DNA methylation patterns expose variations in enhancer-chromatin modifications during embryonic stem cell differentiation.
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ABSTRACT:
SUBMITTER: Alajem A
PROVIDER: S-EPMC8062104 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Epigenetic regulation serves as the basis for stem cell differentiation into distinct cell types, but it is unclear how global epigenetic changes are regulated during this process. Here, we tested the hypothesis that global chromatin organization affects the lineage potential of stem cells and that manipulation of chromatin dynamics influences stem cell function. Using nuclease sensitivity assays, we found a progressive decrease in chromatin digestion among pluripotent embryonic stem cells (ESCs), multipotent hematopoietic stem cells (HSCs), and mature hematopoietic cells. Quantitative high-resolution microscopy revealed that ESCs contain significantly more euchromatin than HSCs, with a further reduction in mature cells. Increased cellular maturation also led to heterochromatin localization to the nuclear periphery. Functionally, prevention of heterochromatin formation by inhibition of the histone methyltransferase G9A resulted in delayed HSC differentiation. Our results demonstrate global chromatin rearrangements during stem cell differentiation and that heterochromatin formation by H3K9 methylation regulates HSC differentiation.
| S-EPMC4649257 | biostudies-literature
Project description:Transcription factors and chromatin modifiers are important in the programming and reprogramming of cellular states during development. Transcription factors bind to enhancer elements and recruit coactivators and chromatin-modifying enzymes to facilitate transcription initiation. During differentiation a subset of these enhancers must be silenced, but the mechanisms underlying enhancer silencing are poorly understood. Here we show that the histone demethylase lysine-specific demethylase 1 (LSD1; ref. 5), which demethylates histone H3 on Lys?4 or Lys?9 (H3K4/K9), is essential in decommissioning enhancers during the differentiation of mouse embryonic stem cells (ESCs). LSD1 occupies enhancers of active genes that are critical for control of the state of ESCs. However, LSD1 is not essential for the maintenance of ESC identity. Instead, ESCs lacking LSD1 activity fail to differentiate fully, and ESC-specific enhancers fail to undergo the histone demethylation events associated with differentiation. At active enhancers, LSD1 is a component of the NuRD (nucleosome remodelling and histone deacetylase) complex, which contains additional subunits that are necessary for ESC differentiation. We propose that the LSD1-NuRD complex decommissions enhancers of the pluripotency program during differentiation, which is essential for the complete shutdown of the ESC gene expression program and the transition to new cell states.
| S-EPMC4144424 | biostudies-literature
Project description:BackgroundEnvironmental challenges during development affect the fetal epigenome, but the period(s) vulnerable to epigenetic dysregulation is(are) not clear. By employing a soy phytoestrogen, genistein, that is known to alter the epigenetic states of the A(vy) allele during embryogenesis, we have explored the sensitive period for epigenetic regulation. The post-implantation period, when de novo DNA methylation actively proceeds, is amenable to in vitro analysis using a mouse embryonic stem (ES) cell differentiation system.Methods and findingsMouse ES cells were differentiated in the presence or absence of genistein, and DNA methylation patterns on day 10 were compared by microarray-based promoter methylation analysis coupled with a methylation-sensitive endonuclease (HpaII/McrBC)-dependent enrichment procedure. Moderate changes in methylation levels were observed in a subset of promoters following genistein treatment. Detailed investigation of the Ucp1 and Sytl1 promoters further revealed that genistein does not affect de novo methylation occurring between day 0 and day 4, but interferes with subsequent regulatory processes and leads to a decrease in methylation level for both promoters.ConclusionGenistein perturbed the methylation pattern of differentiated ES cells after de novo methylation. Our observations suggest that, for a subset of genes, regulation after de novo DNA methylation in the early embryo may be sensitive to genistein.
| S-EPMC3084807 | biostudies-literature
Project description:The Sox2 transcription factor must be robustly transcribed in embryonic stem (ES) cells to maintain pluripotency. Two gene-proximal enhancers, Sox2 regulatory region 1 (SRR1) and SRR2, display activity in reporter assays, but deleting SRR1 has no effect on pluripotency. We identified and functionally validated the sequences required for Sox2 transcription based on a computational model that predicted transcriptional enhancer elements within 130 kb of Sox2. Our reporter assays revealed three novel enhancers--SRR18, SRR107, and SRR111--that, through the formation of chromatin loops, form a chromatin complex with the Sox2 promoter in ES cells. Using the CRISPR/Cas9 system and F1 ES cells (Mus musculus(129) × Mus castaneus), we generated heterozygous deletions of each enhancer region, revealing that only the distal cluster containing SRR107 and SRR111, located >100 kb downstream from Sox2, is required for cis-regulation of Sox2 in ES cells. Furthermore, homozygous deletion of this distal Sox2 control region (SCR) caused significant reduction in Sox2 mRNA and protein levels, loss of ES cell colony morphology, genome-wide changes in gene expression, and impaired neuroectodermal formation upon spontaneous differentiation to embryoid bodies. Together, these data identify a distal control region essential for Sox2 transcription in ES cells.
