Epigenetics & Chromatin最新文献

Background: Topologically associating domains (TADs) are believed to play a role in the regulation of gene expression by constraining or guiding interactions between the regulatory elements. While the impact of TAD perturbations is typically studied in developmental genes with highly cell-type-specific expression patterns, this study examines genes with broad expression profiles separated by a strong insulator boundary. We focused on the mouse Slc29a3/Unc5b locus, which encompasses two distinct TADs containing ubiquitously expressed and essential for viability genes. We disrupted the CTCF-boundary between these TADs and analyzed the resulting changes in gene expression.

Results: Deletion of four CTCF binding sites at the TAD boundary altered local chromatin architecture, abolishing pre‑existing loops and creating novel long‑range interactions that spanned the original TAD boundary. Using UMI-assisted targeted RNA-seq we evaluated transcriptional changes of Unc5b, Slc29a3, Psap, Vsir, Cdh23, and Sgpl1 across various organs. We found that TAD boundary disruption led to variable transcriptional responses, where not only the magnitude but also the direction of gene expression changes were tissue-specific. Current hypotheses on genome architecture function, such as enhancer competition and hijacking, as well as genomic deep learning models, only partially explain these transcriptional changes, highlighting the need for further investigation into the mechanisms underlying TAD function and gene regulation.

Conclusions: Disrupting the insulator element between broadly expressed genes resulted in moderate, tissue-dependent transcriptional alterations, rather than uniformly activating or silencing the target genes. These findings show that TAD boundaries contribute to context‑specific regulation even at housekeeping loci and underscore the need for refined models to predict the effects of non‑coding structural variants.

Introduction: Skeletal muscle stem cells (MuSCs) have strong regenerative abilities, but as we age, their ability to regenerate decreases, leading to a decline in muscle function. Although the methylation reprogramming of super-enhancers (SEs) plays a pivotal role in regulating gene expression associated with the aging process, our understanding of the molecular diversity of stem cells during aging remains limited. This study aimed to identify the methylation profile of SEs in MuSCs and explore potential therapeutic molecular targets associated with aging.

Methods: The ROSE software was employed to identify super enhancers from the ChIP-seq data obtained from the ENCODE database. Additionally, the ALLCools and Methylpy packages were applied to analyze the methylation profile of SEs and to identify differentially methylated regions (DMRs) between aged and control samples using single-cell bisulfite sequencing (scBS-seq) data from the Gene Expression Omnibus (GEO) database. Overlap analysis was used to assess the regions of SEs and DMRs. The target genes and motifs were analyzed using KEGG, GO, and HOMER to identify key biological pathways and functions, followed by validation through snATAC-seq and immunofluorescence techniques.

Results: In conclusion, we conducted a multi-omics and cross-species analysis of MuSCs, creating a detailed methylation profile of SEs during aging. We identified key motifs and genes affected by SE methylation reprogramming, revealing important molecular pathways involved in aging. Notably, further analysis of the key gene PLXND1 revealed a decreasing expression trend in aged MuSCs, which appears to be linked to the hypermethylation of SE Rank 869. This epigenetic alteration is likely to contribute to the dysregulation of the SEMA3 signaling pathway, with profound implications for muscle regeneration in MuSCs during aging.

Conclusion: These findings suggest that epigenetic alterations in the methylation reprogramming of SEs are closely linked to the disruption of transcriptional networks during MuSCs aging. Moreover, our results offer valuable insights into the mechanisms driving SE methylation reprogramming, shedding light on how these epigenetic changes contribute to the molecular processes underlying aging.

Background: Ten-Eleven Translocation (TET1-3) dioxygenases oxidize 5-methylcytosine (5mC) in DNA to generate 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC), initiating DNA demethylation. Since their discovery in 2009, there have been contradictory reports regarding the roles of TET proteins in cancer. TET genes have been characterized as tumor suppressors because their expression levels are reduced in many human cancers including lymphoma, prostate, and pancreas, and TET2 gene mutations are common in hematological cancers. In contrast, TET1 was recently reported to be overexpressed in triple negative breast cancer and to act as a protooncogene in lung cancer.

