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1
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Barman P, Kaja A, Chakraborty P, Bhaumik SR. Chromatin and non-chromatin immunoprecipitations to capture protein-protein and protein-nucleic acid interactions in living cells. Methods 2023; 218:158-166. [PMID: 37611837 PMCID: PMC10528071 DOI: 10.1016/j.ymeth.2023.08.013] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/06/2023] [Revised: 08/18/2023] [Accepted: 08/19/2023] [Indexed: 08/25/2023] Open
Abstract
Proteins are expressed from genes via sequential biological processes of transcription, mRNA processing, export and translation, and play their roles in maintaining cellular functions via interactions with proteins, DNAs or RNAs. Thus, it is important to study the protein interactions during biological processes in living cells towards understanding their mechanisms-of-action in real time. Methodologies have been developed over the years to study protein interactions in vivo. One state-of-the-art approach is formaldehyde crosslinking-based immuno- or chemi-precipitation to analyze selective as well as genome/proteome-wide interactions in living cells. It is a popular and widely used methodology for cellular analysis of the protein-protein and protein-nucleic acid interactions. Here, we describe this approach to analyze protein-protein/nucleic acid interactions in vivo.
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Affiliation(s)
- Priyanka Barman
- Department of Biochemistry and Molecular Biology, Southern Illinois University School of Medicine, Carbondale, IL 62901, USA
| | - Amala Kaja
- Department of Biochemistry and Molecular Biology, Southern Illinois University School of Medicine, Carbondale, IL 62901, USA
| | - Pritam Chakraborty
- Department of Biochemistry and Molecular Biology, Southern Illinois University School of Medicine, Carbondale, IL 62901, USA
| | - Sukesh R Bhaumik
- Department of Biochemistry and Molecular Biology, Southern Illinois University School of Medicine, Carbondale, IL 62901, USA.
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2
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Barman P, Kaja A, Chakraborty P, Guha S, Roy A, Ferdoush J, Bhaumik SR. A novel ubiquitin-proteasome system regulation of Sgf73/ataxin-7 that maintains the integrity of the coactivator SAGA in orchestrating transcription. Genetics 2023; 224:iyad071. [PMID: 37075097 PMCID: PMC10324951 DOI: 10.1093/genetics/iyad071] [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: 01/31/2023] [Revised: 01/31/2023] [Accepted: 03/15/2023] [Indexed: 04/20/2023] Open
Abstract
Ataxin-7 maintains the integrity of Spt-Ada-Gcn5-Acetyltransferase (SAGA), an evolutionarily conserved coactivator in stimulating preinitiation complex (PIC) formation for transcription initiation, and thus, its upregulation or downregulation is associated with various diseases. However, it remains unknown how ataxin-7 is regulated that could provide new insights into disease pathogenesis and therapeutic interventions. Here, we show that ataxin-7's yeast homologue, Sgf73, undergoes ubiquitylation and proteasomal degradation. Impairment of such regulation increases Sgf73's abundance, which enhances recruitment of TATA box-binding protein (TBP) (that nucleates PIC formation) to the promoter but impairs transcription elongation. Further, decreased Sgf73 level reduces PIC formation and transcription. Thus, Sgf73 is fine-tuned by ubiquitin-proteasome system (UPS) in orchestrating transcription. Likewise, ataxin-7 undergoes ubiquitylation and proteasomal degradation, alteration of which changes ataxin-7's abundance that is associated with altered transcription and cellular pathologies/diseases. Collectively, our results unveil a novel UPS regulation of Sgf73/ataxin-7 for normal cellular health and implicate alteration of such regulation in diseases.
