RNA sequencing (RNA-seq) has become key to complementing exome and genome sequencing for variant interpretation. We present a minimally invasive RNA-seq protocol using short-term cultured peripheral blood mononuclear cells (PBMCs) with and without cycloheximide treatment, enabling detection of transcripts subject to nonsense-mediated decay. While broadly applicable, this protocol is particularly suited for neurodevelopmental disorders, as up to 80% of the genes in our intellectual disability and epilepsy gene panel are expressed in PBMCs. Applied to 46 affected individuals and 15 parents, RNA-seq revealed splicing defects in six of nine individuals with splice variants, allowing reclassification of seven variants. Targeted cDNA analysis confirmed aberrant splicing in four individuals but missed intron retention in two. Global analyses (FRASER, OUTRIDER, and monoallelic expression) supported findings but did not yield new diagnoses. We propose a flowchart integrating RNA-seq into diagnostic workflows. Overall, our protocol is easily implementable, captures complex splicing events, and enhances variant classification.

The expression of imprinted genes in the Dlk1-Dio3 domain is regulated by Dlk1-Meg3 intergenic DMR (IG-DMR), which is methylated in a parental-of-origin-specific manner. An unmethylated 4.1-kb region in the IG-DMR is essential for the maternal allele. Several molecular mechanisms have been proposed for the 4.1-kb region of IG-DMR; however, the sequence in the 4.1-kb region essential for imprinted gene expression is still unknown. To explore the sequence responsible for the IG-DMR in vivo, we generated mutant mice with a series of IG-DMR deletions. We observed that a deletion of the 2.7-kb region, including the IG-DMR transcriptional regulatory element (IGTRE), on the maternal allele causes IG-DMR dysfunction, resulting in perinatal lethality. At least two functional sequences exist in IGTRE that are functionally redundant in vivo, and the paternal transmission of a mutant allele, in which IGTRE was deleted together with a tandem repeat sequence in IG-DMR (IGRep), rescued embryonic lethality due to a lack of paternal IGRep. Our findings revealed that a sequence responsible for the lethal phenotype of the maternally inherited 4.1-kb deletion of IG-DMR is in the IGTRE domain.

Genetic variation and 3D chromatin structure have major roles in gene regulation. Due to challenges in mapping chromatin conformation with haplotype-specific resolution, the effects of genetic sequence variation on 3D genome structure and gene expression imbalance remain understudied. Here, we applied Genome Architecture Mapping (GAM) to a hybrid mouse embryonic stem cell (mESC) line with high density of single-nucleotide polymorphisms (SNPs). GAM resolved haplotype-specific 3D genome structures with high sensitivity, revealing extensive allelic differences in chromatin compartments, topologically associating domains (TADs), long-range enhancer-promoter contacts, and CTCF loops. Architectural differences often coincide with allele-specific differences in gene expression, and with Polycomb occupancy. We show that histone genes are expressed with allelic imbalance in mESCs, and are involved in haplotype-specific chromatin contacts marked by H3K27me3. Conditional knockouts of Polycomb enzymatic subunits, Ezh2 or Ring1, show that one-third of ASE genes, including histone genes, is regulated through Polycomb repression. Our work reveals highly distinct 3D folding structures between homologous chromosomes, and highlights their intricate connections with allelic gene expression.

Sex differences in physiological and disease traits are pervasive and begin during early development, but the genetic architecture of these differences is largely unknown. Here, we leverage the human placenta, a transient organ during pregnancy critical to fetal development, to investigate the impact of sex in the regulatory landscape of placental autosomal methylome and transcriptome, and its relevance to health and disease. We find that placental methylation and its genetic regulation are extensively impacted by fetal sex, whereas sex differences in placental gene expression and its genetic regulation are limited. We identify molecular processes and regulatory targets that are enriched in a sex-specific manner, and find enrichment of imprinted genes in sex-differentiated placental methylation, including female-biased methylation within the well-known KCNQ1OT1/CDKN1C imprinting cluster of genes expressed in a parent-of-origin dependent manner. We establish that several sex-differentiated genetic effects on placental methylation and gene expression colocalize with birthweight and adult disease genetic associations, facilitating mechanistic insights on early life origins of health and disease outcomes shaped by sex.

Genomic imprinting is an epigenetic phenomenon in mammals that affects brain development and behavior. Imprinting involves the regulation of allelic expression for some genes in offspring that depends on whether alleles are inherited from mothers compared to fathers, and is thought to provide parental control over offspring social behavior phenotypes. Imprinted gene expression is prevalent in the mammalian brain, and human imprinted gene mutations are associated with neurodevelopmental disorders and neurodivergent social behavior in Prader-Willi Syndrome, Angelman Syndrome, and autism. Here, we provide a review of the evidence that imprinted genes influence social behaviors across major neurodevelopmental stages in humans and mouse animal models that include parent-infant interactions, juvenile sociability, and adult aggression, dominance, and sexual behavior.

Cells preserve and convey certain gene expression patterns to their progeny through the mechanism called epigenetic memory. Epigenetic memory, encoded by epigenetic markers and components, determines germline inheritance, genomic imprinting, and X chromosome inactivation. First discovered long non coding RNAs were implicated in genomic imprinting and X-inactivation and these two phenomena clearly demonstrate the role of lncRNAs in epigenetic memory regulation. Undoubtedly, lncRNAs are well-suited for regulating genes in close proximity at imprinted loci. Due to prolonged association with the transcription site, lncRNAs are able to guide chromatin modifiers to certain locations, thereby enabling accurate temporal and spatial regulation. Nevertheless, the current state of knowledge regarding lncRNA biology and imprinting processes is still in its nascent phase. Herein, we provide a synopsis of recent scientific advancements to enhance our comprehension of lncRNAs and their functions in epigenetic memory, with a particular emphasis on genomic imprinting and X chromosome inactivation, thus gaining a deeper understanding of the role of lncRNAs in epigenetic regulatory networks.