| S-EPMC4265674 | biostudies-literature
Project description:Embryonic stem cell (ESC) differentiation is an excellent model to study chromatin changes at developmentally regulated loci. Differentiating mouse and human ESCs increase genome-wide acetylation (euchromatic) and tri-methylation (heterochromatic) of lysine 9 on histone H3. The Oct4 locus is euchromatic when expressed in undifferentiated ESCs and heterochromatic after differentiation. Brachyury T, a mesoderm-specific transcription factor, is not yet expressed in undifferentiated cells, where its locus has "bivalent" tri-methyl lysine 4 and lysine 27 modifications. During directed differentiation to pre-cardiac mesoderm, the activated brachyury locus has high levels of tri-methyl lysine 4 (euchromatin), switching to heterochromatin after gene silencing. Thus, ESC differentiation is accompanied by genome-wide commitment to euchromatin or heterochromatin. Undifferentiated hESCs bivalently modify the brachyury locus, activate it to euchromatin during mesoderm induction, and subsequently repress it to heterochromatin, demonstrating, to our knowledge, the first analysis of chromatin dynamics at a locus essential for mesoderm and endoderm differentiation.
| S-EPMC3075915 | biostudies-literature
Project description:BACKGROUND:Super-enhancer RNAs (seRNAs) are a kind of noncoding RNA transcribed from super-enhancer regions. The regulation mechanism and functional role of seRNAs are still unclear. Although super-enhancers play a critical role in the core transcriptional regulatory circuity of embryonic stem cell (ESC) differentiation, whether seRNAs have similar properties should be further investigated. RESULTS:We analyzed cap analysis gene expression sequencing (CAGE-seq) datasets collected during the differentiation of embryonic stem cells (ESCs) to cardiomyocytes to identify the seRNAs. A non-negative matrix factorization algorithm was applied to decompose the seRNA profiles and reveal two hidden stages during the ESC differentiation. We further identified 95 and 78 seRNAs associated with early- and late-stage ESC differentiation, respectively. We found that the binding sites of master regulators of ESC differentiation, including NANOG, FOXA2, and MYC, were significantly observed in the loci of the stage-specific seRNAs. Based on the investigation of genes coexpressed with seRNA, these stage-specific seRNAs might be involved in cardiac-related functions such as myofibril assembly and heart development and act in trans to regulate the co-expressed genes. CONCLUSIONS:In this study, we used a computational approach to demonstrate the possible role of seRNAs during ESC differentiation.
| S-EPMC6936076 | biostudies-literature
Project description:Enhancers play a pivotal role in regulating the transcription of distal genes. Although certain chromatin features, such as the histone acetyltransferase P300 and the histone modification H3K4me1, indicate the presence of enhancers, only a fraction of enhancers are functionally active. Individual chromatin marks, such as H3K27ac and H3K27me3, have been identified to distinguish active from inactive enhancers. However, the systematic identification of the most informative single modification, or combination thereof, is still lacking. Furthermore, the discovery of enhancer RNAs (eRNAs) provides an alternative approach to directly predicting enhancer activity. However, it remains challenging to link chromatin modifications to eRNA transcription. Herein, we develop a logistic regression model to unravel the relationship between chromatin modifications and eRNA synthesis. We perform a systematic assessment of 24 chromatin modifications in fetal lung fibroblast and demonstrate that a combination of four modifications is sufficient to accurately predict eRNA transcription. Furthermore, we compare the ability of eRNAs and H3K27ac to discriminate enhancer activity. We demonstrate that eRNA is more indicative of enhancer activity. Finally, we apply our fibroblast trained model to six other cell-types and successfully predict eRNA synthesis. Thus, we demonstrate the learned relationships are general and independent of cell-type. We provided a powerful tool to identify active enhancers and reveal the relationship between chromatin modifications, eRNA production and enhancer activity.
