Epigenetic silencing of HIV-1 by the histone H3 lysine 27 methyltransferase enhancer of Zeste 2

Latent HIV proviruses are silenced as the result of deacetylation and methylation of histones located at the viral long terminal repeat (LTR). Inhibition of histone deacetylases (HDACs) leads to the reemergence of HIV-1 from latency, but the contribution of histone lysine methyltransferases (HKMTs) to maintaining HIV latency remains uncertain. Chromatin immunoprecipitation experiments using latently infected Jurkat T-cell lines demonstrated that the HKMT enhancer of Zeste 2 (EZH2) was present at high levels at the LTR of silenced HIV proviruses and was rapidly displaced following proviral reactivation. Knockdown of EZH2, a key component of the Polycomb repressive complex 2 (PRC2) silencing machinery, and the enzyme which is required for trimethyl histone lysine 27 (H3K27me3) synthesis induced up to 40% of the latent HIV proviruses. In contrast, there was less than 5% induction of latent proviruses following knockdown of SUV39H1, which is required for H3K9me3 synthesis. Knockdown of EZH2 also sensitized latent proviruses to external stimuli, such as T-cell receptor stimulation, and slowed the reversion of reactivated proviruses to latency. Similarly, cell populations that responded poorly to external stimuli carried HIV proviruses that were enriched in H3K27me3 and relatively depleted in H3K9me3. Treating latently infected cells with the HKMT inhibitor 3-deazaneplanocin A, which targets EZH2, led to the reactivation of silenced proviruses, whereas chaetocin and BIX01294 showed only minimal reactivation activities. These findings suggest that PRC2-mediated silencing is an important feature of HIV latency and that inhibitors of histone methylation may play a useful role in induction strategies designed to eradicate latent HIV pools.     

Dynamics of H3K27me3 methylation and demethylation in plant development

Epigenetic regulation controls multiple aspects of the plant development. The N-terminal tail of histone can be differently modified to regulate various chromatin activities. One of them, the trimethylation of histone H3 lysine 27 (H3K27me3) confers a repressive chromatin state with gene silencing. H3K27me3 is dynamically deposited and removed throughout development. While components of the H3K27me3 writer, Polycomb repressive complex 2 (PRC2), have been reported for almost 2 decades, it is only recently that JUMONJI (JMJ) proteins are reported as H3K27me3 demethylases, affirming the dynamic nature of histone modifications. This review highlights recent progress in plant epigenetic research, focusing on the H3K27me3 demethylases.     

Histone Demethylase Jumonji D3 (JMJD3) as a Tumor Suppressor by Regulating p53 Protein Nuclear Stabilization

Histone methylation regulates normal stem cell fate decisions through a coordinated interplay between histone methyltransferases and demethylases at lineage specific genes. Malignant transformation is associated with aberrant accumulation of repressive histone modifications, such as polycomb mediated histone 3 lysine 27 (H3K27me3) resulting in a histone methylation mediated block to differentiation. The relevance, however, of histone demethylases in cancer remains less clear. We report that JMJD3, a H3K27me3 demethylase, is induced during differentiation of glioblastoma stem cells (GSCs), where it promotes a differentiation-like phenotype via chromatin dependent (INK4A/ARF locus activation) and chromatin independent (nuclear p53 protein stabilization) mechanisms. Our findings indicate that deregulation of JMJD3 may contribute to gliomagenesis via inhibition of the p53 pathway resulting in a block to terminal differentiation.     

Structural basis for the recognition of methylated histone H3 by the Arabidopsis LHP1 chromodomain

