H3K9 methylation regulates heterochromatin silencing through incoherent feedforward loops.

Histone H3 lysine-9 methylation (H3K9me) is a hallmark of the condensed and transcriptionally silent heterochromatin. It remains unclear how H3K9me controls transcription silencing and how cells delimit H3K9me domains to avoid silencing essential genes. Here, using Arabidopsis genetic systems that induce H3K9me2 in genes and transposons de novo, we show that H3K9me2 accumulation paradoxically also causes the deposition of the euchromatic mark H3K36me3 by a SET domain methyltransferase, ASHH3. ASHH3-induced H3K36me3 confers anti-silencing by preventing the demethylation of H3K4me1 by LDL2, which mediates transcriptional silencing downstream of H3K9me2. These results demonstrate that H3K9me2 not only facilitates but orchestrates silencing by actuating antagonistic silencing and anti-silencing pathways, providing insights into the molecular basis underlying proper partitioning of chromatin domains and the creation of metastable epigenetic variation.

TET1 upregulation drives cancer cell growth through aberrant enhancer hydroxymethylation of HMGA2 in hepatocellular carcinoma.

Ten-eleven translocation 1 (TET1) is an essential methylcytosine dioxygenase of the DNA demethylation pathway. Despite its dysregulation being known to occur in human cancer, the role of TET1 remains poorly understood. In this study, we report that TET1 promotes cell growth in human liver cancer. The transcriptome analysis of 68 clinical liver samples revealed a subgroup of TET1-upregulated hepatocellular carcinoma (HCC), demonstrating hepatoblast-like gene expression signatures. We performed comprehensive cytosine methylation and hydroxymethylation (5-hmC) profiling and found that 5-hmC was aberrantly deposited preferentially in active enhancers. TET1 knockdown in hepatoma cell lines decreased hmC deposition with cell growth suppression. HMGA2 was highly expressed in a TET1high subgroup of HCC, associated with the hyperhydroxymethylation of its intronic region, marked as histone H3K4-monomethylated, where the H3K27-acetylated active enhancer chromatin state induced interactions with its promoter. Collectively, our findings point to a novel type of epigenetic dysregulation, methylcytosine dioxygenase TET1, which promotes cell proliferation via the ectopic enhancer of its oncogenic targets, HMGA2, in hepatoblast-like HCC.

Dynamics of transcription-mediated conversion from euchromatin to facultative heterochromatin at the Xist promoter by Tsix.

The fine-scale dynamics from euchromatin (EC) to facultative heterochromatin (fHC) has remained largely unclear. Here, we focus on Xist and its silencing initiator Tsix as a paradigm of transcription-mediated conversion from EC to fHC. In mouse epiblast stem cells, induction of Tsix recapitulates the conversion at the Xist promoter. Investigating the dynamics reveals that the conversion proceeds in a stepwise manner. Initially, a transient opened chromatin structure is observed. In the second step, gene silencing is initiated and dependent on Tsix, which is reversible and accompanied by simultaneous changes in multiple histone modifications. At the last step, maintenance of silencing becomes independent of Tsix and irreversible, which correlates with occupation of the -1 position of the transcription start site by a nucleosome and initiation of DNA methylation introduction. This study highlights the hierarchy of multiple chromatin events upon stepwise gene silencing establishment.

Comprehensive epigenome characterization reveals diverse transcriptional regulation across human vascular endothelial cells.

