Drought‐inducible changes in the histone modification H3K9ac are associated with drought‐responsive gene expression in Brachypodium distachyon

• H3K9ac, an epigenetic marker, is widely distributed in plant genomes. H3K9ac enhances gene expression, which is highly conserved in eukaryotes. However, genome‐wide studies of H3K9ac in monocot species are limited, and the changes in H3K9ac under drought stress for individual genes are still not clear.
• We analysed changes in the H3K9ac level of Brachypodium distachyon under 20% PEG‐6000‐simulated drought stress conditions. We also performed chromatin immunoprecipitation, followed by next generation sequencing (ChIP‐seq) on H3K9ac to reveal changes in H3K9ac for individual genes at the genome‐wide level.
• Our study showed that H3K9ac was mainly enriched in gene exon regions. Drought increased or decreased the H3K9ac level at specific genomic loci. We identified 40 genes associated with increased H3K9ac levels and 36 genes associated with decreased H3K9ac levels under drought stress. Further, RT‐qPCR analyses showed that H3K9ac was positively associated with gene expression of those drought‐responsive genes.
• We conclude that H3K9ac enhances the expression level of a large number of drought‐responsive genes under drought stress in B. distachyon. The data presented here will help to reveal the correlation of some specific drought‐responsive genes and their enriched H3K9ac levels in the model plant B. distachyon.

     

JMJD5 cleaves monomethylated histone H3 N‐tail under DNA damaging stress

The histone H3 N‐terminal protein domain (N‐tail) is regulated by multiple posttranslational modifications, including methylation, acetylation, phosphorylation, and by proteolytic cleavage. However, the mechanism underlying H3 N‐tail proteolytic cleavage is largely elusive. Here, we report that JMJD5, a Jumonji C (JmjC) domain‐containing protein, is a Cathepsin L‐type protease that mediates histone H3 N‐tail proteolytic cleavage under stress conditions that cause a DNA damage response. JMJD5 clips the H3 N‐tail at the carboxyl side of monomethyl‐lysine (Kme1) residues. In vitro H3 peptide digestion reveals that JMJD5 exclusively cleaves Kme1 H3 peptides, while little or no cleavage effect of JMJD5 on dimethyl‐lysine (Kme2), trimethyl‐lysine (Kme3), or unmethyl‐lysine (Kme0) H3 peptides is observed. Although H3 Kme1 peptides of K4, K9, K27, and K36 can all be cleaved by JMJD5 in vitro, K9 of H3 is the major cleavage site in vivo, and H3.3 is the major H3 target of JMJD5 cleavage. Cleavage is enhanced at gene promoters bound and repressed by JMJD5 suggesting a role for H3 N‐tail cleavage in gene expression regulation.     

DNA methyltransferase inhibitor RG108 and histone deacetylase inhibitors cooperate to enhance NB4 cell differentiation and E‐cadherin re‐expression by chromatin remodelling

Epigenetic silencing of cancer‐related genes by abnormal methylation and the reversal of this process by DNA methylation inhibitors represents a promising strategy in cancer therapy. As DNA methylation affects gene expression and chromatin structure, we investigated the effects of novel DNMT (DNA methyltransferase) inhibitor, RG108, alone and in its combinations with structurally several HDAC (histone deacetylase) inhibitors [sodium PB (phenyl butyrate) or BML‐210 (N‐(2‐aminophenyl)‐N′phenyloctanol diamine), and all‐trans RA (retinoic acid)] in the human PML (promyelocytic leukaemia) NB4 cells. RG108 at different doses from 20 to 100 μM caused time‐, but not a dose‐dependent inhibition of NB4 cell proliferation without cytotoxicity. Temporal pretreatment with RG108 before RA resulted in a dose‐dependent cell growth inhibition and remarkable acceleration of granulocytic differentiation. Prolonged treatments with RG108 and RA in the presence of HDAC inhibitors significantly increased differentiation. RG108 caused time‐dependent re‐expression of methylation‐silenced E‐cadherin, with increase after temporal or continuous treatments with RG108 and RA, or RA together with PB in parallel, in cell maturation, suggesting the role of E‐cadherin as a possible therapeutic marker. These processes required both PB‐induced hyperacetylation of histone H4 and trimethylation of histone H3 at lysine 4, indicating the cooperative action of histone modifications and DNA methylation/demethylation in derepression of E‐cadherin. This work provides novel experimental evidence of the beneficial role of the DNMT inhibitor RG108 in combinations with RA and HDACIs in the effective differentiation of human PML based on epigenetics.     