Arabidopsis LHP1 (LIKE HETEROCHROMATIN PROTEIN 1), a unique homolog of HP1 in Drosophila, plays important roles in plant development, growth, and architecture. In contrast to specific binding of the HP1 chromodomain to methylated H3K9 histone tails, the chromodomain of LHP1 has been shown to bind to both methylated H3K9 and H3K27 histone tails, and LHP1 carries out its function mainly via its interaction with these two epigenetic marks. However, the molecular mechanism for the recognition of methylated histone H3K9/27 by the LHP1 chromodomain is still unknown. In this study, we characterized the binding ability of LHP1 to histone H3K9 and H3K27 peptides and found that the chromodomain of LHP1 binds to histone H3K9me2/3 and H3K27me2/3 peptides with comparable affinities, although it exhibited no binding or weak binding to unmodified or monomethylated H3K9/K27 peptides. Our crystal structures of the LHP1 chromodomain in peptide-free and peptide-bound forms coupled with mutagenesis studies reveal that the chromodomain of LHP1 bears a slightly different chromodomain architecture and recognizes methylated H3K9 and H3K27 peptides via a hydrophobic clasp, similar to the chromodomains of human Polycomb proteins, which could not be explained only based on primary structure analysis. Our binding and structural studies of the LHP1 chromodomain illuminate a conserved ligand interaction mode between chromodomains of both animals and plants, and shed light on further functional study of the LHP1 protein.     

Histone H3 lysine 4 monomethylation (H3K4me1) and H3 lysine 9 monomethylation (H3K9me1): Distribution and their association in regulating gene expression under hyperglycaemic/hyperinsulinemic conditions in 3T3 cells

Hyperglycemia/hyperinsulinemia are leading cause for the induction type 2 diabetes and the role of post-translational histone modifications in dysregulating the expression of genes has emerged as potential important contributor in the progression of disease. The paradoxical nature of histone H3-Lysine 4 and Lysine 9 mono-methylation (H3K4me1 and H3K9me1) in both gene activation and repression motivated us to elucidate the functional relationship of these histone modifications in regulating expression of genes under hyperglycaemic/hyperinsulinemic condition. Chromatin immunoprecipitation–microarray analysis (ChIP-chip) was performed with H3 acetylation, H3K4me1 and H3K9me1 antibody. CLUSTER analysis of ChIP-chip (Chromatin immunoprecipitation–microarray analysis) data showed that mRNA expression and H3 acetylation/H3K4me1 levels on genes were inversely correlated with H3K9me1 levels on the transcribed regions, after 30 min of insulin stimulation under hyperglycaemic condition. Interestingly, we provide first evidence regarding regulation of histone de/acetylases and de/methylases; Myst4, Jmjd2b, Aof1 and Set by H3Ac, H3K4me1 and H3K9me1 under hyperinsulinemic/hyperglycaemic condition. ChIP–qPCR analysis shows association of increased H3Ac/H3K4me1 and decreased levels of H3K9me1 in up regulation of Myst4, Jmjd2, Set and Aof1 genes. We further analyse promoter occupancy of histone modifications by ChIP walking and observed increased occupancy of H3Ac/H3K4me1 on promoter region (−1000 to −1) of active genes and H3K9me1 on inactive genes under hyperglycemic/hyperinsulinemic condition. To best of our knowledge this is the first report that shows regulation of chromatin remodelling genes by alteration in the occupancy of histone H3Ac/H3K4/K9me on both promoter and transcribed regions.     

Hypoxia causes epigenetic gene regulation in macrophages by attenuating Jumonji histone demethylase activity

Epigenetic processes elicit changes in gene expression by modifying DNA bases or histone side chains without altering DNA sequences. Recently discovered Jumonji histone demethylases (JHDMs) affect gene expression by demethylating lysine residues of histone tails. JHDMs belong to a family of dioxygenases and share similarities with prolyl hydroxylases (PHDs). Therefore, we investigated the influence of hypoxia in macrophages on histone methylation. All JHDM family members JMJD1A–C and JMJD2A–D are expressed in macrophages. Thus, we analyzed the methylation status of histone H3 residues not only under hypoxia but also after treatment with the dioxygenase-inhibitors DMOG, NO and ROS. Western analysis revealed increased methylations in H3K9me2/me3 and H3K36me3 at pO₂ below 3%, DMOG, NO and ROS treatment. Chromatin immunoprecipitation (ChIP) assays demonstrated increased repressive marks H3K9me2 and H3K9me3 in specific promoter regions of the chemokine Ccl2 and the chemokine receptors Ccr1 and Ccr5, which correlated with a downregulation of their mRNA expression under hypoxic conditions. In contrasts, the hypoxia-inducible factor (HIF) target gene adrenomedullin (ADM) mRNA was upregulated and no increase in its histone modification was observed. We suggest that hypoxia and a concomitant loss of JHDM activity increases H3K9 methylation and decreases chemokine expression.     