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Affiliation(s)
- Priyanka Barman
- Department of Biochemistry and Molecular Biology, Southern Illinois University School of Medicine, Carbondale, IL 62901, USA
| | - Amala Kaja
- Department of Biochemistry and Molecular Biology, Southern Illinois University School of Medicine, Carbondale, IL 62901, USA
- Department of Medicine, Baylor College of Medicine, Houston, TX-77030, USA
| | - Pritam Chakraborty
- Department of Biochemistry and Molecular Biology, Southern Illinois University School of Medicine, Carbondale, IL 62901, USA
| | - Shalini Guha
- Department of Biochemistry and Molecular Biology, Southern Illinois University School of Medicine, Carbondale, IL 62901, USA
| | - Arpan Roy
- Department of Biochemistry and Molecular Biology, Southern Illinois University School of Medicine, Carbondale, IL 62901, USA
| | - Jannatul Ferdoush
- Department of Biochemistry and Molecular Biology, Southern Illinois University School of Medicine, Carbondale, IL 62901, USA
- Department of Biology, Geology, and Environmental Science, University of Tennessee at Chattanooga, 615 McCallie Ave, Chattanooga, TN 37403, USA
| | - Sukesh R Bhaumik
- Department of Biochemistry and Molecular Biology, Southern Illinois University School of Medicine, Carbondale, IL 62901, USA
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3
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Haile ST, Rahman S, Fields JK, Orsburn BC, Bumpus NN, Wolberger C. The SAGA HAT module is tethered by its SWIRM domain and modulates activity of the SAGA DUB module. BIOCHIMICA ET BIOPHYSICA ACTA. GENE REGULATORY MECHANISMS 2023; 1866:194929. [PMID: 36965704 PMCID: PMC10226619 DOI: 10.1016/j.bbagrm.2023.194929] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/22/2022] [Revised: 03/12/2023] [Accepted: 03/19/2023] [Indexed: 03/27/2023]
Abstract
The SAGA (Spt-Ada-Gcn5 acetyltransferase) complex is a transcriptional co-activator that both acetylates and deubiquitinates histones. The histone acetyltransferase (HAT) subunit, Gcn5, is part of a subcomplex of SAGA called the HAT module. A minimal HAT module complex containing Gcn5 bound to Ada2 and Ada3 is required for full Gcn5 activity on nucleosomes. Deletion studies have suggested that the Ada2 SWIRM domain plays a role in tethering the HAT module to the remainder of SAGA. While recent cryo-EM studies have resolved the structure of the core of the SAGA complex, the HAT module subunits and molecular details of its interactions with the SAGA core could not be resolved. Here we show that the SWIRM domain is required for incorporation of the HAT module into the yeast SAGA complex, but not the ADA complex, a distinct six-protein acetyltransferase complex that includes the SAGA HAT module proteins. In the isolated Gcn5/Ada2/Ada3 HAT module, deletion of the SWIRM domain modestly increased activity but had negligible effect on nucleosome binding. Loss of the HAT module due to deletion of the SWIRM domain decreases the H2B deubiquitinating activity of SAGA, indicating a role for the HAT module in regulating SAGA DUB module activity. A model of the HAT module created with Alphafold Multimer provides insights into the structural basis for our biochemical data, as well as prior deletion studies.
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Affiliation(s)
- Sara T Haile
- Department of Biophysics and Biophysical Chemistry, The Johns Hopkins University School of Medicine, 725 N. Wolfe Street, Baltimore, MD 21205, United States of America
| | - Sanim Rahman
- Department of Biophysics and Biophysical Chemistry, The Johns Hopkins University School of Medicine, 725 N. Wolfe Street, Baltimore, MD 21205, United States of America
| | - James K Fields
- Department of Biophysics and Biophysical Chemistry, The Johns Hopkins University School of Medicine, 725 N. Wolfe Street, Baltimore, MD 21205, United States of America
| | - Benjamin C Orsburn
- Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, 725 N. Wolfe Street, Baltimore, MD 21205, United States of America
| | - Namandjé N Bumpus
- Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, 725 N. Wolfe Street, Baltimore, MD 21205, United States of America
| | - Cynthia Wolberger
- Department of Biophysics and Biophysical Chemistry, The Johns Hopkins University School of Medicine, 725 N. Wolfe Street, Baltimore, MD 21205, United States of America.