| S-EPMC3905895 | biostudies-other
Project description:Coordinated changes of DNA (de)methylation, nucleosome positioning, and chromatin binding of the architectural protein CTCF play an important role for establishing cell-type-specific chromatin states during differentiation. To elucidate molecular mechanisms that link these processes, we studied the perturbed DNA modification landscape in mouse embryonic stem cells (ESCs) carrying a double knockout (DKO) of the Tet1 and Tet2 dioxygenases. These enzymes are responsible for the conversion of 5-methylcytosine (5mC) into its hydroxymethylated (5hmC), formylated (5fC), or carboxylated (5caC) forms. We determined changes in nucleosome positioning, CTCF binding, DNA methylation, and gene expression in DKO ESCs and developed biophysical models to predict differential CTCF binding. Methylation-sensitive nucleosome repositioning accounted for a significant portion of CTCF binding loss in DKO ESCs, whereas unmethylated and nucleosome-depleted CpG islands were enriched for CTCF sites that remained occupied. A number of CTCF sites also displayed direct correlations with the CpG modification state: CTCF was preferentially lost from sites that were marked with 5hmC in wild-type (WT) cells but not from 5fC-enriched sites. In addition, we found that some CTCF sites can act as bifurcation points defining the differential methylation landscape. CTCF loss from such sites, for example, at promoters, boundaries of chromatin loops, and topologically associated domains (TADs), was correlated with DNA methylation/demethylation spreading and can be linked to down-regulation of neighboring genes. Our results reveal a hierarchical interplay between cytosine modifications, nucleosome positions, and DNA sequence that determines differential CTCF binding and regulates gene expression.
| S-EPMC6499307 | biostudies-literature
Project description:BackgroundEpigenetic modifications, transcription factor (TF) availability and differences in chromatin folding influence how the genome is interpreted by the transcriptional machinery responsible for gene expression. Enhancers buried in non-coding regions are found to be associated with significant differences in histone marks between different cell types. In contrast, gene promoters show more uniform modifications across cell types. Here we used histone modification and chromatin-associated protein ChIP-Seq data sets in mouse embryonic stem (ES) cells as well as genomic features to identify functional enhancer regions. Using co-bound sites of OCT4, SOX2 and NANOG (co-OSN, validated enhancers) and co-bound sites of MYC and MYCN (limited enhancer activity) as enhancer positive and negative training sets, we performed multinomial logistic regression with LASSO regularization to identify key features.ResultsCross validations reveal that a combination of p300, H3K4me1, MED12 and NIPBL features to be top signatures of co-OSN regions. Using a model from 10 signatures, 83% of top 1277 putative 1 kb enhancer regions (probability greater than or equal to 0.8) overlapped with at least one TF peak from 7 mouse ES cell ChIP-Seq data sets. These putative enhancers are associated with increased gene expression of neighbouring genes and significantly enriched in multiple TF bound loci in agreement with combinatorial models of TF binding. Furthermore, we identified several motifs of known TFs significantly enriched in putative enhancer regions compared to random promoter regions and background. Comparison with an active H3K27ac mark in various cell types confirmed cell type-specificity of these enhancers.ConclusionsThe top enhancer signatures we identified (p300, H3K4me1, MED12 and NIPBL) will allow for the identification of cell type-specific enhancer regions in diverse cell types.
| S-EPMC3406964 | biostudies-literature
Project description:DNA methylation is related closely to sequence contexts and chromatin modifications; however, their potential differences in different genomic regions across cell types remain largely unexplored. We used publicly available genome-scale DNA methylation and histone modification profiles to study their relationships among different genomic regions in human embryonic stem cells (H1), H1-derived neuronal progenitor cultured cells (NPC), and foetal fibroblasts (IMR90) using the Random forests classifier. Histone modifications achieved high accuracy in modelling DNA methylation patterns on a genome scale in the three cell types. The inclusion of sequence features helped improve accuracy only in non-promoter regions of IMR90. Furthermore, the top six feature combinations obtained by mean decrease Gini were important indicators of different DNA methylation patterns, suggesting that H3K4me2 and H3K4me3 are important indicators that are independent of genomic regions and cell types. H3K9me3 was IMR90-specific and exhibited a genomic region-specific correlation with DNA methylation. Variations of essential chromatin modification signals may effectively discriminate changes of DNA methylation between H1 and IMR90. Genes with different co-variations of epigenetic marks exhibited genomic region-specific biological relevance. This study provides an integrated strategy to identify systematically essential epigenetic and genetic elements of genomic region-specific and cell type-specific DNA methylation patterns.
| S-EPMC4325317 | biostudies-literature