Epigenetic modifications provide mechanisms for influencing gene expression, regulating cell differentiation and maintaining long-term memory of cellular identity and function. As oocytes transmit epigenetic information to offspring, correct establishment of the oocyte epigenome is important for normal offspring development. Oocyte epigenetic programming is highly complex, involving a range of epigenetic modifiers which interact to establish a specific distribution of DNA methylation and histone modifications. Disruptions to oocyte epigenetic programming can alter epigenetic memory and prevent normal developmental outcomes in the next generation. Therefore, it is critical that we further our understanding of the interdependent relationships between various epigenetic modifiers and modifications during oogenesis.

In this study we investigated the spatial and temporal distribution of a range of epigenetic modifiers and modifications in growing oocytes of primordial to antral follicles. We provide comprehensive immunofluorescent profiles of SETD2, H3K36me3, KDM6A, RBBP7, H3K27me3, DNMT3A and DNMT3L and compare these profiles to our previously published profiles of the Polycomb Repressive Complex 2 components EED, EZH2 and SUZ12 in growing oocytes of wildtype mice. In addition, we examined the nuclear levels and spatial distribution of these epigenetic modifiers and modifications in oocytes that lacked the essential Polycomb Repressive Complex 2 subunit, EED. Notably, histone remodelling in primary-secondary follicle oocytes preceded upregulation of DNMT3A and DNMT3L in secondary-antral follicle oocytes. Moreover, loss of EED and H3K27me3 led to significantly increased levels of the H3K36me3 methyltransferase SETD2 during early-mid oocyte growth, although the average levels of H3K36me3 were unchanged.

Overall, these data demonstrate that oocyte epigenetic programming is a highly ordered process, with histone remodelling in early growing oocytes preceding de novo DNA methylation in secondary-antral follicle oocytes. These results indicate that tight temporal and spatial regulation of histone modifiers and modifications is essential to ensure correct establishment of the unique epigenome present in fully grown oocytes. Further understanding of the temporal and spatial relationships between different epigenetic modifications and how they interact is essential for understanding how germline epigenetic programming affects inheritance and offspring development in mammals, including humans.

Extraembryonic tissues have pivotal roles in morphogenesis and patterning of the early mammalian embryo. Developmental programmes mediated through signalling pathways and gene regulatory networks determine the sequence in which fate determination and lineage commitment of extraembryonic tissues take place, and epigenetic processes allow the memory of cell identity and state to be sustained throughout and beyond embryo development, even extending across generations. In this Review, we discuss the molecular and cellular mechanisms necessary for the different extraembryonic tissues to develop and function, from their initial specification up until the end of gastrulation, when the body plan of the embryo and the anatomical organization of its supporting extraembryonic structures are established. We examine the interaction between extraembryonic and embryonic tissues during early patterning and morphogenesis, and outline how epigenetic memory supports extraembryonic tissue development.

Genomic imprinting is the parent-of-origin dependent monoallelic expression of genes often associated with regions of germline-derived DNA methylation that are maintained as differentially methylated regions (gDMRs) in somatic tissues. This form of epigenetic regulation is highly conserved in mammals and is thought to have co-evolved with placentation. Tissue-specific gDMRs have been identified in human placenta, suggesting that species-specific imprinting dependent on unorthodox epigenetic establishment or maintenance may be more widespread than previously anticipated. Non-canonical imprinting, reliant on differential allelic H3K27me3 enrichment, has been reported in mouse and rat pre-implantation embryos, often overlapping long terminal repeat (LTR)-derived promoters. These non-canonical imprints lose parental allele-specific H3K27me3 specificity, subsequently gaining DNA methylation on the same allele in extra-embryonic tissues resulting in placenta-specific, somatically acquired maternal DMRs. To determine if similar non-canonical imprinting is present in the human placenta, we interrogated allelic DNA methylation for a selected number of loci, including (i) the human orthologues of non-canonical imprinted regions in mouse and rat, (ii) promoters of human LTR-derived transcripts, and (iii) CpG islands with intermediate placenta-specific methylation that are unmethylated in gametes and pre-implantation embryos. We failed to identify any non-canonical imprints in the human placenta whole villi samples. Furthermore, the assayed genes were shown to be biallelically expressed in human pre-implantation embryos, indicating they are not imprinted at earlier time points. Together, our work reiterates the continued evolution of placenta-specific imprinting in mammals, which we suggest is linked to epigenetic differences during the maternal-to-embryo transition and species-specific integration of retrotransposable elements.

DNA methylation at cytosine bases (5-methylcytosine, 5mC) is a heritable epigenetic mark regulating gene expression. While enzymes that metabolize 5mC are well-characterized, endogenous signaling molecules that regulate DNA methylation machinery have not been described. We report that physiological nitric oxide (NO) concentrations reversibly inhibit the DNA demethylases TET and ALKBH2 by binding to the mononuclear non-heme iron atom forming a dinitrosyliron complex (DNIC) and preventing cosubstrates from binding. In cancer cells treated with exogenous NO, or endogenously synthesizing NO, 5mC and 5-hydroxymethylcytosine (5hmC) increase, with no changes in DNA methyltransferase activity. 5mC is also significantly increased in NO-producing patient-derived xenograft tumors from mice. Genome-wide methylome analysis of cells chronically treated with NO (10 days) shows enrichment of 5mC and 5hmC at gene-regulatory loci, correlating with altered expression of NO-regulated tumor-associated genes. Regulation of DNA methylation is distinctly different from canonical NO signaling and represents a unique epigenetic role for NO.

