MLL3/MLL4 methyltransferase activities control early embryonic development and embryonic stem cell differentiation in a lineage-selective manner.

H3K4me1 methyltransferases MLL3 (KMT2C) and MLL4 (KMT2D) are critical for enhancer activation, cell differentiation and development. However, roles of MLL3/4 enzymatic activities and MLL3/4-mediated enhancer H3K4me1 in these processes remain unclear. Here we report that constitutive elimination of both MLL3 and MLL4 enzymatic activities prevents initiation of gastrulation and leads to early embryonic lethality in mice. However, selective elimination of MLL3/4 enzymatic activities in embryonic, but not extraembryonic, lineages leaves gastrulation largely intact. Consistent with this, embryonic stem cells (ESCs) lacking MLL3/4 enzymatic activities can differentiate toward the three embryonic germ layers but show aberrant differentiation to extraembryonic endoderm (ExEn) and trophectoderm. The failure in ExEn differentiation can be attributed to markedly reduced enhancer-binding of the lineage-determining transcription factor GATA6. Furthermore, we show that MLL3/4-catalyzed H3K4me1 is largely dispensable for enhancer activation during ESC differentiation. Together, our findings suggest a lineage-selective, but enhancer activation-independent, role of MLL3/4 methyltransferase activities in early embryonic development and ESC differentiation.

An epigenome atlas of neural progenitors within the embryonic mouse forebrain.

A comprehensive characterization of epigenomic organization in the embryonic mouse forebrain will enhance our understanding of neurodevelopment and provide insight into mechanisms of neurological disease. Here we collected single-cell chromatin accessibility profiles from four distinct neurogenic regions of the embryonic mouse forebrain using single nuclei ATAC-Seq (snATAC-Seq). We identified thousands of differentially accessible peaks, many restricted to distinct progenitor cell types or brain regions. We integrated snATAC-Seq and single cell transcriptome data to characterize changes of chromatin accessibility at enhancers and promoters with associated transcript abundance. Multi-modal integration of histone modifications (CUT&Tag and CUT&RUN), promoter-enhancer interactions (Capture-C) and high-order chromatin structure (Hi-C) extended these initial observations. This dataset reveals a diverse chromatin landscape with region-specific regulatory mechanisms and genomic interactions in distinct neurogenic regions of the embryonic mouse brain and represents an extensive public resource of a 'ground truth' epigenomic landscape at this critical stage of neurogenesis.

Extensive co-binding and rapid redistribution of NANOG and GATA6 during emergence of divergent lineages.

Fate-determining transcription factors (TFs) can promote lineage-restricted transcriptional programs from common progenitor states. The inner cell mass (ICM) of mouse blastocysts co-expresses the TFs NANOG and GATA6, which drive the bifurcation of the ICM into either the epiblast (Epi) or the primitive endoderm (PrE), respectively. Here, we induce GATA6 in embryonic stem cells-that also express NANOG-to characterize how a state of co-expression of opposing TFs resolves into divergent lineages. Surprisingly, we find that GATA6 and NANOG co-bind at the vast majority of Epi and PrE enhancers, a phenomenon we also observe in blastocysts. The co-bound state is followed by eviction and repression of Epi TFs, and quick remodeling of chromatin and enhancer-promoter contacts thus establishing the PrE lineage while repressing the Epi fate. We propose that co-binding of GATA6 and NANOG at shared enhancers maintains ICM plasticity and promotes the rapid establishment of Epi- and PrE-specific transcriptional programs.

Linkage-specific deubiquitylation by OTUD5 defines an embryonic pathway intolerant to genomic variation.

Reversible modification of proteins with linkage-specific ubiquitin chains is critical for intracellular signaling. Information on physiological roles and underlying mechanisms of particular ubiquitin linkages during human development are limited. Here, relying on genomic constraint scores, we identify 10 patients with multiple congenital anomalies caused by hemizygous variants in OTUD5, encoding a K48/K63 linkage-specific deubiquitylase. By studying these mutations, we find that OTUD5 controls neuroectodermal differentiation through cleaving K48-linked ubiquitin chains to counteract degradation of select chromatin regulators (e.g., ARID1A/B, histone deacetylase 2, and HCF1), mutations of which underlie diseases that exhibit phenotypic overlap with OTUD5 patients. Loss of OTUD5 during differentiation leads to less accessible chromatin at neuroectodermal enhancers and aberrant gene expression. Our study describes a previously unidentified disorder we name LINKED (LINKage-specific deubiquitylation deficiency-induced Embryonic Defects) syndrome and reveals linkage-specific ubiquitin cleavage from chromatin remodelers as an essential signaling mode that coordinates chromatin remodeling during embryogenesis.

Integrating the Epigenome to Identify Drivers of Hepatocellular Carcinoma.

Disruption of epigenetic mechanisms has been intimately linked to the etiology of human cancer. Understanding how these epigenetic mechanisms (including DNA methylation [5mC], hydroxymethylation [5hmC], and histone post-translational modifications) work in concert to drive cancer initiation and progression remains unknown. Hepatocellular carcinoma (HCC) is increasing in frequency in Western countries but lacks efficacious treatments. The epigenome of HCC remains understudied. To better understand the epigenetic underpinnings of HCC, we performed a genome-wide assessment of 5mC, 5hmC, four histone modifications linked to promoter/enhancer function (H3K4me1, H3K27ac, H3K4me3, and H3K27me3), and transcription across normal, cirrhotic, and HCC liver tissue. Implementation of bioinformatic strategies integrated these epigenetic marks with each other and with transcription to provide a comprehensive epigenetic profile of how and when the liver epigenome is perturbed during progression to HCC. Our data demonstrate significant deregulation of epigenetic regulators combined with disruptions in the epigenome hallmarked by profound loss of 5hmC, locus-specific gains in 5mC and 5hmC, and markedly altered histone modification profiles, particularly remodeling of enhancers. Data integration demonstrates that these marks collaborate to influence transcription (e.g., hyper-5hmC in HCC-gained active enhancers is linked to elevated expression) of genes regulating HCC proliferation. Two such putative epigenetic driver loci identified through our integrative approach, COMT and FMO3, increase apoptosis and decrease cell viability in liver-derived cancer cell lines when ectopically re-expressed. Conclusion: Altogether, integration of multiple epigenetic parameters is a powerful tool for identifying epigenetically regulated drivers of HCC and elucidating how epigenome deregulation contributes to liver disease and HCC.

