HP1a targets the Drosophila KDM4A demethylase to a subset of heterochromatic genes to regulate H3K36me3 levels

The KDM4 subfamily of JmjC domain-containing demethylases mediates demethylation of histone H3K36me3/me2 and H3K9me3/me2. Several studies have shown that human and yeast KDM4 proteins bind to specific gene promoters and regulate gene expression. However, the genome-wide distribution of KDM4 proteins and the mechanism of genomic-targeting remain elusive. We have previously identified Drosophila KDM4A (dKDM4A) as a histone H3K36me3 demethylase that directly interacts with HP1a. Here, we performed H3K36me3 ChIP-chip analysis in wild type and dkdm4a mutant embryos to identify genes regulated by dKDM4A demethylase activity in vivo. A subset of heterochromatic genes that show increased H3K36me3 levels in dkdm4a mutant embryos overlap with HP1a target genes. More importantly, binding to HP1a is required for dKDM4A-mediated H3K36me3 demethylation at a subset of heterochromatic genes. Collectively, these results show that HP1a functions to target the H3K36 demethylase dKDM4A to heterochromatic genes in Drosophila.     

RNA-dependent chromatin localization of KDM4D lysine demethylase promotes H3K9me3 demethylation

The JmjC-containing lysine demethylase, KDM4D, demethylates di-and tri-methylation of histone H3 on lysine 9 (H3K9me3). How KDM4D is recruited to chromatin and recognizes its histone substrates remains unknown. Here, we show that KDM4D binds RNA independently of its demethylase activity. We mapped two non-canonical RNA binding domains: the first is within the N-terminal spanning amino acids 115 to 236, and the second is within the C-terminal spanning amino acids 348 to 523 of KDM4D. We also demonstrate that RNA interactions with KDM4D N-terminal region are critical for its association with chromatin and subsequently for demethylating H3K9me3 in cells. This study implicates, for the first time, RNA molecules in regulating the levels of H3K9 methylation by affecting KDM4D association with chromatin.     

JMJD3 is a histone H3K27 demethylase

Histone methylation is an important epigenetic phenomenon that participates in a diverse array of cellular processes and has been found to be associated with cancer. Recent identification of several histone demethylases has proved that histone methylation is a reversible process. Through a candidate approach, we have biochemically identified JMJD3 as an H3K27 demethylase. Transfection of JMJD3 into HeLa cells caused a specific reduction oftrimethyl H3K27, but had no effect on di-and monomethyl H3K27, or histone lysine methylations on H3K4 and H3K9. The enzymatic activity requires the JmjC domain and the conserved histidine that has been suggested to be important for a cofactor binding. In vitro biochemical experiments demonstrated that JMJD3 directly catalyzes the demethylation. In addition, we found that JMJD3 is upregulated in prostate cancer, and its expression is higher in metastatic prostate cancer. Thus, we identified JMJD3 as a demethylase capable of removing the trimethyl group from histone H3 lysine 27 and upregulated in prostate cancer.     

The localization of histone H3K27me3 demethylase Jmjd3 is dynamically regulated

Jmjd3 is required for cellular differentiation and senescence, and inhibits the induction of pluripotent stem cells by demethylating histone 3 lysine 27 trimethylation (H3K27me3). Although recent studies reveal crucial biological roles for Jmjd3, it is unclear how its demethylase activity is controlled. Here, we show that nuclear localization of Jmjd3 is required for effective demethylation of H3K27me3. Our subcellular localization analysis of Jmjd3 shows that the N-terminal region of the protein is responsible for its nuclear placement, whereas the C-terminal region harboring the catalytic Jumonji C (JmjC) domain cannot situate into the nucleus. We identify two classical nuclear localization signals (cNLSs) in the N-terminal domain of Jmjd3. Forced nuclear emplacement of the catalytic domain of Jmjd3 by fusion with a heterologous cNLS significantly enhances its H3K27me3 demethylation activity. A dynamic nucleocytoplasmic shuttling of endogenous Jmjd3 occurs in mouse embryonic fibroblasts. Jmjd3 is localized both into the cytoplasm and the nucleus, and its nuclear export is dependent on Exportin-1, as treatment with leptomycin B triggers nuclear accumulation of Jmjd3. These results suggest that the subcellular localization of Jmjd3 is dynamically regulated and has pivotal roles for H3K27me3 status.     

