Regulation of mammalian 3D genome organization and histone H3K9 dimethylation by H3K9 methyltransferases

Histone H3 lysine 9 dimethylation (H3K9me2) is a highly conserved silencing epigenetic mark. Chromatin marked with H3K9me2 forms large domains in mammalian cells and overlaps well with lamina-associated domains and the B compartment defined by Hi-C. However, the role of H3K9me2 in 3-dimensional (3D) genome organization remains unclear. Here, we investigated genome-wide H3K9me2 distribution, transcriptome, and 3D genome organization in mouse embryonic stem cells following the inhibition or depletion of H3K9 methyltransferases (MTases): G9a, GLP, SETDB1, SUV39H1, and SUV39H2. We show that H3K9me2 is regulated by all five MTases; however, H3K9me2 and transcription in the A and B compartments are regulated by different MTases. H3K9me2 in the A compartments is primarily regulated by G9a/GLP and SETDB1, while H3K9me2 in the B compartments is regulated by all five MTases. Furthermore, decreased H3K9me2 correlates with changes to more active compartmental state that accompanied transcriptional activation. Thus, H3K9me2 contributes to inactive compartment setting.

Dysregulation of NRSF/REST via EHMT1 is associated with psychiatric disorders

SummaryGenetic evidence indicates disrupted epigenetic regulation as a major risk factor for psychiatric disorders, but the molecular mechanisms that drive this association are undetermined. EHMT1 is an epigenetic repressor that is causal for Kleefstra Syndrome (KS), a neurodevelopmental disorder (NDD) leading to ID, and is associated with schizophrenia. Here, we show that reduced EHMT1 activity decreases NRSF/REST protein leading to abnormal neuronal gene expression and progression of neurodevelopment in human iPSC. We further show that EHMT1 regulates NRSF/REST indirectly via repression of miRNA leading to aberrant neuronal gene regulation and neurodevelopment timing. Expression of a NRSF/REST mRNA that lacks the miRNA-binding sites restores neuronal gene regulation to EHMT1 deficient cells. Importantly, the EHMT1-regulated miRNA gene set with elevated expression is enriched for NRSF/REST regulators with an association for ID and schizophrenia. This reveals a molecular interaction between H3K9 dimethylation and NSRF/REST contributing to the aetiology of psychiatric disorders.

Sequential enrichment at the nuclear periphery of H2A.Zac and H3K9me2 accompanies pluripotency loss in human embryonic stem cells

During the transition from pluripotency to a lineage-committed state, chromatin undergoes large-scale changes in structure to effect the required changes to the transcriptional program. This involves covalent modification of histone tails, replacement of histone variants, and alteration in the subnuclear position of genes, including associations with the nuclear periphery. Here, using high-resolution microscopy and quantitative image analysis, we surveyed a panel of histone variants and covalent modifications for changes in nuclear periphery association during differentiation of human embryonic stem cells to a trophoblast-like lineage. This differentiation process is rapid and homogeneous, facilitating the use of a relatively fine timecourse (12h, 24h, and 48h post-initiation) to enable detection of transient changes. With this scheme, we detected two modifications with significant changes in enrichment at the nuclear periphery: acetylation of histone variant H2A.Z, and dimethylation of histone H3 at lysine 9. We show that these chromatin marks increase specifically at the nuclear periphery in a sequential, complementary manner, with a H2A.Z acetylation preceding H3K9 dimethylation. The increase of H3K9 dimethylation occurred coincidentally with but independently of accumulation of Lamin A, since Lamin A-/- hES cells showed no changes in the localization pattern of H3K9 dimethylation. Inhibition of histone deacetylases led to persistent and increased H2A.Z acetylation at the periphery, and failure to differentiate. Our results show that a concerted dynamic change in the nature of peripheral chromatin is required for differentiation into the trophoblast state.

Heterochromatic foci and transcriptional repression by an unstructured MET-2/SETDB1 co-factor LIN-65

The segregation of the genome into accessible euchromatin and histone H3K9-methylated heterochromatin helps silence repetitive elements and tissue-specific genes. In Caenorhabditis elegans, MET-2, the homologue of mammalian SETDB1, catalyzes H3K9me1 and me2, yet like SETDB1, its regulation is enigmatic. Contrary to the cytosolic enrichment of overexpressed MET-2, we show that endogenous MET-2 is nuclear throughout development, forming perinuclear foci in a cell cycle–dependent manner. Mass spectrometry identified two cofactors that bind MET-2: LIN-65, a highly unstructured protein, and ARLE-14, a conserved GTPase effector. All three factors colocalize in heterochromatic foci. Ablation of lin-65, but not arle-14, mislocalizes and destabilizes MET-2, resulting in decreased H3K9 dimethylation, dispersion of heterochromatic foci, and derepression of MET-2 targets. Mutation of met-2 or lin-65 also disrupts the perinuclear anchoring of genomic heterochromatin. Loss of LIN-65, like that of MET-2, compromises temperature stress resistance and germline integrity, which are both linked to promiscuous repeat transcription and gene expression.