways. This paper describes the application of Skyline software for the identification and quantification of the biotinylated form of the biotin acceptor peptide (BAP) tag, which is a marker of in vivo PPIs. The tag was used in the Proximity Utilizing Biotinylation (PUB) method, which is based on the co-expression of BAP-X and BirA-Y in mammalian cells, where X or Y are interacting proteins of interest. A high level of biotinylation was detected in the model experiments where X and Y were pluripotency transcription factors Sox2 and Oct4, or heterochromatin protein HP1γ. MRM data processed by Skyline were normalized and recalculated. Ratios of biotinylation levels in experiment versus controls were 86 ± 6 (3 h biotinylation time) and 71 ± 5 (9 h biotinylation time) for BAP-Sox2 + BirA-Oct4 and 32 ± 3 (4 h biotinylation time) for BAP-HP1γ + BirA-HP1γ experiments. Skyline can also be applied for the analysis and identification of PPIs from shotgun proteomics data downloaded from publicly available datasets and repositories.
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Affiliation(s)
- Arman Kulyyassov
- Republican State Enterprise "National Center for Biotechnology" under the Science Committee of Ministry of Education and Science of the Republic of Kazakhstan, 13/5, Kurgalzhynskoye Road, Nur-Sultan 010000, Kazakhstan
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Deciphering the generating rules and functionalities of complex networks. Sci Rep 2021; 11:22964. [PMID: 34824290 PMCID: PMC8616909 DOI: 10.1038/s41598-021-02203-4] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/30/2021] [Accepted: 11/08/2021] [Indexed: 11/08/2022] Open
Abstract
Network theory helps us understand, analyze, model, and design various complex systems. Complex networks encode the complex topology and structural interactions of various systems in nature. To mine the multiscale coupling, heterogeneity, and complexity of natural and technological systems, we need expressive and rigorous mathematical tools that can help us understand the growth, topology, dynamics, multiscale structures, and functionalities of complex networks and their interrelationships. Towards this end, we construct the node-based fractal dimension (NFD) and the node-based multifractal analysis (NMFA) framework to reveal the generating rules and quantify the scale-dependent topology and multifractal features of a dynamic complex network. We propose novel indicators for measuring the degree of complexity, heterogeneity, and asymmetry of network structures, as well as the structure distance between networks. This formalism provides new insights on learning the energy and phase transitions in the networked systems and can help us understand the multiple generating mechanisms governing the network evolution.
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LSD1: Expanding Functions in Stem Cells and Differentiation. Cells 2021; 10:cells10113252. [PMID: 34831474 PMCID: PMC8624367 DOI: 10.3390/cells10113252] [Citation(s) in RCA: 26] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/11/2021] [Revised: 11/12/2021] [Accepted: 11/16/2021] [Indexed: 12/23/2022] Open
Abstract
Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSC) provide a powerful model system to uncover fundamental mechanisms that control cellular identity during mammalian development. Histone methylation governs gene expression programs that play a key role in the regulation of the balance between self-renewal and differentiation of ESCs. Lysine-specific demethylase 1 (LSD1, also known as KDM1A), the first identified histone lysine demethylase, demethylates H3K4me1/2 and H3K9me1/2 at target loci in a context-dependent manner. Moreover, it has also been shown to demethylate non-histone substrates playing a central role in the regulation of numerous cellular processes. In this review, we summarize current knowledge about LSD1 and the molecular mechanism by which LSD1 influences the stem cells state, including the regulatory circuitry underlying self-renewal and pluripotency.
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Cell division- and DNA replication-free reprogramming of somatic nuclei for embryonic transcription. iScience 2021; 24:103290. [PMID: 34849463 PMCID: PMC8609233 DOI: 10.1016/j.isci.2021.103290] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/16/2021] [Revised: 09/03/2021] [Accepted: 10/13/2021] [Indexed: 01/01/2023] Open
Abstract
Nuclear transfer systems represent the efficient means to reprogram a cell and in theory provide a basis for investigating the development of endangered species. However, conventional nuclear transfer using oocytes of laboratory animals does not allow reprogramming of cross-species nuclei owing to defects in cell divisions and activation of embryonic genes. Here, we show that somatic nuclei transferred into mouse four-cell embryos arrested at the G2/M phase undergo reprogramming toward the embryonic state. Remarkably, genome-wide transcriptional reprogramming is induced within a day, and ZFP281 is important for this replication-free reprogramming. This system further enables transcriptional reprogramming of cells from Oryx dammah, now extinct in the wild. Thus, our findings indicate that arrested mouse embryos are competent to induce intra- and cross-species reprogramming. The direct induction of embryonic transcripts from diverse genomes paves a unique approach for identifying mechanisms of transcriptional reprogramming and genome activation from a diverse range of species.
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