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4
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Geng Q, Li H, Wang D, Sheng RC, Zhu H, Klosterman SJ, Subbarao KV, Chen JY, Chen FM, Zhang DD. The Verticillium dahliae Spt-Ada-Gcn5 Acetyltransferase Complex Subunit Ada1 Is Essential for Conidia and Microsclerotia Production and Contributes to Virulence. Front Microbiol 2022; 13:852571. [PMID: 35283850 PMCID: PMC8905346 DOI: 10.3389/fmicb.2022.852571] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/11/2022] [Accepted: 01/31/2022] [Indexed: 12/16/2022] Open
Abstract
Verticillium dahliae is a destructive soil-borne pathogen of many economically important dicots. The genetics of pathogenesis in V. dahliae has been extensively studied. Spt-Ada-Gcn5 acetyltransferase complex (SAGA) is an ATP-independent multifunctional chromatin remodeling complex that contributes to diverse transcriptional regulatory functions. As members of the core module in the SAGA complex in Saccharomyces cerevisiae, Ada1, together with Spt7 and Spt20, play an important role in maintaining the integrity of the complex. In this study, we identified homologs of the SAGA complex in V. dahliae and found that deletion of the Ada1 subunit (VdAda1) causes severe defects in the formation of conidia and microsclerotia, and in melanin biosynthesis and virulence. The effect of VdAda1 on histone acetylation in V. dahliae was confirmed by western blot analysis. The deletion of VdAda1 resulted in genome-wide alteration of the V. dahliae transcriptome, including genes encoding transcription factors and secreted proteins, suggesting its prominent role in the regulation of transcription and virulence. Overall, we demonstrated that VdAda1, a member of the SAGA complex, modulates multiple physiological processes by regulating global gene expression that impinge on virulence and survival in V. dahliae.
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Affiliation(s)
- Qi Geng
- Co-Innovation Center for Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing, China
| | - Huan Li
- Co-Innovation Center for Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing, China
| | - Dan Wang
- Team of Crop Verticillium Wilt, State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China
| | - Ruo-Cheng Sheng
- Co-Innovation Center for Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing, China
| | - He Zhu
- National Cotton Industry Technology System Liaohe Comprehensive Experimental Station, The Cotton Research Center of Liaoning Academy of Agricultural Sciences, Liaoning Provincial Institute of Economic Crops, Liaoyang, China
| | - Steven J Klosterman
- United States Department of Agriculture, Agricultural Research Service, Crop Improvement and Protection Research Unit, Salinas, CA, United States
| | - Krishna V Subbarao
- Department of Plant Pathology, c/o U.S. Agricultural Research Station, University of California, Davis, Salinas, CA, United States
| | - Jie-Yin Chen
- Team of Crop Verticillium Wilt, State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China
| | - Feng-Mao Chen
- Co-Innovation Center for Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing, China
| | - Dan-Dan Zhang
- Team of Crop Verticillium Wilt, State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China
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5
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Fluorescence resonance energy transfer in revealing protein-protein interactions in living cells. Emerg Top Life Sci 2021; 5:49-59. [PMID: 33856021 DOI: 10.1042/etls20200337] [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: 11/29/2020] [Revised: 02/22/2021] [Accepted: 03/04/2021] [Indexed: 11/17/2022]
Abstract
Genes are expressed to proteins for a wide variety of fundamental biological processes at the cellular and organismal levels. However, a protein rarely functions alone, but rather acts through interactions with other proteins to maintain normal cellular and organismal functions. Therefore, it is important to analyze the protein-protein interactions to determine functional mechanisms of proteins, which can also guide to develop therapeutic targets for treatment of diseases caused by altered protein-protein interactions leading to cellular/organismal dysfunctions. There is a large number of methodologies to study protein interactions in vitro, in vivo and in silico, which led to the development of many protein interaction databases, and thus, have enriched our knowledge about protein-protein interactions and functions. However, many of these interactions were identified in vitro, but need to be verified/validated in living cells. Furthermore, it is unclear whether these interactions are direct or mediated via other proteins. Moreover, these interactions are representative of cell- and time-average, but not a single cell in real time. Therefore, it is crucial to detect direct protein-protein interactions in a single cell during biological processes in vivo, towards understanding the functional mechanisms of proteins in living cells. Importantly, a fluorescence resonance energy transfer (FRET)-based methodology has emerged as a powerful technique to decipher direct protein-protein interactions at a single cell resolution in living cells, which is briefly described in a limited available space in this mini-review.