Epigenome editing with DNA-targeting technologies such as CRISPR-dCas9 can be used to dissect gene regulatory mechanisms and potentially treat associated disorders. For example, Prader-Willi syndrome (PWS) results from loss of paternally expressed imprinted genes on chromosome 15q11.2-q13.3, although the maternal allele is intact but epigenetically silenced. Using CRISPR repression and activation screens in human induced pluripotent stem cells (iPSCs), we identified genomic elements that control the expression of the PWS gene SNRPN from the paternal and maternal chromosomes. We showed that either targeted transcriptional activation or DNA demethylation can activate the silenced maternal SNRPN and downstream PWS transcripts. However, these two approaches function at unique regions, preferentially activating different transcript variants and involving distinct epigenetic reprogramming mechanisms. Remarkably, transient expression of the targeted demethylase leads to stable, long-term maternal SNRPN expression in PWS iPSCs. This work uncovers targeted epigenetic manipulations to reprogram a disease-associated imprinted locus and suggests possible therapeutic interventions.

In the germ line and during early embryogenesis, DNA methylation (DNAme) undergoes global erasure and re-establishment to support germ cell and embryonic development. While DNAme acquisition during male germ cell development is essential for setting genomic DNA methylation imprints, other intergenerational roles for paternal DNAme in defining embryonic chromatin are unknown. Through conditional gene deletion of the de novo DNA methyltransferases Dnmt3a and/or Dnmt3b, we observe that DNMT3A primarily safeguards against DNA hypomethylation in undifferentiated spermatogonia, while DNMT3B catalyzes de novo DNAme during spermatogonial differentiation. Failing de novo DNAme in Dnmt3a/Dnmt3b double deficient spermatogonia is associated with increased nucleosome occupancy in mature sperm, preferentially at sites with higher CpG content, supporting the model that DNAme modulates nucleosome retention in sperm. To assess the impact of altered sperm chromatin in formatting embryonic chromatin, we measure H3K4me3 occupancy at paternal and maternal alleles in 2-cell embryos using a transposon-based tagging approach. Our data show that reduced DNAme in sperm renders paternal alleles permissive for H3K4me3 establishment in early embryos, independently of possible paternal inheritance of sperm born H3K4me3. Together, this study provides evidence that paternally inherited DNAme directs chromatin formation during early embryonic development.

Human puberty is a dynamic biological process determined by the increase in the pulsatile secretion of GnRH triggered by distinct factors not fully understood. Current knowledge reveals fine tuning between an increase in stimulatory factors and a decrease in inhibitory factors, where genetic and epigenetic factors have been indicated as key players in the regulation of puberty onset by distinct lines of evidence. Central precocious puberty (CPP) results from the premature reactivation of pulsatile secretion of GnRH. In the past decade, the identification of genetic causes of CPP has largely expanded, revealing hypothalamic regulatory factors of pubertal timing. Among them, 3 genes associated with CPP are linked to mechanisms involving DNA methylation, reinforcing the strong role of epigenetics underlying this disorder. Loss-of-function mutations in Makorin Ring-Finger Protein 3 (MKRN3) and Delta-Like Non-Canonical Notch Ligand 1 (DLK1), 2 autosomal maternally imprinted genes, have been described as relevant monogenic causes of CPP with the phenotype exclusively associated with paternal transmission. MKRN3 has proven to be a key component of the hypothalamic inhibitory input on GnRH neurons through different mechanisms. Additionally, rare heterozygous variants in the Methyl-CpG-Binding Protein 2 (MECP2), an X-linked gene that is a key factor of DNA methylation machinery, were identified in girls with sporadic CPP with or without neurodevelopmental disorders. In this mini-review, we focus on how the identification of genetic causes of CPP has revealed epigenetic regulators of human pubertal timing, summarizing the latest knowledge on the associations of puberty with MKRN3, DLK1, and MECP2.

Epigenetic factors are crucial for ensuring proper chromatin dynamics during the initial stages of embryo development. Among these factors, the Polycomb group (PcG) of proteins plays a key role in establishing correct transcriptional programmes during mouse embryogenesis. PcG proteins are classified into two complexes: Polycomb repressive complex 1 (PRC1) and PRC2. Both complexes decorate histone proteins with distinct post-translational modifications (PTMs) that are predictive of a silent transcriptional chromatin state. In recent years, a critical adaptation of the classical techniques to analyse chromatin profiles and to study biochemical interactions at low-input resolution has allowed us to deeply explore PcG molecular mechanisms in the very early stages of mouse embryo development- from fertilisation to gastrulation, and from zygotic genome activation (ZGA) to specific lineages differentiation. These advancements provide a foundation for a deeper understanding of the fundamental role Polycomb complexes play in early development and have elucidated the mechanistic dynamics of PRC1 and PRC2. In this review, we discuss the functions and molecular mechanisms of both PRC1 and PRC2 during early mouse embryo development, integrating new studies with existing knowledge. Furthermore, we highlight the molecular functionality of Polycomb complexes from ZGA through gastrulation, with a particular focus on non-canonical imprinted and bivalent genes, and Hox cluster regulation.