Holocarboxylase synthetase is a chromatin protein and interacts directly with histone H3 to mediate biotinylation of K9 and K18

Holocarboxylase synthetase (HCS) mediates the binding of biotin to lysine (K) residues in histones H2A, H3 and H4; HCS knockdown disturbs gene regulation and decreases stress resistance and lifespan in eukaryotes. We tested the hypothesis that HCS interacts physically with histone H3 for subsequent biotinylation. Co-immunoprecipitation experiments were conducted and provided evidence that HCS co-localizes with histone H3 in human cells; physical interactions between HCS and H3 were confirmed using limited proteolysis assays. Yeast two-hybrid (Y2H) studies revealed that the N-terminal and C-terminal domains in HCS participate in H3 binding. Recombinant human HCS was produced and exhibited biological activity, as evidenced by biotinylation of its known substrate, recombinant p67. Recombinant histone H3.2 and synthetic H3-based peptides were also good targets for biotinylation by recombinant HCS (rHCS) in vitro, based on tracing histone-bound biotin with [³H]biotin, streptavidin and anti-biotin antibody. Biotinylation site-specific antibodies were generated and revealed that both K9 and K18 in H3 were biotinylated by HCS. Collectively, these studies provide conclusive evidence that HCS interacts directly with histone H3, causing biotinylation of K9 and K18. We speculate that the targeting of HCS to distinct regions in human chromatin is mediated by DNA sequence, biotin, RNA, epigenetic marks or chromatin proteins.     

HDAC1 and HDAC3 underlie dynamic H3K9 acetylation during embryonic neurogenesis and in schizophrenia‐like animals

Although histone acetylation is one of the most widely studied epigenetic modifications, there is still a lack of information regarding how the acetylome is regulated during brain development and pathophysiological processes. We demonstrate that the embryonic brain (E15) is characterized by an increase in H3K9 acetylation as well as decreases in the levels of HDAC1 and HDAC3. Moreover, experimental induction of H3K9 hyperacetylation led to the overexpression of NCAM in the embryonic cortex and depletion of Sox2 in the subventricular ependyma, which mimicked the differentiation processes. Inducing differentiation in HDAC1‐deficient mouse ESCs resulted in early H3K9 deacetylation, Sox2 downregulation, and enhanced astrogliogenesis, whereas neuro‐differentiation was almost suppressed. Neuro‐differentiation of (wt) ESCs was characterized by H3K9 hyperacetylation that was associated with HDAC1 and HDAC3 depletion. Conversely, the hippocampi of schizophrenia‐like animals showed H3K9 deacetylation that was regulated by an increase in both HDAC1 and HDAC3. The hippocampi of schizophrenia‐like brains that were treated with the cannabinoid receptor‐1 inverse antagonist AM251 expressed H3K9ac at the level observed in normal brains. Together, the results indicate that co‐regulation of H3K9ac by HDAC1 and HDAC3 is important to both embryonic brain development and neuro‐differentiation as well as the pathophysiology of a schizophrenia‐like phenotype.     

The histone methyltransferase ESET is required for the survival of spermatogonial stem/progenitor cells in mice