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6
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Grant PA, Winston F, Berger SL. The biochemical and genetic discovery of the SAGA complex. BIOCHIMICA ET BIOPHYSICA ACTA-GENE REGULATORY MECHANISMS 2020; 1864:194669. [PMID: 33338653 DOI: 10.1016/j.bbagrm.2020.194669] [Citation(s) in RCA: 17] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Subscribe] [Scholar Register] [Received: 10/26/2020] [Revised: 12/10/2020] [Accepted: 12/11/2020] [Indexed: 12/12/2022]
Abstract
One of the major advances in our understanding of gene regulation in eukaryotes was the discovery of factors that regulate transcription by controlling chromatin structure. Prominent among these discoveries was the demonstration that Gcn5 is a histone acetyltransferase, establishing a direct connection between transcriptional activation and histone acetylation. This breakthrough was soon followed by the purification of a protein complex that contains Gcn5, the SAGA complex. In this article, we review the early genetic and biochemical experiments that led to the discovery of SAGA and the elucidation of its multiple activities.
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Affiliation(s)
- Patrick A Grant
- Department of Biomedical Science, Charles E. Schmidt College of Medicine, Florida Atlantic University, Boca Raton, FL 33431, United States of America
| | - Fred Winston
- Department of Genetics, Blavatnik Institute, Harvard Medical School, Boston, MA 02115, United States of America.
| | - Shelley L Berger
- Department of Cell and Developmental Biology, Penn Epigenetics Institute, Department of Biology, Department of Genetics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, United States of America
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7
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Barman P, Reddy D, Bhaumik SR. Mechanisms of Antisense Transcription Initiation with Implications in Gene Expression, Genomic Integrity and Disease Pathogenesis. Noncoding RNA 2019; 5:ncrna5010011. [PMID: 30669611 PMCID: PMC6468509 DOI: 10.3390/ncrna5010011] [Citation(s) in RCA: 20] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/15/2018] [Revised: 01/01/2019] [Accepted: 01/15/2019] [Indexed: 02/07/2023] Open
Abstract
Non-coding antisense transcripts arise from the strand opposite the sense strand. Over 70% of the human genome generates non-coding antisense transcripts while less than 2% of the genome codes for proteins. Antisense transcripts and/or the act of antisense transcription regulate gene expression and genome integrity by interfering with sense transcription and modulating histone modifications or DNA methylation. Hence, they have significant pathological and physiological relevance. Indeed, antisense transcripts were found to be associated with various diseases including cancer, diabetes, cardiac and neurodegenerative disorders, and, thus, have promising potentials for prognostic and diagnostic markers and therapeutic development. However, it is not clearly understood how antisense transcription is initiated and epigenetically regulated. Such knowledge would provide new insights into the regulation of antisense transcription, and hence disease pathogenesis with therapeutic development. The recent studies on antisense transcription initiation and its epigenetic regulation, which are limited, are discussed here. Furthermore, we concisely describe how antisense transcription/transcripts regulate gene expression and genome integrity with implications in disease pathogenesis and therapeutic development.
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Affiliation(s)
- Priyanka Barman
- Department of Biochemistry and Molecular Biology, Southern Illinois University School of Medicine, Carbondale, IL 62901, USA.
| | - Divya Reddy
- Department of Biochemistry and Molecular Biology, Southern Illinois University School of Medicine, Carbondale, IL 62901, USA.
| | - Sukesh R Bhaumik
- Department of Biochemistry and Molecular Biology, Southern Illinois University School of Medicine, Carbondale, IL 62901, USA.
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8
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Arras SDM, Ormerod KL, Erpf PE, Espinosa MI, Carpenter AC, Blundell RD, Stowasser SR, Schulz BL, Tanurdzic M, Fraser JA. Convergent microevolution of Cryptococcus neoformans hypervirulence in the laboratory and the clinic. Sci Rep 2017; 7:17918. [PMID: 29263343 PMCID: PMC5738413 DOI: 10.1038/s41598-017-18106-2] [Citation(s) in RCA: 27] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/18/2017] [Accepted: 12/05/2017] [Indexed: 12/30/2022] Open
Abstract
Reference strains are a key component of laboratory research, providing a common background allowing for comparisons across a community of researchers. However, laboratory passage of these strains has been shown to lead to reduced fitness and the attenuation of virulence in some species. In this study we show the opposite in the fungal pathogen Cryptococcus neoformans, with analysis of a collection of type strain H99 subcultures revealing that the most commonly used laboratory subcultures belong to a mutant lineage of the type strain that is hypervirulent. The pleiotropic mutant phenotypes in this H99L (for “Laboratory”) lineage are the result of a deletion in the gene encoding the SAGA Associated Factor Sgf29, a mutation that is also present in the widely-used H99L-derived KN99a/α congenic pair. At a molecular level, loss of this gene results in a reduction in histone H3K9 acetylation. Remarkably, analysis of clinical isolates identified loss of function SGF29 mutations in C. neoformans strains infecting two of fourteen patients, demonstrating not only the first example of hypervirulence in clinical C. neoformans samples, but also parallels between in vitro and in vivo microevolution for hypervirulence in this important pathogen.