Genomic imprinting is an epigenetic process that results in parent-of-origin effects on mammalian development and growth. Research on genomic imprinting in domesticated animals has lagged due to a primary focus on orthologs of mouse and human imprinted genes. This emphasis has limited the discovery of imprinted genes specific to livestock. To identify genomic imprinting in pigs, we generated parthenogenetic porcine embryos alongside biparental normal embryos, and then performed whole-genome bisulfite sequencing and RNA sequencing on these samples. In our analyses, we discovered a maternally methylated differentially methylated region within the orthologous ZNF791 locus in pigs. Additionally, we identified both a major imprinted isoform of the ZNF791-like gene and an unannotated antisense transcript that has not been previously annotated. Importantly, our comparative analyses of the orthologous ZNF791 gene in various eutherian mammals, including humans, non-human primates, rodents, artiodactyls, and dogs, revealed that this gene is subjected to genomic imprinting exclusively in domesticated animals, thereby highlighting lineage-specific imprinting. Furthermore, we explored the potential mechanisms behind the establishment of maternal DNA methylation imprints in porcine and bovine oocytes, supporting the notion that integration of transposable elements, active transcription, and histone modification may collectively contribute to the methylation of embedded intragenic CpG island promoters. Our findings convey fundamental insights into molecular and evolutionary aspects of livestock species-specific genomic imprinting and provide critical agricultural implications.

DNA methylation is an essential epigenetic modification that plays a crucial role in regulating gene expression and maintaining genomic stability. With the advancement in sequencing technology, methylation studies have provided valuable insights into the diagnosis of rare diseases through the various identification of episignatures, epivariation, epioutliers, and allele-specific methylation. However, current methylation studies are not without limitations. This mini-review explores the current understanding of DNA methylation in rare diseases, highlighting the key mechanisms and diagnostic potential, and emphasizing the need for advanced methodologies and integrative approaches to enhance the understanding of disease progression and design more personable treatment for patients, given the nature of rare diseases.

Early embryonic arrest (EEA) is a critical impediment in assisted reproductive technology (ART), affecting 40% of infertile patients by halting the development of early embryos from the zygote to blastocyst stage, resulting in a lack of viable embryos for successful pregnancy. Despite its prevalence, the molecular mechanism underlying EEA remains elusive. This review synthesizes the latest research on the genetic and molecular factors contributing to EEA, with a focus on maternal, paternal, and embryonic factors. Maternal factors such as irregularities in follicular development and endometrial environment, along with mutations in genes like NLRP5, PADI6, KPNA7, IGF2, and TUBB8, have been implicated in EEA. Specifically, PATL2 mutations are hypothesized to disrupt the maternal-zygotic transition, impairing embryo development. Paternal contributions to EEA are linked to chromosomal variations, epigenetic modifications, and mutations in genes such as CFAP69, ACTL7A, and M1AP, which interfere with sperm development and lead to infertility. Aneuploidy may disrupt spindle assembly checkpoints and pathways including Wnt, MAPK, and Hippo signaling, thereby contributing to EEA. Additionally, key genes involved in embryonic genome activation-such as ZSCAN4, DUXB, DUXA, NANOGNB, DPPA4, GATA6, ARGFX, RBP7, and KLF5-alongside functional disruptions in epigenetic modifications, mitochondrial DNA, and small non-coding RNAs, play critical roles in the onset of EEA. This review provides a comprehensive understanding of the genetic and molecular underpinnings of EEA, offering a theoretical foundation for the diagnosis and potential therapeutic strategies aimed at improving pregnancy outcomes.

Genomic imprinting is essential for mammalian growth and embryogenesis. High-throughput bisulfite sequencing accompanied with parental haplotype-specific information allows analysis of imprinted genes and imprinting control regions (ICRs) on a large scale. Currently, although several allelic methylated regions (AMRs) detection software were developed, methods for detecting imprinted AMRs is still limited. Here, we developed a SNP-independent statistical approach, AIMER, to detect imprinting-like AMRs. By using the mouse frontal cortex methylome as input, we demonstrated that AIMER performs very well in detecting known germline ICRs compared with other methods. Furthermore, we found the putative parental AMRs AIMER detected could be distinguished from sequence-dependent AMRs. Finally, we found a novel germline imprinting-like AMR using WGBS data from 17 distinct mouse tissue samples. The results indicate that AIMER is a good choice for detecting imprinting-like (parent-of-origin-dependent) AMRs. We hope this method will be helpful for future genomic imprinting studies. The Python source code for our project is now publicly available on both GitHub (https://github.com/ZhaoLab-TMU/AIMER) and Gitee (https://gitee.com/zhaolab_tmu/AIMER).