Results: In the present study, we employed genetic approaches to directly address the function of TET1 protein in lung adenocarcinoma. We found that overexpression (OE) of TET1 in human lung adenocarcinoma (H441, H1975) cells decreased their proliferation and inhibited colony formation, cell migration, and 3D spheroid tumorigenesis. In contrast, TET1 knockout (KO) accelerated lung adenocarcinoma cell growth and promoted colony formation, cell migration, and 3D spheroid tumorigenesis. Transcriptomics and proteomics analyses revealed that TET1 overexpression was associated with increased prevalence of immune markers, primarily via activation of the TNF and NF-kB signaling pathways. Conversely, TET1 knockout in lung adenocarcinoma cells induced the expression of genes involved in cellular metabolism and cell growth.

Conclusions: Our results are consistent with tumor suppressor role of TET1 gene in lung adenocarcinoma cells (H441, H1975) and reveal its possible role in activating antitumor immunity.

Morphogenesis and development of hair follicle fundamentally depend on the interaction between the epidermis and dermis, with dermal papilla cells (DPCs) playing a critical role in these processes. H3K4me3, one of the key histone modifications, is essential for coordinating gene expression. However, the epigenetic modification profile of H3K4me3 in cashmere goat DPCs and its mechanism of action in hair follicle development remain unexplored. In this study, the apparent regulation map of H3K4me3 was drawn by CUT&Tag technology. DPCs were exogenously treated with the H3K4me3 inhibitor BCL-121 and the agonist PBIT. Functional experiment results showed that increasing H3K4me3 levels significantly enhanced the proliferation capacity of DPCs and promoted the expression of Wnt signaling pathway-related genes. Subsequently, the regulatory mechanism of H3K4me3 was explored, and the differentially expressed gene RSPO3 in the embryonic stage regulated by H3K4me3 was screened through CUT&Tag and RNA-seq correlation analysis. Functional studies demonstrated that RSPO3 could promote DPCs proliferation, inhibit apoptosis, and increase the expression of genes related to the Wnt signaling pathway. In summary, our findings indicated that H3K4me3 regulates the transcription of RSPO3 in DPCs, which would lay the foundation for the molecular mechanism of hair follicle development.

Background: INO80 and SWR1 are evolutionarily related ATP-dependent chromatin remodeling complexes that regulate the chromatin occupancy of the histone variant H2A.Z, playing critical roles in transcriptional regulation, genome replication, and DNA repair. While the H2A.Z-related functions of INO80 and SWR1 are well characterized in budding yeast and metazoans, much less is known about their composition and chromatin-targeting mechanisms outside of the Opisthokonts. We previously found that a distinct bromodomain-containing protein, IBD1, is involved in multiple chromatin-related complexes, including the SWR1-complex, in the ciliate protozoan Tetrahymena thermophila.

Results: Here, we report that a closely related bromodomain-containing protein, IBD2, functions as an acetyl lysine reader module within a putative INO80 complex. Through iterative proteomic analyses, we show that the Tetrahymena INO80 complex retains several conserved subunits found in its yeast and metazoan counterparts. In vitro binding assays reveal that recombinant IBD2 preferentially recognizes acetylated histone H3 tails. Chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) demonstrates that IBD2 is enriched near transcription start sites and promoter regions. Notably, the IBD1 and IBD2 genomic binding profiles strongly correlate with that of H2A.Z (Hv1), supporting their functional association with the SWRI- and INO80-complexes.

Conclusions: Together, our findings support a model in which H2A.Z chromatin dynamics are modulated by SWR1- and INO80-complexes that are differentially recruited to chromatin via distinct bromodomain proteins that recognize specific histone acetylation marks.

Background: The Pacific white shrimp (Litopenaeus vannamei) is the most widely farmed shrimp species globally, yet the epigenetic regulation underlying its embryonic development remains largely unexplored. Histone modifications are known to orchestrate gene expression during early development in model organisms, but their role in crustaceans is poorly understood.