Histone H3K27me3 demethylases KDM6A and KDM6B modulate definitive endoderm differentiation from human ESCs by regulating WNT signaling pathway

Definitive endoderm differentiation is crucial for generating respiratory and gastrointestinal organs including pancreas and liver. However, whether epigenetic regulation contributes to this process is unknown. Here, we show that the H3K27me3 demethylases KDM6A and KDM6B play an important role in endoderm differentiation from human ESCs. Knockdown of KDM6A or KDM6B impairs endoderm differentiation, which can be rescued by sequential treatment with WNT agonist and antagonist. KDM6A and KDM6B contribute to the activation of WNT3 and DKK1 at different differentiation stages when WNT3 and DKK1 are required for mesendoderm and definitive endoderm differentiation, respectively. Our study not only uncovers an important role of the H3K27me3 demethylases in definitive endoderm differentiation, but also reveals that they achieve this through modulating the WNT signaling pathway.     

The localization of histone H3K27me3 demethylase Jmjd3 is dynamically regulated

Jmjd3 is required for cellular differentiation and senescence, and inhibits the induction of pluripotent stem cells by demethylating histone 3 lysine 27 trimethylation (H3K27me3). Although recent studies reveal crucial biological roles for Jmjd3, it is unclear how its demethylase activity is controlled. Here, we show that nuclear localization of Jmjd3 is required for effective demethylation of H3K27me3. Our subcellular localization analysis of Jmjd3 shows that the N-terminal region of the protein is responsible for its nuclear placement, whereas the C-terminal region harboring the catalytic Jumonji C (JmjC) domain cannot situate into the nucleus. We identify two classical nuclear localization signals (cNLSs) in the N-terminal domain of Jmjd3. Forced nuclear emplacement of the catalytic domain of Jmjd3 by fusion with a heterologous cNLS significantly enhances its H3K27me3 demethylation activity. A dynamic nucleocytoplasmic shuttling of endogenous Jmjd3 occurs in mouse embryonic fibroblasts. Jmjd3 is localized both into the cytoplasm and the nucleus, and its nuclear export is dependent on Exportin-1, as treatment with leptomycin B triggers nuclear accumulation of Jmjd3. These results suggest that the subcellular localization of Jmjd3 is dynamically regulated and has pivotal roles for H3K27me3 status.     

H3K4me3 demethylation by the histone demethylase KDM5C/JARID1C promotes DNA replication origin firing

DNA replication is a tightly regulated process that initiates from multiple replication origins and leads to the faithful transmission of the genetic material. For proper DNA replication, the chromatin surrounding origins needs to be remodeled. However, remarkably little is known on which epigenetic changes are required to allow the firing of replication origins. Here, we show that the histone demethylase KDM5C/JARID1C is required for proper DNA replication at early origins. JARID1C dictates the assembly of the pre-initiation complex, driving the binding to chromatin of the pre-initiation proteins CDC45 and PCNA, through the demethylation of the histone mark H3K4me3. Fork activation and histone H4 acetylation, additional early events involved in DNA replication, are not affected by JARID1C downregulation. All together, these data point to a prominent role for JARID1C in a specific phase of DNA replication in mammalian cells, through its demethylase activity on H3K4me3.     

KDM4A regulates myogenesis by demethylating H3K9me3 of myogenic regulatory factors

Histone lysine demethylase 4A (KDM4A) plays a crucial role in regulating cell proliferation, cell differentiation, development and tumorigenesis. However, little is known about the function of KDM4A in muscle development and regeneration. Here, we found that the conditional ablation of KDM4A in skeletal muscle caused impairment of embryonic and postnatal muscle formation. The loss of KDM4A in satellite cells led to defective muscle regeneration and blocked the proliferation and differentiation of satellite cells. Myogenic differentiation and myotube formation in KDM4A-deficient myoblasts were inhibited. Chromatin immunoprecipitation assay revealed that KDM4A promoted myogenesis by removing the histone methylation mark H3K9me3 at MyoD, MyoG and Myf5 locus. Furthermore, inactivation of KDM4A in myoblasts suppressed myoblast differentiation and accelerated H3K9me3 level. Knockdown of KDM4A in vitro reduced myoblast proliferation through enhancing the expression of the cyclin-dependent kinase inhibitor P21 and decreasing the expression of cell cycle regulator Cyclin D1. Together, our findings identify KDM4A as an important regulator for skeletal muscle development and regeneration, orchestrating myogenic cell proliferation and differentiation.Subject terms: Differentiation, Muscle stem cells, Epigenetics