ZBTB24 is a transcriptional regulator that coordinates with DNMT3B to control DNA methylation.

The interplay between transcription factors and epigenetic writers like the DNA methyltransferases (DNMTs), and the role of this interplay in gene expression, is being increasingly appreciated. ZBTB24, a poorly characterized zinc-finger protein, or the de novo methyltransferase DNMT3B, when mutated, cause Immunodeficiency, Centromere Instability, and Facial anomalies (ICF) syndrome, suggesting an underlying mechanistic link. Chromatin immunoprecipitation coupled with loss-of-function approaches in model systems revealed common loci bound by ZBTB24 and DNMT3B, where they function to regulate gene body methylation. Genes coordinately regulated by ZBTB24 and DNMT3B are enriched for molecular mechanisms essential for cellular homeostasis, highlighting the importance of the ZBTB24-DNMT3B interplay in maintaining epigenetic patterns required for normal cellular function. We identify a ZBTB24 DNA binding motif, which is contained within the promoters of most of its transcriptional targets, including CDCA7, AXIN2, and OSTC. Direct binding of ZBTB24 at the promoters of these genes targets them for transcriptional activation. ZBTB24 binding at the promoters of RNF169 and CAMKMT, however, targets them for transcriptional repression. The involvement of ZBTB24 targets in diverse cellular programs, including the VDR/RXR and interferon regulatory pathways, suggest that ZBTB24's role as a transcriptional regulator is not restricted to immune cells.

Distinctive epigenomes characterize glioma stem cells and their response to differentiation cues.

BACKGROUND: Glioma stem cells (GSCs) are a subpopulation of stem-like cells that contribute to glioblastoma (GBM) aggressiveness, recurrence, and resistance to radiation and chemotherapy. Therapeutically targeting the GSC population may improve patient survival, but unique vulnerabilities need to be identified. RESULTS: We isolate GSCs from well-characterized GBM patient-derived xenografts (PDX), characterize their stemness properties using immunofluorescence staining, profile their epigenome including 5mC, 5hmC, 5fC/5caC, and two enhancer marks, and define their transcriptome. Fetal brain-derived neural stem/progenitor cells are used as a comparison to define potential unique and common molecular features between these different brain-derived cells with stem properties. Our integrative study reveals that abnormal expression of ten-eleven-translocation (TET) family members correlates with global levels of 5mC and 5fC/5caC and may be responsible for the distinct levels of these marks between glioma and neural stem cells. Heterogenous transcriptome and epigenome signatures among GSCs converge on several genes and pathways, including DNA damage response and cell proliferation, which are highly correlated with TET expression. Distinct enhancer landscapes are also strongly associated with differential gene regulation between glioma and neural stem cells; they exhibit unique co-localization patterns with DNA epigenetic mark switching events. Upon differentiation, glioma and neural stem cells exhibit distinct responses with regard to TET expression and DNA mark changes in the genome and GSCs fail to properly remodel their epigenome. CONCLUSIONS: Our integrative epigenomic and transcriptomic characterization reveals fundamentally distinct yet potentially targetable biologic features of GSCs that result from their distinct epigenomic landscapes.

Distinct and overlapping control of 5-methylcytosine and 5-hydroxymethylcytosine by the TET proteins in human cancer cells.

BACKGROUND: The TET family of dioxygenases catalyze conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), but their involvement in establishing normal 5mC patterns during mammalian development and their contributions to aberrant control of 5mC during cellular transformation remain largely unknown. We depleted TET1, TET2, and TET3 in a pluripotent embryonic carcinoma cell model and examined the impact on genome-wide 5mC, 5hmC, and transcriptional patterns. RESULTS: TET1 depletion yields widespread reduction of 5hmC, while depletion of TET2 and TET3 reduces 5hmC at a subset of TET1 targets suggesting functional co-dependence. TET2 or TET3 depletion also causes increased 5hmC, suggesting these proteins play a major role in 5hmC removal. All TETs prevent hypermethylation throughout the genome, a finding dramatically illustrated in CpG island shores, where TET depletion results in prolific hypermethylation. Surprisingly, TETs also promote methylation, as hypomethylation was associated with 5hmC reduction. TET function is highly specific to chromatin environment: 5hmC maintenance by all TETs occurs at polycomb-marked chromatin and genes expressed at moderate levels; 5hmC removal by TET2 is associated with highly transcribed genes enriched for H3K4me3 and H3K36me3. Importantly, genes prone to hypermethylation in cancer become depleted of 5hmC with TET deficiency, suggesting that TETs normally promote 5hmC at these loci. Finally, all three TETs, but especially TET2, are required for 5hmC enrichment at enhancers, a condition necessary for expression of adjacent genes. CONCLUSIONS: These results provide novel insight into the division of labor among TET proteins and reveal important connections between TET activity, the chromatin landscape, and gene expression.