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Affiliation(s)
- Samantha D M Arras
- Australian Infectious Diseases Research Centre, The University of Queensland, Brisbane, Queensland, Australia.,School of Chemistry & Molecular Biosciences, The University of Queensland, Brisbane, Queensland, Australia
| | - Kate L Ormerod
- Australian Infectious Diseases Research Centre, The University of Queensland, Brisbane, Queensland, Australia.,School of Chemistry & Molecular Biosciences, The University of Queensland, Brisbane, Queensland, Australia
| | - Paige E Erpf
- Australian Infectious Diseases Research Centre, The University of Queensland, Brisbane, Queensland, Australia.,School of Chemistry & Molecular Biosciences, The University of Queensland, Brisbane, Queensland, Australia
| | - Monica I Espinosa
- Australian Infectious Diseases Research Centre, The University of Queensland, Brisbane, Queensland, Australia.,School of Chemistry & Molecular Biosciences, The University of Queensland, Brisbane, Queensland, Australia
| | - Alex C Carpenter
- Australian Infectious Diseases Research Centre, The University of Queensland, Brisbane, Queensland, Australia.,School of Chemistry & Molecular Biosciences, The University of Queensland, Brisbane, Queensland, Australia
| | - Ross D Blundell
- Australian Infectious Diseases Research Centre, The University of Queensland, Brisbane, Queensland, Australia.,School of Chemistry & Molecular Biosciences, The University of Queensland, Brisbane, Queensland, Australia
| | - Samantha R Stowasser
- Australian Infectious Diseases Research Centre, The University of Queensland, Brisbane, Queensland, Australia.,School of Chemistry & Molecular Biosciences, The University of Queensland, Brisbane, Queensland, Australia
| | - Benjamin L Schulz
- Australian Infectious Diseases Research Centre, The University of Queensland, Brisbane, Queensland, Australia.,School of Chemistry & Molecular Biosciences, The University of Queensland, Brisbane, Queensland, Australia
| | - Milos Tanurdzic
- School of Biological Sciences, The University of Queensland, Brisbane, Queensland, Australia
| | - James A Fraser
- Australian Infectious Diseases Research Centre, The University of Queensland, Brisbane, Queensland, Australia. .,School of Chemistry & Molecular Biosciences, The University of Queensland, Brisbane, Queensland, Australia.
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9
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Two Distinct Regulatory Mechanisms of Transcriptional Initiation in Response to Nutrient Signaling. Genetics 2017; 208:191-205. [PMID: 29141908 DOI: 10.1534/genetics.117.300518] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/30/2016] [Accepted: 10/26/2017] [Indexed: 12/19/2022] Open
Abstract
SAGA (Spt-Ada-Gcn5-Acetyltransferase) and TFIID (transcription factor IID) have been previously shown to facilitate the formation of the PIC (pre-initiation complex) at the promoters of two distinct sets of genes. Here, we demonstrate that TFIID and SAGA differentially participate in the stimulation of PIC formation (and hence transcriptional initiation) at the promoter of PHO84, a gene for the high-affinity inorganic phosphate (Pi) transporter for crucial cellular functions, in response to nutrient signaling. We show that transcriptional initiation of PHO84 occurs predominantly in a TFIID-dependent manner in the absence of Pi in the growth medium. Such TFIID dependency is mediated via the NuA4 (nucleosome acetyltransferase of H4) histone acetyltransferase (HAT). Intriguingly, transcriptional initiation of PHO84 also occurs in the presence of Pi in the growth medium, predominantly via the SAGA complex, but independently of NuA4 HAT. Thus, Pi in the growth medium switches transcriptional initiation of PHO84 from NuA4-TFIID to SAGA dependency. Further, we find that both NuA4-TFIID- and SAGA-dependent transcriptional initiations of PHO84 are facilitated by the 19S proteasome subcomplex or regulatory particle (RP) via enhanced recruitment of the coactivators SAGA and NuA4 HAT, which promote TFIID-independent and -dependent PIC formation for transcriptional initiation, respectively. NuA4 HAT does not regulate activator binding to PHO84, but rather facilitates PIC formation for transcriptional initiation in the absence of Pi in the growth medium. On the other hand, SAGA promotes activator recruitment to PHO84 for transcriptional initiation in the growth medium containing Pi. Collectively, our results demonstrate two distinct stimulatory pathways for PIC formation (and hence transcriptional initiation) at PHO84 by TFIID, SAGA, NuA4, and 19S RP in the presence and absence of an essential nutrient, Pi, in the growth media, thus providing new regulatory mechanisms of transcriptional initiation in response to nutrient signaling.
