We previously showed that histone variant H2A.Z is critical for the self-renewal and differentiation by maintaining an open chromatin structure at key regulatory regions of transcription. It is also known that pluripotency of embryonic stem (ES) cells is controlled in part by chromatin-modifying factors that regulate histone H3 lysine 4 (H3K4) methylation. However, it remains unclear how H3K4 demethylation contributes to ES cell function. We showed that KDM5B, which demethylates lysine 4 of histone H3, co-localizes with H3K4me3 near promoters and enhancers of active genes in ES cells; its depletion leads to spreading of H3K4 methylation into gene bodies and enhancer shores, indicating that KDM5B functions to focus H3K4 methylation at promoters and enhancers. Spreading of H3K4 methylation to gene bodies and enhancer shores leads to differential gene expression during self-renewal and differentiation of Kdm5b depleted ES cells, and decreased enhancer activity, as measured by H3K27ac levels. KDM5B critically regulates H3K4 methylation at bivalent genes during differentiation in the absence of LIF or Oct4. We also show that KDM5B and LSD1, another H3K4 demethylase, co-regulate H3K4 methylation at active promoters but they retain distinct roles in demethylating gene body regions and bivalent genes. Our results provide global and functional insight into the role of KDM5B in regulating H3K4 methylation marks near promoters, gene bodies, and enhancers in ES cells and during differentiation. We have recently addressed the regulation of histone variant H3.3 deposition in chromatin. We developed a Tet-inhibited expression system of epitope-tagged H3.3 and examined the relationship between H3.3-nucleosome turnover and ESC-specific transcription factors, chromatin modifiers and epigenetic marks. Our comprehensive analysis of H3.3 dissociation rates revealed distinct H3.3 dissociation dynamics at various functional chromatin domains. To understand how H3.3 dynamics is regulated, we have recently used proteomics approach to investigate H3.3-interacting proteins. We data indicate that a variety of chromatin-regulating proteins are associated with H3.3, which may regulate the deposition and turnover of H3.3 on chromatin. Chromatin states are regulated by both active and repressive marks. However, the roles of repressive histone modifications such as trimethylated histone lysine 20 (H4K20me3) in pluripotency and development are largely unknown. We now show that the histone lysine methyltransferase SMYD5 (SET and MYND domain-containing protein 5) mediates H4K20me3 modification at LTR/LINE repetitive DNA sequences. Depletion of SMYD5 leads to compromised self-renewal, including dysregulated expression of OCT4 targets, and perturbed differentiation. SMYD5 bound regions are enriched with repetitive LINE and LTR elements. Knockdown of SMYD5 results in a global decrease of H4K20me3 levels, a redistribution of heterochromatin constituents including H3K9me3/2, G9a, and HP1, and de-repression of endogenous LTR/LINE elements. A combined depletion of SMYD5 and Suv420h1/h2 leads to even greater decreases of H4K20me3 levels globally. Moreover, a loss of SMYD5-dependent silencing of LTR/LINE repeats nearby genic regions leads to upregulated expression of lineage-specific genes, thus contributing to the decreased self-renewal and accelerated differentiation of SMYD5-depeleted ES cells. These findings implicate an important role for SMYD5 in regulating heterochromatin and repressing endogenous repetitive DNA elements.
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