Results: In this study, we present the first comprehensive histone modification landscape during L. vannamei embryogenesis using CUT&Tag (Cleavage Under Targets and Tagmentation). We profiled high-resolution landscapes of four histone marks (H3K4me1, H3K4me3, H3K27ac, H3K27me3) across seven developmental stages from blastula to nauplius, revealing dynamic chromatin state transitions associated with developmental progression. Integration with transcriptomic data uncovered a strong temporal correlation between chromatin states and gene expression, particularly during zygotic genome activation (ZGA). Furthermore, our analysis uncovered key developmental genes associated with critical biological processes such as molting, body segmentation, and neurogenesis, providing novel insights into the epigenetic regulation of these events. Functional annotation of cis-regulatory elements based on histone marks identified candidate enhancers and regulatory loci linked to these key genes.

Conclusions: Our study provides the first epigenomic framework of shrimp embryogenesis, uncovering chromatin-based regulatory mechanisms during early development. The identification of stage-specific enhancers and active chromatin regions offers valuable resources for functional genomics in crustaceans and sheds light on conserved and divergent aspects of ZGA regulation beyond model systems.

Background: Insulators are the multifunctional DNA binding proteins that perform architectural functions and regulate gene transcription. Although insulators have a well-established role in genome organization, it is still unclear how insulator proteins affect the control of tissue-specific processes. The Drosophila insulator BEAF32 (Boundary Element-Associated Factor of 32 kD) is a component of chromatin complexes found in open chromatin regions containing promoters of housekeeping genes. BEAF32 knockout impairs oogenesis and female fertility suggesting its specific functions during oogenesis.

Results: To get a better understanding of BEAF32 roles in oogenesis, we first examined its ovarian binding targets and discovered an enrichment of its localization sites in the promoters of both housekeeping and tissue-specific genes. Differential expression gene analysis revealed that BEAF32 knockout resulted in abnormal activation of non-ovarian tissue-specific genes in the ovaries, implying that BEAF32 regulates tissue-specific patterns of gene expression. We discovered that BEAF32 occupied many ovary-specific gene promoters and acted as a positive regulator of expression for the cell-cycle regulatory kinase, Polo. To investigate the possible role of BEAF32 in the Piwi-interacting RNAs (piRNAs) pathway we analyzed ovarian small RNAs in BEAF32 null mutants and found a strong decrease in the production of piRNAs from the 3R subtelomeric region. Our data suggest that the BEAF32-containing chromatin complex located upstream of the subtelomeric repeats preserves transcriptional and chromatin integrity of this domain in the germline. BEAF32 was also found to localize upstream of flamenco, a major piRNA source locus in follicular cells, and to be required for cell-specific transcription of the flamenco locus.

Conclusions: Our findings suggest that BEAF32 coordinates multiple transcriptional regulatory functions important for Drosophila oogenesis. BEAF32 represses the ectopic expression of developmental and tissue-specific genes in the ovaries. BEAF32 regulates polo kinase and other oogenesis-related genes. We demonstrate here that BEAF32 play a specific ovarian role in the maintenance of piRNA-producing loci. Our results support an important role for the BEAF32 insulator protein in determining the proper landscape of tissue-specific gene expression.

Background: Methylation of H4K20 has been implicated in the regulation of gene expression but also plays essential roles in numerous cellular functions, making studies of its effects on transcription challenging. To gain insights into the role of H4K20 methylation in regulating gene expression, we studied H4K20me1 and H4K20me3 in the context of the well-characterized erythroid differentiation of human hematopoietic stem and progenitor cells.

Results: H4K20me1 enrichment over the gene body was strongly correlated with expression changes. During erythroid differentiation, there was a dramatic decline in the level of RNA Polymerase II (Pol II); H4K20me1 was lost where Pol II was lost, and gained at genes where Pol II occupancy was maintained and transcripts were upregulated. We did identify a small subset of highly expressed genes, including beta-globin, that had a dramatic loss of H4K20me1 during erythroid differentiation, despite a substantial gain of Pol II. The histone demethylase PHF8 was present at these genes, as well as at the transcription start site of many active genes. In contrast to H4K20me1 over gene bodies correlating with transcription, enrichment at the transcription start site occurred at genes with low levels of Pol II occupancy and RNA expression throughout erythroid differentiation. The majority of H4K20me3 was present over intergenic regions, consistent with its well-established role as a repressor of repetitive elements. Intriguingly, H4K20me3 was also present at the transcription start site of genes with H4K20me1 over the gene body. At these genes, H4K20me3 levels were highly correlated with chromatin accessibility at the transcription start site, and an elevated Pol II pausing index. There was a dramatic loss of H4K20me3 occupancy in genic, but not intergenic, regions during erythroid differentiation.