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10
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Nucleosome competition reveals processive acetylation by the SAGA HAT module. Proc Natl Acad Sci U S A 2015; 112:E5461-70. [PMID: 26401015 DOI: 10.1073/pnas.1508449112] [Citation(s) in RCA: 40] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
The Spt-Ada-Gcn5 acetyltransferase (SAGA) coactivator complex hyperacetylates histone tails in vivo in a manner that depends upon histone 3 lysine 4 trimethylation (H3K4me3), a histone mark enriched at promoters of actively transcribed genes. SAGA contains a separable subcomplex known as the histone acetyltransferase (HAT) module that contains the HAT, Gcn5, bound to Sgf29, Ada2, and Ada3. Sgf29 contains a tandem Tudor domain that recognizes H3K4me3-containing peptides and is required for histone hyperacetylation in vivo. However, the mechanism by which H3K4me3 recognition leads to lysine hyperacetylation is unknown, as in vitro studies show no effect of the H3K4me3 modification on histone peptide acetylation by Gcn5. To determine how H3K4me3 binding by Sgf29 leads to histone hyperacetylation by Gcn5, we used differential fluorescent labeling of histones to monitor acetylation of individual subpopulations of methylated and unmodified nucleosomes in a mixture. We find that the SAGA HAT module preferentially acetylates H3K4me3 nucleosomes in a mixture containing excess unmodified nucleosomes and that this effect requires the Tudor domain of Sgf29. The H3K4me3 mark promotes processive, multisite acetylation of histone H3 by Gcn5 that can account for the different acetylation patterns established by SAGA at promoters versus coding regions. Our results establish a model for Sgf29 function at gene promoters and define a mechanism governing crosstalk between histone modifications.
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11
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Kurabe N, Murakami S, Tashiro F. SGF29 and Sry pathway in hepatocarcinogenesis. World J Biol Chem 2015; 6:139-147. [PMID: 26322172 PMCID: PMC4549758 DOI: 10.4331/wjbc.v6.i3.139] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/27/2015] [Revised: 05/31/2015] [Accepted: 07/02/2015] [Indexed: 02/05/2023] Open
Abstract
Deregulated c-Myc expression is a hallmark of many human cancers. We have recently identified a role of mammalian homolog of yeast SPT-ADA-GCN5-acetyltransferas (SAGA) complex component, SAGA-associated factor 29 (SGF29), in regulating the c-Myc overexpression. Here, we discuss the molecular nature of SFG29 in SPT3-TAF9-GCN5-acetyltransferase complex, a counterpart of yeast SAGA complex, and the mechanism through which the elevated SGF29 expression contribute to oncogenic potential of c-Myc in hepatocellularcarcinoma (HCC). We propose that the upstream regulation of SGF29 elicited by sex-determining region Y (Sry) is also augmented in HCC. We hypothesize that c-Myc elevation driven by the deregulated Sry and SGF29 pathway is implicated in the male specific acquisition of human HCCs.