BACKGROUND: Endothelial cells (ECs) make up the innermost layer throughout the entire vasculature. Their phenotypes and physiological functions are initially regulated by developmental signals and extracellular stimuli. The underlying molecular mechanisms responsible for the diverse phenotypes of ECs from different organs are not well understood. RESULTS: To characterize the transcriptomic and epigenomic landscape in the vascular system, we cataloged gene expression and active histone marks in nine types of human ECs (generating 148 genome-wide datasets) and carried out a comprehensive analysis with chromatin interaction data. We developed a robust procedure for comparative epigenome analysis that circumvents variations at the level of the individual and technical noise derived from sample preparation under various conditions. Through this approach, we identified 3765 EC-specific enhancers, some of which were associated with disease-associated genetic variations. We also identified various candidate marker genes for each EC type. We found that the nine EC types can be divided into two subgroups, corresponding to those with upper-body origins and lower-body origins, based on their epigenomic landscape. Epigenomic variations were highly correlated with gene expression patterns, but also provided unique information. Most of the deferentially expressed genes and enhancers were cooperatively enriched in more than one EC type, suggesting that the distinct combinations of multiple genes play key roles in the diverse phenotypes across EC types. Notably, many homeobox genes were differentially expressed across EC types, and their expression was correlated with the relative position of each organ in the body. This reflects the developmental origins of ECs and their roles in angiogenesis, vasculogenesis and wound healing. CONCLUSIONS: This comprehensive analysis of epigenome characterization of EC types reveals diverse transcriptional regulation across human vascular systems. These datasets provide a valuable resource for understanding the vascular system and associated diseases.

Sex-specific histone modifications in mouse fetal and neonatal germ cells.

AIMS: Epigenetic signatures of germline cells are dynamically reprogrammed to induce appropriate differentiation, development and sex specification. We investigated sex-specific epigenetic changes in mouse fetal germ cells (FGCs) and neonatal germ cells. MATERIALS & METHODS: Six histone marks in mouse E13.5 FGCs and P1 neonatal germ cells were analyzed by chromatin immunoprecipitation and sequencing. These datasets were compared with transposase-accessible chromatin sites, DNA methylation and transcriptome. RESULTS: Different patterns of each histone mark were detected in female and male FGCs, and H3K4me3/H3K27me3 bivalent marks were enriched in different chromosomal regions of female and male FGCs. CONCLUSION: Our results suggest that histone modifications may affect FGC gene expression following DNA methylation erasure, contributing to the differentiation into female and male germ cells.

SETDB1 is essential for mouse primordial germ cell fate determination by ensuring BMP signaling.

In mouse embryos, primordial germ cells (PGCs) are fate-determined from epiblast cells. Signaling pathways involved in PGC formation have been identified, but their epigenetic mechanisms remain poorly understood. Here, we show that the histone methyltransferase SETDB1 is an epigenetic regulator of PGC fate determination. Setdb1-deficient embryos exhibit drastic reduction of nascent PGCs. Dppa2, Otx2 and Utf1 are de-repressed whereas mesoderm development-related genes, including BMP4 signaling-related genes, are downregulated by Setdb1 knockdown during PGC-like cell (PGCLC) induction. In addition, binding of SETDB1 is observed at the flanking regions of Dppa2, Otx2 and Utf1 in cell aggregates containing PGCLCs, and trimethylation of lysine 9 of histone H3 is reduced by Setdb1 knockdown at those regions. Furthermore, DPPA2, OTX2 and UTF1 binding is increased in genes encoding BMP4 signaling-related proteins, including SMAD1. Finally, overexpression of Dppa2, Otx2 and Utf1 in cell aggregates containing PGCLCs results in the repression of BMP4 signaling-related genes and PGC determinant genes. We propose that the localization of SETDB1 to Dppa2, Otx2 and Utf1, and subsequent repression of their expression, are crucial for PGC determination by ensuring BMP4 signaling.

LTR retrotransposons transcribed in oocytes drive species-specific and heritable changes in DNA methylation.

De novo DNA methylation (DNAme) during mouse oogenesis occurs within transcribed regions enriched for H3K36me3. As many oocyte transcripts originate in long terminal repeats (LTRs), which are heterogeneous even between closely related mammals, we examined whether species-specific LTR-initiated transcription units (LITs) shape the oocyte methylome. Here we identify thousands of syntenic regions in mouse, rat, and human that show divergent DNAme associated with private LITs, many of which initiate in lineage-specific LTR retrotransposons. Furthermore, CpG island (CGI) promoters methylated in mouse and/or rat, but not human oocytes, are embedded within rodent-specific LITs and vice versa. Notably, at a subset of such CGI promoters, DNAme persists on the maternal genome in fertilized and parthenogenetic mouse blastocysts or in human placenta, indicative of species-specific epigenetic inheritance. Polymorphic LITs are also responsible for disparate DNAme at promoter CGIs in distantly related mouse strains, revealing that LITs also promote intra-species divergence in CGI DNAme.