Epigenetic modifications regulate chromatin accessibility, gene expression, cell differentiation and tissue development. As epigenetic modifications can be inherited via mitotic and meiotic cell divisions, they enable a heritable memory of cell identity and function and can alter inherited characteristics in the next generation. Tight regulation of epigenetic information is critical for normal cell function and is often disrupted in diseases including cancer, metabolic, neurological and inherited congenital conditions. The ovary performs critical functions in female reproductive health and fertility, including oocyte and sex-hormone production. Oocytes undergo extensive epigenetic programming including the establishment of maternal genomic imprints, which are critical for offspring health and development. Epigenetic modifiers also regulate ovarian somatic cells, such as granulosa and theca cells which support oocytes and produce hormones. While ovarian dysfunction contributes to serious ovarian conditions such as primary ovarian insufficiency (POI), polycystic ovary syndrome (PCOS) and ovarian cancers, the roles of epigenetic modifications in the ovary and their contribution to ovarian dysfunction are not properly understood. Here we review recent advancements in understanding Polycomb proteins, important epigenetic modifiers that have emerging roles in ovarian development and maternal epigenetic inheritance. Polycomb group proteins (PcGs) contribute to the faithful establishment of epigenetic information in oocytes, a process essential for normal offspring development in mice. Emerging evidence also indicates that PcGs regulate ovarian function and female fertility. Understanding these and similar mechanisms will provide greater insight into the epigenetic regulation of ovarian and oocyte function, and how its disruption can impact reproductive health and maternal inheritance.

Plants can program and reprogram their genomes to create genetic variation and epigenetic modifications, leading to phenotypic plasticity. Although consequences of genetic changes are comprehensible, the basis for transgenerational inheritance of epigenetic variation is elusive. This review addresses contributions of external (environmental) and internal (genomic) factors to the establishment and maintenance of epigenetic memory during plant evolution, crop domestication, and modern breeding. Dynamic and pervasive changes in DNA methylation and chromatin modifications provide a diverse repertoire of epigenetic variation potentially for transgenerational inheritance. Elucidating and harnessing epigenetic inheritance will help us develop innovative breeding strategies and biotechnological tools to improve crop yield and resilience in the face of environmental challenges. Beyond plants, epigenetic principles are shared across sexually reproducing organisms including humans with relevance to medicine and public health.

The mammalian imprinted Dlk1-Dio3 domain contains multiple lncRNAs, mRNAs, the largest miRNA cluster in the genome and four differentially methylated regions (DMRs), and deletion of maternally expressed RNA within this locus results in embryonic lethality, but the mechanism by which this occurs is not clear. Here, we optimized the model of maternally expressed RNAs transcription termination in the domain and found that the cause of embryonic death was apoptosis in the embryo, particularly in the liver. We generated a mouse model of maternally expressed RNAs silencing in the Dlk1-Dio3 domain by inserting a 3 × polyA termination sequence into the Gtl2 locus. By analyzing RNA-seq data of mouse embryos combined with histological analysis, we found that silencing of maternally expressed RNAs in the domain activated apoptosis, causing vascular rupture of the fetal liver, resulting in hemorrhage and injury. Mechanistically, termination of Gtl2 transcription results in the silencing of maternally expressed RNAs and activation of paternally expressed genes in the interval, and it is the gene itself rather than the IG-DMR and Gtl2-DMR that causes the aforementioned phenotypes. In conclusion, these findings illuminate a novel mechanism by which the silencing of maternally expressed RNAs within Dlk1-Dio3 domain leads to hepatic hemorrhage and embryonic death through the activation of apoptosis.

The maintenance of normal pregnancy requires appropriate maturation and transformation of various cells, which constitute the microenvironmental regulatory network at the maternal-fetal interface. Interestingly, changes in the cellular components of the maternal-fetal immune microenvironment and the regulation of epigenetic modifications of the genome have attracted much attention. With the development of epigenetics (DNA and RNA methylation, histone modifications, etc.), new insights have been gained into early embryonic developmental stages (e.g., maternal-to-zygotic transition, MZT). Understanding the various appropriate modes of transcriptional regulation required for the early embryonic developmental process from the perspective of epigenetic modifications will help us to provide new targets and insights into the pathogenesis of embryonic failure during further natural fertilization. This review focuses on the loci of action of epigenetic modifications from the perspectives of female germ cell development and embryo development to provide new insights for personalized diagnosis and treatment of abortion.

Neuroblastoma (NB) is a complex disease, and the current understanding of NB biology is limited. Deregulation in genomic imprinting is a common event in malignancy. Since imprinted genes play crucial roles in early fetal growth and development, their role in NB pathogenesis could be suggested.

We examined alterations in DNA methylation patterns of 369 NB tumours at 49 imprinted differentially methylated regions (DMRs) and assessed its association with overall survival probabilities and selected clinical and genomic features of the tumours. In addition, an integrated analysis of DNA methylation and allele-specific copy number alterations (CNAs) was performed, to understand the correlation between the two molecular events.

Several imprinted regions with aberrant methylation patterns in NB were identified. Regions that underwent loss of methylation in > 30% of NB samples were DMRs annotated to the genes NDN, SNRPN, IGF2, MAGEL2 and HTR5A and regions with gain of methylation were NNAT, RB1 and GPR1. Methylation alterations at six of the 49 imprinted DMRs were statistically significantly associated with reduced overall survival: MIR886, RB1, NNAT/BLCAP, MAGEL2, MKRN3 and INPP5F. RB1, NNAT/BLCAP and MKRN3 were further able to stratify low-risk NB tumours i.e. tumours that lacked MYCN amplification and 11q deletion into risk groups. Methylation alterations at NNAT/BLCAP, MAGEL2 and MIR886 predicted risk independently of MYCN amplification or 11q deletion and age at diagnosis. Investigation of the allele-specific CNAs demonstrated that the imprinted regions that displayed most alterations in NB tumours harbor true epigenetic changes and are not result of the underlying CNAs.

Aberrant methylation in imprinted regions is frequently occurring in NB tumours and several of these regions have independent prognostic value. Thus, these could serve as potentially important clinical epigenetic markers to identify individuals with adverse prognosis. Incorporation of methylation status of these regions together with the established risk predictors may further refine the prognostication of NB patients.