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12
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Moraga F, Aquea F. Composition of the SAGA complex in plants and its role in controlling gene expression in response to abiotic stresses. FRONTIERS IN PLANT SCIENCE 2015; 6:865. [PMID: 26528322 PMCID: PMC4604261 DOI: 10.3389/fpls.2015.00865] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/30/2015] [Accepted: 09/30/2015] [Indexed: 05/07/2023]
Abstract
Protein complexes involved in epigenetic regulation of transcription have evolved as molecular strategies to face environmental stress in plants. SAGA (Spt-Ada-Gcn5 Acetyltransferase) is a transcriptional co-activator complex that regulates numerous cellular processes through the coordination of multiple post-translational histone modifications, including acetylation, deubiquitination, and chromatin recognition. The diverse functions of the SAGA complex involve distinct modules that are highly conserved between yeast, flies, and mammals. In this review, the composition of the SAGA complex in plants is described and its role in gene expression regulation under stress conditions summarized. Some of these proteins are likely involved in the regulation of the inducible expression of genes under light, cold, drought, salt, and iron stress, although the functions of several of its components remain unknown.
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Affiliation(s)
- Felipe Moraga
- Laboratorio de Bioingeniería, Facultad de Ingeniería y Ciencias, Universidad Adolfo IbáñezSantiago, Chile
| | - Felipe Aquea
- Laboratorio de Bioingeniería, Facultad de Ingeniería y Ciencias, Universidad Adolfo IbáñezSantiago, Chile
- Center for Applied Ecology and SustainabilitySantiago, Chile
- *Correspondence: Felipe Aquea
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13
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Durairaj G, Sen R, Uprety B, Shukla A, Bhaumik SR. Sus1p facilitates pre-initiation complex formation at the SAGA-regulated genes independently of histone H2B de-ubiquitylation. J Mol Biol 2014; 426:2928-2941. [PMID: 24911582 DOI: 10.1016/j.jmb.2014.05.028] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/18/2014] [Revised: 04/29/2014] [Accepted: 05/31/2014] [Indexed: 12/23/2022]
Abstract
Sus1p is a common component of transcriptional co-activator, SAGA (Spt-Ada-Gcn5-Acetyltransferase), and mRNA export complex, TREX-2 (Transcription-export 2), and is involved in promoting transcription and mRNA export. However, it is not clearly understood how Sus1p promotes transcription. Here, we show that Sus1p is predominantly recruited to the upstream activating sequence of a SAGA-dependent gene, GAL1, under transcriptionally active conditions as a component of SAGA to promote the formation of pre-initiation complex (PIC) at the core promoter and, consequently, transcriptional initiation. Likewise, Sus1p promotes the PIC formation at other SAGA-dependent genes and hence transcriptional initiation. Such function of Sus1p in promoting PIC formation and transcriptional initiation is not mediated via its role in regulation of SAGA's histone H2B de-ubiquitylation activity. However, Sus1p's function in regulation of histone H2B ubiquitylation is associated with transcriptional elongation, DNA repair and replication. Collectively, our results support that Sus1p promotes PIC formation (and hence transcriptional initiation) at the SAGA-regulated genes independently of histone H2B de-ubiquitylation and further controls transcriptional elongation, DNA repair and replication via orchestration of histone H2B ubiquitylation, thus providing distinct functional insights of Sus1p in regulation of DNA transacting processes.
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Affiliation(s)
- Geetha Durairaj
- Department of Biochemistry and Molecular Biology Southern Illinois University School of Medicine Carbondale, IL-62901 USA
| | - Rwik Sen
- Department of Biochemistry and Molecular Biology Southern Illinois University School of Medicine Carbondale, IL-62901 USA
| | - Bhawana Uprety
- Department of Biochemistry and Molecular Biology Southern Illinois University School of Medicine Carbondale, IL-62901 USA
| | - Abhijit Shukla
- Department of Biochemistry and Molecular Biology Southern Illinois University School of Medicine Carbondale, IL-62901 USA
| | - Sukesh R Bhaumik
- Department of Biochemistry and Molecular Biology Southern Illinois University School of Medicine Carbondale, IL-62901 USA
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Kamata K, Goswami G, Kashio S, Urano T, Nakagawa R, Uchida H, Oki M. The N-terminus and Tudor domains of Sgf29 are important for its heterochromatin boundary formation function. ACTA ACUST UNITED AC 2013; 155:159-71. [DOI: 10.1093/jb/mvt108] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022]