Base-Resolution Analysis of 5-Hydroxymethylcytosine by One-Pot Bisulfite-Free Chemical Conversion with Peroxotungstate.

5-Hydroxymethylcytosine (hmC) is an essential intermediate in the active DNA demethylation pathway. Here we report a new base-resolution method for measuring hmC by combining peroxotungstate-mediated oxidation and sequencing analysis. We reveal that an oxidized product of hmC, trihydroxylated thymine (thT), tolerated the incorporation of dATP as a substrate in the process of DNA polymerase elongation. By comparing the results of Sanger sequencing before and after the oxidation, we observed that hmC sites on single-stranded DNAs could be discriminated from unmethylated cytosines. We found that a thermal cycle condition during peroxotungstate treatment enhanced the oxidation reaction of hmC in double-stranded DNA. Furthermore, Illumina sequencing analysis of hmC-containing synthetic genome fragments enabled us to identify simultaneously the positions of hmC in base resolution. This bisulfite-free simple hmC detection technique could facilitate the acquisition of epigenomic information.

Concurrent loss of Ezh2 and Tet2 cooperates in the pathogenesis of myelodysplastic disorders.

Polycomb group (PcG) proteins are essential regulators of hematopoietic stem cells. Recent extensive mutation analyses of the myeloid malignancies have revealed that inactivating somatic mutations in PcG genes such as EZH2 and ASXL1 occur frequently in patients with myelodysplastic disorders including myelodysplastic syndromes (MDSs) and MDS/myeloproliferative neoplasm (MPN) overlap disorders (MDS/MPN). In our patient cohort, EZH2 mutations were also found and often coincided with tet methylcytosine dioxygenase 2 (TET2) mutations. Consistent with these findings, deletion of Ezh2 alone was enough to induce MDS/MPN-like diseases in mice. Furthermore, concurrent depletion of Ezh2 and Tet2 established more advanced myelodysplasia and markedly accelerated the development of myelodysplastic disorders including both MDS and MDS/MPN. Comprehensive genome-wide analyses in hematopoietic progenitor cells revealed that upon deletion of Ezh2, key developmental regulator genes were kept transcriptionally repressed, suggesting compensation by Ezh1, whereas a cohort of oncogenic direct and indirect polycomb targets became derepressed. Our findings provide the first evidence of the tumor suppressor function of EZH2 in myeloid malignancies and highlight the cooperative effect of concurrent gene mutations in the pathogenesis of myelodysplastic disorders.

Autocatalytic differentiation of epigenetic modifications within the Arabidopsis genome.

In diverse eukaryotes, constitutively silent sequences, such as transposons and repeats, are marked by methylation at histone H3 lysine 9 (H3K9me). Although selective H3K9me is critical for maintaining genome integrity, mechanisms to exclude H3K9me from active genes remain largely unexplored. Here, we show in Arabidopsis that the exclusion depends on a histone demethylase gene, IBM1 (increase in BONSAI methylation). Loss-of-function ibm1 mutation results in ectopic H3K9me and non-CG methylation in thousands of genes. The ibm1-induced genic H3K9me depends on both histone methylase KYP/SUVH4 and DNA methylase CMT3, suggesting interdependence of two epigenetic marks--H3K9me and non-CG methylation. Notably, IBM1 enhances loss of H3K9me in transcriptionally de-repressed sequences. Furthermore, disruption of transcription in genes induces ectopic non-CG methylation, which mimics the loss of IBM1 function. We propose that active chromatin is stabilized by an autocatalytic loop of transcription and H3K9 demethylation. This process counteracts a similarly autocatalytic accumulation of silent epigenetic marks, H3K9me and non-CG methylation.