Imprinted genes provide an attractive paradigm to unravel links between transcription and genome architecture. The parental allele-specific expression of these essential genes - which are clustered in chromosomal domains - is mediated by parental methylation imprints at key regulatory DNA sequences. Recent chromatin conformation capture (3C)-based studies show differential organization of topologically associating domains between the parental chromosomes at imprinted domains, in embryonic stem and differentiated cells. At several imprinted domains, differentially methylated regions show allelic binding of the insulator protein CTCF, and linked focal retention of cohesin, at the non-methylated allele only. This generates differential patterns of chromatin looping between the parental chromosomes, already in the early embryo, and thereby facilitates the allelic gene expression. Recent research evokes also the opposite scenario, in which allelic transcription contributes to the differential genome organization, similarly as reported for imprinted X chromosome inactivation. This may occur through epigenetic effects on CTCF binding, through structural effects of RNA Polymerase II, or through imprinted long non-coding RNAs that have chromatin repressive functions. The emerging picture is that epigenetically-controlled differential genome architecture precedes and facilitates imprinted gene expression during development, and that at some domains, conversely, the mono-allelic gene expression also influences genome architecture.

The imprinted Dlk1-Dio3 domain comprises the developmental genes Dlk1 and Rtl1, which are silenced on the maternal chromosome in different cell types. On this parental chromosome, the domain's imprinting control region activates a polycistron that produces the lncRNA Meg3 and many miRNAs (Mirg) and C/D-box snoRNAs (Rian). Although Meg3 lncRNA is nuclear and associates with the maternal chromosome, it is unknown whether it controls gene repression in cis. We created mouse embryonic stem cells (mESCs) that carry an ectopic poly(A) signal, reducing RNA levels along the polycistron, and generated Rian-/- mESCs as well. Upon ESC differentiation, we found that Meg3 lncRNA (but not Rian) is required for Dlk1 repression on the maternal chromosome. Biallelic Meg3 expression acquired through CRISPR-mediated demethylation of the paternal Meg3 promoter led to biallelic Dlk1 repression, and to loss of Rtl1 expression. lncRNA expression also correlated with DNA hypomethylation and CTCF binding at the 5'-side of Meg3. Using Capture Hi-C, we found that this creates a Topologically Associating Domain (TAD) organization that brings Meg3 close to Dlk1 on the maternal chromosome. The requirement of Meg3 for gene repression and TAD structure may explain how aberrant MEG3 expression at the human DLK1-DIO3 locus associates with imprinting disorders.

Beta cells within the pancreatic islet represent a heterogenous population wherein individual sub-groups of cells make distinct contributions to the overall control of insulin secretion. These include a subpopulation of highly connected 'hub' cells, important for the propagation of intercellular Ca2+ waves. Functional subpopulations have also been demonstrated in human beta cells, with an altered subtype distribution apparent in type 2 diabetes. At present, the molecular mechanisms through which beta cell hierarchy is established are poorly understood. Changes at the level of the epigenome provide one such possibility, which we explore here by focusing on the imprinted gene Nnat (encoding neuronatin [NNAT]), which is required for normal insulin synthesis and secretion.

Single-cell RNA-seq datasets were examined using Seurat 4.0 and ClusterProfiler running under R. Transgenic mice expressing enhanced GFP under the control of the Nnat enhancer/promoter regions were generated for FACS of beta cells and downstream analysis of CpG methylation by bisulphite sequencing and RNA-seq, respectively. Animals deleted for the de novo methyltransferase DNA methyltransferase 3 alpha (DNMT3A) from the pancreatic progenitor stage were used to explore control of promoter methylation. Proteomics was performed using affinity purification mass spectrometry and Ca2+ dynamics explored by rapid confocal imaging of Cal-520 AM and Cal-590 AM. Insulin secretion was measured using homogeneous time-resolved fluorescence imaging.

Nnat mRNA was differentially expressed in a discrete beta cell population in a developmental stage- and DNA methylation (DNMT3A)-dependent manner. Thus, pseudo-time analysis of embryonic datasets demonstrated the early establishment of Nnat-positive and -negative subpopulations during embryogenesis. NNAT expression is also restricted to a subset of beta cells across the human islet that is maintained throughout adult life. NNAT+ beta cells also displayed a discrete transcriptome at adult stages, representing a subpopulation specialised for insulin production, and were diminished in db/db mice. 'Hub' cells were less abundant in the NNAT+ population, consistent with epigenetic control of this functional specialisation.

These findings demonstrate that differential DNA methylation at Nnat represents a novel means through which beta cell heterogeneity is established during development. We therefore hypothesise that changes in methylation at this locus may contribute to a loss of beta cell hierarchy and connectivity, potentially contributing to defective insulin secretion in some forms of diabetes.

The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD048465.

In diploid organisms, half of the chromosomes in each cell come from the father and half from the mother. Through previous studies, it was found that the paternal chromosome and the maternal chromosome can be regulated and expressed independently, leading to the emergence of allele specific expression (ASE). In this study, we analyzed the differential expression of alleles in the high-altitude population and the normal population based on the RNA sequencing data. Through gene cluster analysis and protein interaction network analysis, we found some changes occurred at the gene level, and some negative effects. During the study, we realized that the calmodulin homology domain may have a certain correlation with long-term survival at high altitude. The plateau environment is characterized by hypoxia, low air pressure, strong ultraviolet radiation, and low temperature. Accordingly, the genetic changes in the process of adaptation are mainly reflected in these characteristics. High altitude generation living is also highly related to cancer, immune disease, cardiovascular disease, neurological disease, endocrine disease, and other diseases. Therefore, the medical system in high altitude areas should pay more attention to these diseases.

