H3K36 dimethylation shapes the epigenetic interaction landscape by directing repressive chromatin modifications in embryonic stem cells.

Epigenetic modifications on the chromatin do not occur in isolation. Chromatin-associated proteins and their modification products form a highly interconnected network, and disturbing one component may rearrange the entire system. We see this increasingly clearly in epigenetically dysregulated cancers. It is important to understand the rules governing epigenetic interactions. Here, we use the mouse embryonic stem cell (mESC) model to describe in detail the relationships within the H3K27-H3K36-DNA methylation subnetwork. In particular, we focus on the major epigenetic reorganization caused by deletion of the histone 3 lysine 36 methyltransferase NSD1, which in mESCs deposits nearly all of the intergenic H3K36me2. Although disturbing the H3K27 and DNA methylation (DNAme) components also affects this network to a certain extent, the removal of H3K36me2 has the most drastic effect on the epigenetic landscape, resulting in full intergenic spread of H3K27me3 and a substantial decrease in DNAme. By profiling DNMT3A and CHH methylation (mCHH), we show that H3K36me2 loss upon Nsd1-KO leads to a massive redistribution of DNMT3A and mCHH away from intergenic regions and toward active gene bodies, suggesting that DNAme reduction is at least in part caused by redistribution of de novo methylation. Additionally, we show that pervasive acetylation of H3K27 is regulated by the interplay of H3K36 and H3K27 methylation. Our analysis highlights the importance of H3K36me2 as a major determinant of the developmental epigenome and provides a framework for further consolidating our knowledge of epigenetic networks.

ZMYM2 is essential for methylation of germline genes and active transposons in embryonic development.

ZMYM2 is a transcriptional repressor whose role in development is largely unexplored. We found that Zmym2-/- mice show embryonic lethality by E10.5. Molecular characterization of Zmym2-/- embryos revealed two distinct defects. First, they fail to undergo DNA methylation and silencing of germline gene promoters, resulting in widespread upregulation of germline genes. Second, they fail to methylate and silence the evolutionarily youngest and most active LINE element subclasses in mice. Zmym2-/- embryos show ubiquitous overexpression of LINE-1 protein as well as aberrant expression of transposon-gene fusion transcripts. ZMYM2 homes to sites of PRC1.6 and TRIM28 complex binding, mediating repression of germline genes and transposons respectively. In the absence of ZMYM2, hypermethylation of histone 3 lysine 4 occurs at target sites, creating a chromatin landscape unfavourable for establishment of DNA methylation. ZMYM2-/- human embryonic stem cells also show aberrant upregulation and demethylation of young LINE elements, indicating a conserved role in repression of active transposons. ZMYM2 is thus an important new factor in DNA methylation patterning in early embryonic development.

Distinctive aspects of the placental epigenome and theories as to how they arise.

The placenta has a methylome dramatically unlike that of any somatic cell type. Among other distinctions, it features low global DNA methylation, extensive "partially methylated domains" packed in dense heterochromatin and methylation of hundreds of CpG islands important in somatic development. These features attract interest in part because a substantial fraction of human cancers feature the exact same phenomena, suggesting parallels between epigenome formation in placentation and cancer. Placenta also features an expanded set of imprinted genes, some of which come about by distinctive developmental pathways. Recent discoveries, some from far outside the placental field, shed new light on how the unusual placental epigenetic state may arise. Nonetheless, key questions remain unresolved.

Hypoxia and loss of GCM1 expression prevents differentiation and contact inhibition in human trophoblast stem cells.

The placenta develops alongside the embryo and nurtures fetal development to term. During the first stages of embryonic development, due to low blood circulation, the blood and ambient oxygen supply is very low (∼1-2% O 2 ) and gradually increases upon placental invasion. While a hypoxic environment is associated with stem cell self-renewal and proliferation, persistent hypoxia may have severe effects on differentiating cells and could be the underlying cause of placental disorders. We find that human trophoblast stem cells (hTSC) thrive in low oxygen, whereas differentiation of hTSC to trophoblast to syncytiotrophoblast (STB) and extravillous trophoblast (EVT) is negatively affected by hypoxic conditions. The pro-differentiation factor GCM1 (human Glial Cell Missing-1) is downregulated in low oxygen, and concordantly there is substantial reduction of GCM1-regulated genes in hypoxic conditions. Knockout of GCM1 in hTSC caused impaired EVT and STB formation and function, reduced expression of differentiation-responsive genes, and resulted in maintenance of self-renewal genes. Treatment with a PI3K inhibitor reported to reduce GCM1 protein levels likewise counteracts spontaneous or directed differentiation. Additionally, chromatin immunoprecipitation of GCM1 showed enrichment of GCM1-specific binding near key transcription factors upregulated upon differentiation including the contact inhibition factor CDKN1C. Loss of GCM1 resulted in downregulation of CDKN1C and corresponding loss of contact inhibition, implicating GCM1 in regulation of this critical process.

Naive Human Embryonic Stem Cells Can Give Rise to Cells with a Trophoblast-like Transcriptome and Methylome.

Human embryonic stem cells (hESCs) readily differentiate to somatic or germ lineages but have impaired ability to form extra-embryonic lineages such as placenta or yolk sac. Here, we demonstrate that naive hESCs can be converted into cells that exhibit the cellular and molecular phenotypes of human trophoblast stem cells (hTSCs) derived from human placenta or blastocyst. The resulting "transdifferentiated" hTSCs show reactivation of core placental genes, acquisition of a placenta-like methylome, and the ability to differentiate to extravillous trophoblasts and syncytiotrophoblasts. Modest differences are observed between transdifferentiated and placental hTSCs, most notably in the expression of certain imprinted loci. These results suggest that naive hESCs can differentiate to extra-embryonic lineage and demonstrate a new way of modeling human trophoblast specification and placental methylome establishment.