JMJD3 as an epigenetic regulator in development and disease

Gene expression is epigenetically regulated through DNA methylation and covalent chromatin modifications, such as acetylation, phosphorylation, ubiquitination, sumoylation, and methylation of histones. Histone methylation state is dynamically regulated by different groups of histone methyltransferases and demethylases. The trimethylation of histone 3 (H3K4) at lysine 4 is usually associated with the activation of gene expression, whereas trimethylation of histone 3 at lysine 27 (H3K27) is associated with the repression of gene expression. The polycomb repressive complex contains the H3K27 methyltransferase Ezh2 and controls dimethylation and trimethylation of H3K27 (H3K27me2/3). The Jumonji domain containing-3 (Jmjd3, KDM6B) and ubiquitously transcribed X-chromosome tetratricopeptide repeat protein (UTX, KDM6A) have been identified as H3K27 demethylases that catalyze the demethylation of H3K27me2/3. The role and mechanisms of both JMJD3 and UTX have been extensively studied for their involvement in development, cell plasticity, immune system, neurodegenerative disease, and cancer. In this review, we will focus on recent progresses made on understanding JMJD3 in the regulation of gene expression in development and diseases. This article is part of a Directed Issue entitled: Epigenetics dynamics in development and disease.     

The H3K27 demethylase UTX-1 regulates C. elegans lifespan in a germline-independent, insulin-dependent manner

Aging is accompanied by alterations in epigenetic marks that control chromatin states, including histone acetylation and methylation. Enzymes that reversibly affect histone marks associated with active chromatin have recently been found to regulate aging in Caenorhabditis elegans. However, relatively little is known about the importance for aging of histone marks associated with repressed chromatin. Here, we use a targeted RNAi screen in C. elegans to identify four histone demethylases that significantly regulate worm lifespan, UTX‐1, RBR‐2, LSD‐1, and T26A5.5. Interestingly, UTX‐1 belongs to a conserved family of histone demethylases specific for lysine 27 of histone H3 (H3K27me3), a mark associated with repressed chromatin. Both utx‐1 knockdown and heterozygous mutation of utx‐1 extend lifespan and increase the global levels of the H3K27me3 mark in worms. The H3K27me3 mark significantly drops in somatic cells during the normal aging process. UTX‐1 regulates lifespan independently of the presence of the germline, but in a manner that depends on the insulin‐FoxO signaling pathway. These findings identify the H3K27me3 histone demethylase UTX‐1 as a novel regulator of worm lifespan in somatic cells.     

Lysine 27 dimethylation of Drosophila linker histone dH1 contributes to heterochromatin organization independently of H3K9 methylation

Post-translational modifications (PTMs) of core histones are important epigenetic determinants that correlate with functional chromatin states. However, despite multiple linker histone H1s PTMs have been identified, little is known about their genomic distribution and contribution to the epigenetic regulation of chromatin. Here, we address this question in Drosophila that encodes a single somatic linker histone, dH1. We previously reported that dH1 is dimethylated at K27 (dH1K27me2). Here, we show that dH1K27me2 is a major PTM of Drosophila heterochromatin. At mitosis, dH1K27me2 accumulates at pericentromeric heterochromatin, while, in interphase, it is also detected at intercalary heterochromatin. ChIPseq experiments show that >98% of dH1K27me2 enriched regions map to heterochromatic repetitive DNA elements, including transposable elements, simple DNA repeats and satellite DNAs. Moreover, expression of a mutated dH1K27A form, which impairs dH1K27me2, alters heterochromatin organization, upregulates expression of heterochromatic transposable elements and results in the accumulation of RNA:DNA hybrids (R-loops) in heterochromatin, without affecting H3K9 methylation and HP1a binding. The pattern of dH1K27me2 is H3K9 methylation independent, as it is equally detected in flies carrying a H3K9R mutation, and is not affected by depletion of Su(var)3–9, HP1a or Su(var)4–20. Altogether these results suggest that dH1K27me2 contributes to heterochromatin organization independently of H3K9 methylation.