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Schram AW, Baas R, Jansen PWTC, Riss A, Tora L, Vermeulen M, Timmers HTM. A dual role for SAGA-associated factor 29 (SGF29) in ER stress survival by coordination of both histone H3 acetylation and histone H3 lysine-4 trimethylation. PLoS One 2013; 8:e70035. [PMID: 23894581 PMCID: PMC3720948 DOI: 10.1371/journal.pone.0070035] [Citation(s) in RCA: 35] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/12/2013] [Accepted: 06/14/2013] [Indexed: 02/02/2023] Open
Abstract
The SGF29 protein binds to tri-methylated lysine-4 of histone H3 (H3K4me3), which is a histone modification associated with active promoters. Human SGF29 is a subunit of the histone acetyltransferase module of the SAGA (Spt-Ada-Gcn5 acetyltransferase) and ATAC (Ada-Two-A-containing 2A) co-activator complexes. Previous work revealed that the SAGA complex is recruited to endoplasmic reticulum (ER) stress target genes and required for their induction. Here, we report the involvement of SGF29 in the survival of human cells from ER stress. SGF29 knockdown results in impaired transcription of the ER stress genes GRP78 and CHOP. Besides histone H3K14 acetylation, we find that SGF29 is also required for the maintenance of H3K4me3 at these genes, which is already present prior to ER stress. Reduced levels of H3K4me3 in the absence of SGF29 correlate with a decreased association of ASH2L, which is a core component of the SET1/MLL complexes, to GFP78 and CHOP. In conclusion, our results suggest that the H3K4me3-binding protein SGF29 plays a central and dual role in the ER stress response. Prior to ER stress, the protein coordinates H3K4me3 levels, thereby maintaining a ‘poised’ chromatin state on ER stress target gene promoters. Following ER stress induction, SGF29 is required for increased H3K14 acetylation on these genes, which then results in full transcriptional activation, thereby promoting cell survival.
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Affiliation(s)
- Andrea W. Schram
- Department of Molecular Cancer Research, University Medical Center Utrecht, Utrecht, The Netherlands
| | - Roy Baas
- Department of Molecular Cancer Research, University Medical Center Utrecht, Utrecht, The Netherlands
| | - Pascal W. T. C. Jansen
- Department of Molecular Cancer Research, University Medical Center Utrecht, Utrecht, The Netherlands
| | - Anne Riss
- Cellular Signaling and Nuclear Dynamics Program, Institut de Génétique et de Biologie, Université de Strasbourg, Illkirch, France
| | - Laszlo Tora
- Cellular Signaling and Nuclear Dynamics Program, Institut de Génétique et de Biologie, Université de Strasbourg, Illkirch, France
| | - Michiel Vermeulen
- Department of Molecular Cancer Research, University Medical Center Utrecht, Utrecht, The Netherlands
- * E-mail: (MV); (HTMT)
| | - H. Th. Marc Timmers
- Department of Molecular Cancer Research, University Medical Center Utrecht, Utrecht, The Netherlands
- * E-mail: (MV); (HTMT)
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Cell-cycle perturbations suppress the slow-growth defect of spt10Δ mutants in Saccharomyces cerevisiae. G3-GENES GENOMES GENETICS 2013; 3:573-83. [PMID: 23450643 PMCID: PMC3583463 DOI: 10.1534/g3.112.005389] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 12/18/2012] [Accepted: 01/17/2013] [Indexed: 01/05/2023]
Abstract
Spt10 is a putative acetyltransferase of Saccharomyces cerevisiae that directly activates the transcription of histone genes. Deletion of SPT10 causes a severe slow growth phenotype, showing that Spt10 is critical for normal cell division. To gain insight into the function of Spt10, we identified mutations that impair or improve the growth of spt10 null (spt10Δ) mutants. Mutations that cause lethality in combination with spt10Δ include particular components of the SAGA complex as well as asf1Δ and hir1Δ. Partial suppressors of the spt10Δ growth defect include mutations that perturb cell-cycle progression through the G1/S transition, S phase, and G2/M. Consistent with these results, slowing of cell-cycle progression by treatment with hydroxyurea or growth on medium containing glycerol as the carbon source also partially suppresses the spt10Δ slow-growth defect. In addition, mutations that impair the Lsm1-7-Pat1 complex, which regulates decapping of polyadenylated mRNAs, also partially suppress the spt10Δ growth defect. Interestingly, suppression of the spt10Δ growth defect is not accompanied by a restoration of normal histone mRNA levels. These findings suggest that Spt10 has multiple roles during cell division.
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