As sessile organisms, plants must respond constantly to ever-changing environments to complete their life cycle; this includes the transition from vegetative growth to reproductive development. This process is mediated by photoperiodic response to sensing the length of night or day through circadian regulation of light-signaling molecules, such as phytochromes, to measure the length of night to initiate flowering. Flowering time is the most important trait to optimize crop performance in adaptive regions. In this review, we focus on interplays between circadian and light signaling pathways that allow plants to optimize timing for flowering and seed production in Arabidopsis, rice, soybean, and cotton. Many crops are polyploids and domesticated under natural selection and breeding. In response to adaptation and polyploidization, circadian and flowering pathway genes are epigenetically reprogrammed. Understanding the genetic and epigenetic bases for photoperiodic flowering will help improve crop yield and resilience in response to climate change.

Genetic variation and 3D chromatin structure have major roles in gene regulation. Due to challenges in mapping chromatin conformation with haplotype-specific resolution, the effects of genetic sequence variation on 3D genome structure and gene expression imbalance remain understudied. Here, we applied Genome Architecture Mapping (GAM) to a hybrid mouse embryonic stem cell (mESC) line with high density of single nucleotide polymorphisms (SNPs). GAM resolved haplotype-specific 3D genome structures with high sensitivity, revealing extensive allelic differences in chromatin compartments, topologically associating domains (TADs), long-range enhancer-promoter contacts, and CTCF loops. Architectural differences often coincide with allele-specific differences in gene expression, mediated by Polycomb repression. We show that histone genes are expressed with allelic imbalance in mESCs, are involved in haplotype-specific chromatin contact marked by H3K27me3, and are targets of Polycomb repression through conditional knockouts of Ezh2 or Ring1b. Our work reveals highly distinct 3D folding structures between homologous chromosomes, and highlights their intricate connections with allelic gene expression.

Epigenetic modifications, characterized by changes in gene expression without altering the DNA sequence, play a crucial role in the development and progression of cancer by significantly influencing gene activity and cellular function. This insight has led to the development of a novel class of therapeutic agents, known as epigenetic drugs. These drugs, including histone deacetylase inhibitors, histone acetyltransferase inhibitors, histone methyltransferase inhibitors, and DNA methyltransferase inhibitors, aim to modulate gene expression to curb cancer growth by uniquely altering the epigenetic landscape of cancer cells. Ongoing research and clinical trials are rigorously evaluating the efficacy of these drugs, particularly their ability to improve therapeutic outcomes when used in combination with other treatments. Such combination therapies may more effectively target cancer and potentially overcome the challenge of drug resistance, a significant hurdle in cancer therapy. Additionally, the importance of nutrition, inflammation control, and circadian rhythm regulation in modulating drug responses has been increasingly recognized, highlighting their role as critical modifiers of the epigenetic landscape and thereby influencing the effectiveness of pharmacological interventions and patient outcomes. Epigenetic drugs represent a paradigm shift in cancer treatment, offering targeted therapies that promise a more precise approach to treating a wide spectrum of tumors, potentially with fewer side effects compared to traditional chemotherapy. This progress marks a step towards more personalized and precise interventions, leveraging the unique epigenetic profiles of individual tumors to optimize treatment strategies.

Ten-eleven translocation (TET) enzymes iteratively oxidize 5-methylcytosine (5mC) to generate 5-hydroxymethylcytosine (5hmC), 5-formylcytosine, and 5-carboxylcytosine to facilitate active genome demethylation. Whether these bases are required to promote replication-coupled dilution or activate base excision repair during mammalian germline reprogramming remains unresolved due to the inability to decouple TET activities. Here, we generated two mouse lines expressing catalytically inactive TET1 (Tet1-HxD) and TET1 that stalls oxidation at 5hmC (Tet1-V). Tet1 knockout and catalytic mutant primordial germ cells (PGCs) fail to erase methylation at select imprinting control regions and promoters of meiosis-associated genes, validating the requirement for the iterative oxidation of 5mC for complete germline reprogramming. TET1V and TET1HxD rescue most hypermethylation of Tet1-/- sperm, suggesting the role of TET1 beyond its oxidative capability. We additionally identify a broader class of hypermethylated regions in Tet1 mutant mouse sperm that depend on TET oxidation for reprogramming. Our study demonstrates the link between TET1-mediated germline reprogramming and sperm methylome patterning.

In mammals, dosage compensation involves two parallel processes: (1) X inactivation, which equalizes X chromosome dosage between males and females, and (2) X hyperactivation, which upregulates the active X for X-autosome balance. The field currently favors models whereby dosage compensation initiates "de novo" during mouse development. Here, we develop "So-Smart-seq" to revisit the question and interrogate a comprehensive transcriptome including noncoding genes and repeats in mice. Intriguingly, de novo silencing pertains only to a subset of Xp genes. Evolutionarily older genes and repetitive elements demonstrate constitutive Xp silencing, adopt distinct signatures, and do not require Xist to initiate silencing. We trace Xp silencing backward in developmental time to meiotic sex chromosome inactivation in the male germ line and observe that Xm hyperactivation is timed to Xp silencing on a gene-by-gene basis. Thus, during the gamete-to-embryo transition, older Xp genes are transmitted in a "pre-inactivated" state. These findings have implications for the evolution of imprinting.