Conclusions: There are dynamic changes in H4K20 methylation during cellular differentiation that correlate strongly with Pol II occupancy and activity. These changes occurred in genic regions, with H4K20me3 at the transcription start site correlated with Pol II pausing, and H4K20me1 gene body levels tightly linked with transcriptional changes. Together, these data provide important insights into the role of H4K20 methylation in the regulation of gene expression during cellular differentiation.

Background: Genomic imprinting is required for normal development, and abnormal methylation of differentially methylated regions (iDMRs) controlling the parent of origin-dependent expression of the imprinted genes has been found in congenital disorders affecting growth, metabolism, neurobehavior, and in cancer. In most of these cases the cause of the imprinting abnormalities is unknown. Also, these studies have generally been performed on a limited number of CpGs, and a systematic investigation of iDMR methylation in the general population is lacking.

Results: By analysing a vast number of either in-house generated or online available whole-genome methylation array datasets of unaffected individuals, and patients with complex and rare disorders, we determined the most common iDMR methylation profiles in a large population and identified many genetic and non-genetic factors contributing to their variability in blood DNA. We found that methylation variability was not homogeneous within the iDMRs and that the CpGs closer to the ZFP57 binding sites are less susceptible to methylation changes. We demonstrated the methylation polymorphism of three iDMRs and the atypical behaviour of several others, and reported the association of 25 disease- and 47 non-disease-complex traits as well as 15 Mendelian and chromosomal disorders with iDMR methylation changes. The most significantly associated complex traits included ageing, intracytoplasmic sperm injection, African versus European ancestry, female sex, pre- and postnatal exposure to pollutants and blood cell type compositions, while the associated genetic diseases included Down syndrome and the developmental disorders with molecular defects in the DNA methyltransferases DNMT1 and DNMT3B, H3K36 methyltransferase SETD2, chromatin remodelers SRCAP and SMARCA4 and transcription factor ADNP.

Conclusions: These findings identify several genetic and non-genetic factors including new genes associated with genomic imprinting maintenance in humans, which may have a role in the aetiology of the diseases with imprinting abnormalities and have clear implications in molecular diagnostics.

Background: N-terminal acetylation (Nt-Ac), mediated by N-terminal acetyltransferases (NATs) is one of the most abundant protein modifications occurring approximately in 80% of all eukaryotic proteins. In contrast to the broad spectrum NATs, the human N-alpha-acetyltransferase 40 (NAA40) is highly specific, currently known to Nt-acetylate only the two histone proteins H4 and H2A, which share an Ser(1)-Gly(2)-Arg(3)-Gly(4) N-terminal sequence. Previous work from our lab and others has highlighted the biological and clinical relevance of this NAA40-mediated modification.

Results: In this study, by performing in silico analysis of protein sequences combined with biochemical assays we identify the histone variants H2A.X and H2A.J and the chromatin remodeler SMARCD2 as new potential substrates of human NAA40. Subsequently, focusing on H2A.X, we show for the first time by mass spectrometry analysis that H2A.X is N-terminally acetylated (Nt-acH2A.X) within human cells. Next, we demonstrate that NAA40 specifically interacts and N-terminally acetylates histone H2A.X, in vitro and within cells. Finally, we provide evidence that H2A.X N-terminal acetylation is responsive to Ultraviolet B (UVB)-induced DNA damage and its associated enzyme NAA40 affects the survival of cells exposed to UVB irradiation.

Conclusion: Our findings identify H2A.X as a novel bona fide substrate of NAA40. Moreover, the responsiveness of H2A.X N-terminal acetylation to UV-induced DNA damage indicates that this is a dynamic modification with potential biological functions.