Self-renewal and differentiation of spermatogonial stem cells (SSCs) are the foundation of spermatogenesis throughout a male''s life. SSC transplantation will be a valuable solution for young male patients to preserve their fertility. As SSCs in the collected testis tissue from the patients are very limited, it is necessary to expansion the SSCs in vitro. Previous studies suggested that histone methyltransferase ERG-associated protein with SET domain (ESET) represses gene expression and is essential for the maintenance of the pool of embryonic stem cells and neurons. The objective of this study was to determine the role of ESET in SSCs using in vitrocell culture and germ cell transplantation. Cell transplantation assay showed that knockdown of ESET reduced the number of seminiferous tubules with spermatogenesis when compared with that of the control. Knockdown of ESET also upregulated the expression of apoptosis-associated genes (such as P53, Caspase9, Apaf1), whereas inhibited the expression of apoptosis-suppressing genes (such as Bcl2l1, X-linked inhibitor of apoptosis protein). In addition, suppression of ESET led to increase in expression of Caspase9 and activation of Caspase3 (P17) as well as cleavage of poly (ADP-ribose) polymerase. Among the five ESET-targeting genes (Cox4i2, spermatogenesis and oogenesis Specific Basic Helix-Loop-Helix 2, Nobox, Foxn1 and Dazl) examined by ChIP assay, Cox4i2 was found to regulate SSC apoptosis by the rescue experiment. BSP analyses further showed that DNA methylation in the promoter loci of Cox4i2was influenced by ESET, indicating that ESET also regulated gene expression through DNA methylation in addition to histone methylation. In conclusion, we found that ESET regulated SSC apoptosis by suppressing of Cox4i2 expression through histone H3 lysine 9 tri-methylation and DNA methylation. The results obtained will provide unique insights that would broaden the research on SSC biology and contribute to the treatment of male infertility.     

Identification of small molecules that inhibit the histone chaperone Asf1 and its chromatin function

The eukaryotic genome is packed into chromatin, which is important for the genomic integrity and gene regulation. Chromatin structures are maintained through assembly and disassembly of nucleosomes catalyzed by histone chaperones. Asf1 (anti-silencing function 1) is a highly conserved histone chaperone that mediates histone transfer on/off DNA and promotes histone H3 lysine 56 acetylation at globular core domain of histone H3. To elucidate the role of Asf1 in the modulation of chromatin structure, we screened and identified small molecules that inhibit Asf1 and H3K56 acetylation without affecting other histone modifications. These pyrimidine-2,4,6-trione derivative molecules inhibited the nucleosome assembly mediated by Asf1 in vitro, and reduced the H3K56 acetylation in HeLa cells. Furthermore, production of HSV viral particles was reduced by these compounds. As Asf1 is implicated in genome integrity, cell proliferation, and cancer, current Asf1 inhibitor molecules may offer an opportunity for the therapeutic development for treatment of diseases. [BMB Reports 2015; 48(12): 685-690]     

Dynamic chromatin association of IκBα is regulated by acetylation and cleavage of histone H4

Nuclear IκBα preferentially binds the acetylated N‐terminal tail of histone H4 in vivo, specifically in the skin and intestine stem cell compartments. N‐terminal cleavage of histone H4 facilitates IκBα dissociation and cellular differentiation.     

JHDM3A module as an effector molecule in guide-directed modification of target chromatin

With the objective of returning cells to their undifferentiated state through alteration of epigenetic states, small molecules have been used that specifically inhibit proteins involved in sustaining the epigenetic system. However, this chemical-based approach can cause chaotic epigenomic states due to random actions of the inhibitors. We investigated whether JHDM3A/JMJD2A, a trimethylated histone H3-lysine 9 (H3K9me3)-specific demethylase, could function as an effector molecule to selectively demethylate target chromatin, with the aid of a guide protein to serve as a delivery vehicle. JHDM3A, which normally locates in euchromatin, spread out to heterochromatin when it was fused to heterochromatin protein-1α (HP1α) or HP1β; in these cells, demethylation efficiency was also markedly increased. Two truncated modules, JHDM3A(GFP)(406) and JHDM3A(GFP)(701), had contrasting modes and efficiencies of H3K9me3 demethylation; JHDM3A(GFP)(406) showed a very uniform rate (～80%) of demethylation, whereas JHDM3A(GFP)(701) had a broad methylation range of 4-80%. The methylation values were highly dependent on the presence of the guide proteins OCT4, CTCF, and HP1. Chromatin immunoprecipitation detected reduced H3K9me3 levels at OCT4 regulatory loci in the cells expressing OCT4-tagged JHDM3A(GFP)(701). Derepression of the Sox2 gene was observed in JHDM3A(GFP)(701)OCT4-expressing cells, but not in cells that expressed the JHDM3A(GFP)(701) module alone. JHDM3A(GFP)(701)-assisted OCT4 more efficiently turned on stem cell-related microRNAs than GFP-OCT4 itself. These results suggest that JHDM3A(GFP)(701) is a suitable catalytic module that can be targeted, under the control of a guide protein, to specific loci where the chromatin H3K9me3 status and the milieu of gene expression are to be modified.