Allele-specific methylation (ASM) is an epigenetic modification whereby one parental allele becomes methylated and the other unmethylated at a specific locus. ASM is most often driven by the presence of nearby heterozygous variants that influence methylation, but also occurs somatically in the context of genomic imprinting. In this study, we investigate ASM using publicly available single-cell reduced representation bisulfite sequencing (scRRBS) data on 608 B cells sampled from six healthy B cell samples and 1,230 cells from 11 chronic lymphocytic leukemia (CLL) samples. We developed a likelihood-based criterion to test whether a CpG exhibited ASM, based on the distributions of methylated and unmethylated reads both within and across cells. Applying our likelihood ratio test, 65,998 CpG sites exhibited ASM in healthy B cell samples according to a Bonferroni criterion (p < 8.4 × 10-9), and 32,862 CpG sites exhibited ASM in CLL samples (p < 8.5 × 10-9). We also called ASM at the sample level. To evaluate the accuracy of our method, we called heterozygous variants from the scRRBS data, which enabled variant-based calls of ASM within each cell. Comparing sample-level ASM calls to the variant-based measures of ASM, we observed a positive predictive value of 76%-100% across samples. We observed high concordance of ASM across samples and an overrepresentation of ASM in previously reported imprinted genes and genes with imprinting binding motifs. Our study demonstrates that single-cell bisulfite sequencing is a potentially powerful tool to investigate ASM, especially as studies expand to increase the number of samples and cells sequenced.

Genomic imprinting is an epigenetic regulation mechanism in mammals resulting in the parentally dependent monoallelic expression of genes. Imprinting disorders in humans are associated with several congenital syndromes and cancers and remain the focus of many medical studies. Cattle is a better model organism for investigating human embryo development than mice. Imprinted genes usually cluster on chromosomes and are regulated by different methylation regions (DMRs) located in imprinting control regions that control gene expression in cis. There is an imprinted locus on human chromosome 16q24.1 associated with congenital lethal developmental lung disease in newborns. However, genomic imprinting on bovine chromosome 18, which is homologous with human chromosome 16 has not been systematically studied. The aim of this study was to analyze the allelic expressions of eight genes (CDH13, ATP2C2, TLDC1, COTL1, CRISPLD2, ZDHHC7, KIAA0513, and GSE1) on bovine chromosome 18 and to search the DMRs associated gene allelic expression. Three transcript variants of the ZDHHC7 gene (X1, X2, and X5) showed maternal imprinting in bovine placentas. In addition, the monoallelic expression of X2 and X5 was tissue-specific. Five transcripts of the KIAA0513 gene showed tissue- and isoform-specific monoallelic expression. The CDH13, ATP2C2, and TLDC1 genes exhibited tissue-specific imprinting, however, COTL1, CRISLPLD2, and GSE1 escaped imprinting. Four DMRs, established after fertilization, were found in this region. Two DMRs were located between the ZDHHC7 and KIAA0513 genes, and two were in exon 1 of the CDH13 and ATP2C2 genes, respectively. The results from this study support future studies on the molecular mechanism to regulate the imprinting of candidate genes on bovine chromosome 18.

In recent years, there has been growing concern about the adverse effects of endocrine disrupting chemicals (EDCs) on male fertility. Epigenetic modification is critical for male germline development, and has been suggested as a potential mechanism for impaired fertility induced by EDCs. Bisphenol A (BPA) has been recognized as a typical EDC. BPA and its analogues, which are still widely used in various consumer products, have garnered increasing attention due to their reproductive toxicity and the potential to induce epigenetic alteration. This literature review provides an overview of studies investigating the adverse effects of bisphenol exposures on epigenetic modifications and male fertility. Existing studies provide evidence that exposure to bisphenols can lead to adverse effects on male fertility, including declined semen quality, altered reproductive hormone levels, and adverse reproductive outcomes. Epigenetic patterns, including DNA methylation, histone modification, and non-coding RNA expression, can be altered by bisphenol exposures. Transgenerational effects, which influence the fertility and epigenetic patterns of unexposed generations, have also been identified. However, the magnitude and direction of certain outcomes varied across different studies. Investigations into the dynamics of histopathological and epigenetic alterations associated with bisphenol exposures during developmental stages can enhance the understanding of the epigenetic effects of bisphenols, the implication of epigenetic alteration on male fertility, and the health of successive generation.

Epigenome editing with DNA-targeting technologies such as CRISPR-dCas9 can be used to dissect gene regulatory mechanisms and potentially treat associated disorders. For example, Prader-Willi Syndrome (PWS) is caused by loss of paternally expressed imprinted genes on chromosome 15q11.2-q13.3, although the maternal allele is intact but epigenetically silenced. Using CRISPR repression and activation screens in human induced pluripotent stem cells (iPSCs), we identified genomic elements that control expression of the PWS gene SNRPN from the paternal and maternal chromosomes. We showed that either targeted transcriptional activation or DNA demethylation can activate the silenced maternal SNRPN and downstream PWS transcripts. However, these two approaches function at unique regions, preferentially activating different transcript variants and involving distinct epigenetic reprogramming mechanisms. Remarkably, transient expression of the targeted demethylase leads to stable, long-term maternal SNRPN expression in PWS iPSCs. This work uncovers targeted epigenetic manipulations to reprogram a disease-associated imprinted locus and suggests possible therapeutic interventions.