Studies of H3K4me3 demethylation by KDM5B/Jarid1B/PLU1 reveals strong substrate recognition in vitro and identifies 2,4-pyridine-dicarboxylic acid as an in vitro and in cell inhibitor

Dynamic methylations and demethylations of histone lysine residues are important for gene regulation and are facilitated by histone methyltransferases and histone demethylases (HDMs). KDM5B/Jarid1B/PLU1 is an H3K4me3/me2-specific lysine demethylase belonging to the JmjC domain-containing family of histone demethylases (JHDMs). Several studies have linked KDM5B to breast, prostate and skin cancer, highlighting its potential as a drug target. However, most inhibitor studies have focused on other JHDMs, and inhibitors for KDM5B remain to be explored. Here, we report the expression, purification and characterization of the catalytic core of recombinant KDM5B (ccKDM5B, residues 1-769). We show that ccKDM5B, recombinantly expressed in insect cells, demethylates H3K4me3 and H3K4me2 in vitro. The kinetic characterization showed that ccKDM5B has an apparent Michaelis constant (K(m) (app) ) value of 0.5 μm for its trimethylated substrate H3(1-15)K4me3, a considerably increased apparent substrate affinity than reported for related HDMs. Despite the presence of a PHD domain, the catalytic activity was not affected by additional methylation at the H3K9 position, suggesting that in vitro chromatin cross-talk between H3K4 and H3K9 does not occur for ccKDM5B. Inhibition studies of ccKDM5B showed both in vitro and in cell inhibition of ccKDM5B by 2,4-pyridinedicarboxylic acid (2,4-PDCA) with a potency similar to that reported for the HDM KDM4C. Structure-guided sequence alignment indicated that the binding mode of 2,4-PDCA is conserved between KDM4A/C and KDM5B.     

KDM5B focuses H3K4 methylation near promoters and enhancers during embryonic stem cell self-renewal and differentiation

Background

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.

Results

Here, we show 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 is linked to defects in gene expression programs and enhancer activity, respectively, during self-renewal and differentiation of KDM5B-depleted ES cells. 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.

Conclusions

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.     

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.     

Chromatin Sensing by the Auxiliary Domains of KDM5C Regulates Its Demethylase Activity and Is Disrupted by X-linked Intellectual Disability Mutations

The H3K4me3 chromatin modification, a hallmark of promoters of actively transcribed genes, is dynamically removed by the KDM5 family of histone demethylases. The KDM5 demethylases have a number of accessory domains, two of which, ARID and PHD1, lie between the segments of the catalytic domain. KDM5C, which has a unique role in neural development, harbors a number of mutations adjacent to its accessory domains that cause X-linked intellectual disability (XLID). The roles of these accessory domains remain unknown, limiting an understanding of how XLID mutations affect KDM5C activity. Through in vitro binding and kinetic studies using nucleosomes, we find that while the ARID domain is required for efficient nucleosome demethylation, the PHD1 domain alone has an inhibitory role in KDM5C catalysis. In addition, the unstructured linker region between the ARID and PHD1 domains interacts with PHD1 and is necessary for nucleosome binding. Our data suggests a model in which the PHD1 domain inhibits DNA recognition by KDM5C. This inhibitory effect is relieved by the H3 tail, enabling recognition of flanking DNA on the nucleosome. Importantly, we find that XLID mutations adjacent to the ARID and PHD1 domains break this regulation by enhancing DNA binding, resulting in the loss of specificity of substrate chromatin recognition and rendering demethylase activity lower in the presence of flanking DNA. Our findings suggest a model by which specific XLID mutations could alter chromatin recognition and enable euchromatin-specific dysregulation of demethylation by KDM5C.     

SUMOylation negatively modulates target gene occupancy of the KDM5B,a histone lysine demethylase

The histone lysine demethylase KDM5B plays key roles in gene repression by demethylating trimethylated lysine 4 of histone H3 (H3K4me3), a modification commonly found at the promoter region of actively transcribed genes. KDM5B is known to regulate the expression of genes involved in cell cycle progression; however, little is known about the post-translational modifications that regulate KDM5B. Herein, we report that KDM5B is SUMOylated at lysine residues 242 and 278 and that the ectopic expression of the hPC2 SUMO E3 ligase enhances this SUMOylation. Interestingly, the levels of KDM5B and its SUMOylated forms are regulated during the cell cycle. KDM5B is modulated by RNF4, an E3 ubiquitin ligase that targets SUMO-modified proteins to proteasomal degradation. Digital gene expression analyses showed that cells expressing the SUMOylation-deficient KDM5B harbor repressed mRNA expression profiles of cell cycle and DNA repair genes. Chromatin immunoprecipitations confirmed some of these genes as KDM5B targets, as they displayed reduced H3K4me3 levels in cells ectopically expressing KDM5B. We propose that SUMOylation by hPC2 regulates the activity of KDM5B.     

Investigations on small molecule inhibitors targeting the histone H3K4 tri-methyllysine binding PHD-finger of JmjC histone demethylases

Plant homeodomain (PHD) containing proteins are important epigenetic regulators and are of interest as potential drug targets. Inspired by the amiodarone derivatives reported to inhibit the PHD finger 3 of KDM5A (KDM5A(PHD3)), a set of compounds were synthesised. Amiodarone and its derivatives were observed to weakly disrupt the interactions of a histone H3K4me3 peptide with KDM5A(PHD3). Selected amiodarone derivatives inhibited catalysis of KDM5A, but in a PHD-finger independent manner. Amiodarone derivatives also bind to H3K4me3-binding PHD-fingers from the KDM7 subfamily. Further work is required to develop potent and selective PHD finger inhibitors.     

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.     

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.     

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.     

Metformin directly targets the H3K27me3 demethylase KDM6A/UTX

Metformin, the first drug chosen to be tested in a clinical trial aimed to target the biology of aging per se, has been clinically exploited for decades in the absence of a complete understanding of its therapeutic targets or chemical determinants. We here outline a systematic chemoinformatics approach to computationally predict biomolecular targets of metformin. Using several structure‐ and ligand‐based software tools and reference databases containing 1,300,000 chemical compounds and more than 9,000 binding sites protein cavities, we identified 41 putative metformin targets including several epigenetic modifiers such as the member of the H3K27me3‐specific demethylase subfamily, KDM6A/UTX. AlphaScreen and AlphaLISA assays confirmed the ability of metformin to inhibit the demethylation activity of purified KDM6A/UTX enzyme. Structural studies revealed that metformin might occupy the same set of residues involved in H3K27me3 binding and demethylation within the catalytic pocket of KDM6A/UTX. Millimolar metformin augmented global levels of H3K27me3 in cultured cells, including reversion of global loss of H3K27me3 occurring in premature aging syndromes, irrespective of mitochondrial complex I or AMPK. Pharmacological doses of metformin in drinking water or intraperitoneal injection significantly elevated the global levels of H3K27me3 in the hepatic tissue of low‐density lipoprotein receptor‐deficient mice and in the tumor tissues of highly aggressive breast cancer xenograft‐bearing mice. Moreover, nondiabetic breast cancer patients receiving oral metformin in addition to standard therapy presented an elevated level of circulating H3K27me3. Our biocomputational approach coupled to experimental validation reveals that metformin might directly regulate the biological machinery of aging by targeting core chromatin modifiers of the epigenome.     

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