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.     

Antagonistic functions of SET-2/SET1 and HPL/HP1 proteins in C. elegans development

Cellular identity during metazoan development is maintained by epigenetic modifications of chromatin structure brought about by the activity of specific proteins which mediate histone variant incorporation, histone modifications, and nucleosome remodeling. HP1 proteins directly influence gene expression by modifying chromatin structure. We previously showed that the Caenorhabditis elegans HP1 proteins HPL-1 and HPL-2 are required for several aspects of post-embryonic development. To gain insight into how HPL proteins influence gene expression in a developmental context, we carried out a candidate RNAi screen to identify suppressors of hpl-1 and hpl-2 phenotypes. We identified SET-2, the homologue of yeast and mammalian SET1, as an antagonist of HPL-1 and HPL-2 activity in growth and somatic gonad development. Yeast Set1 and its mammalian counterparts SET1/MLL are H3 lysine 4 (H3K4) histone methyltransferases associated with gene activation as part of large multisubunit complexes. We show that the nematode counterparts of SET1/MLL complex subunits also antagonize HPL function in post-embryonic development. Genetic analysis is consistent with SET1/MLL complex subunits having both shared and unique functions in development. Furthermore, as observed in other species, we find that SET1/MLL complex homologues differentially affect global H3K4 methylation. Our results suggest that HP1 and a SET1/MLL-related complex may play antagonistic roles in the epigenetic regulation of specific developmental programs.     

Jumonji C Domain Protein JMJ705-Mediated Removal of Histone H3 Lysine 27 Trimethylation Is Involved in Defense-Related Gene Activation in Rice

Histone methylation is an important epigenetic modification in chromatin function, genome activity, and gene regulation. Dimethylated or trimethylated histone H3 lysine 27 (H3K27me2/3) marks silent or repressed genes involved in developmental processes and stress responses in plants. However, the role and the mechanism of the dynamic removal of H3K27me2/3 during gene activation remain unclear. Here, we show that the rice (Oryza sativa) Jumonji C (jmjC) protein gene JMJ705 encodes a histone lysine demethylase that specifically reverses H3K27me2/3. The expression of JMJ705 is induced by stress signals and during pathogen infection. Overexpression of the gene reduces the resting level of H3K27me2/3 resulting in preferential activation of H3K27me3-marked biotic stress-responsive genes and enhances rice resistance to the bacterial blight disease pathogen Xanthomonas oryzae pathovar oryzae. Mutation of the gene reduces plant resistance to the pathogen. Further analysis revealed that JMJ705 is involved in methyl jasmonate–induced dynamic removal of H3K27me3 and gene activation. The results suggest that JMJ705 is a biotic stress-responsive H3K27me2/3 demethylase that may remove H3K27me3 from marked defense-related genes and increase their basal and induced expression during pathogen infection.     

The Dnmt3a PWWP Domain Reads Histone 3 Lysine 36 Trimethylation and Guides DNA Methylation

The Dnmt3a DNA methyltransferase contains in its N-terminal part a PWWP domain that is involved in chromatin targeting. Here, we have investigated the interaction of the PWWP domain with modified histone tails using peptide arrays and show that it specifically recognizes the histone 3 lysine 36 trimethylation mark. H3K36me3 is known to be a repressive modification correlated with DNA methylation in mammals and heterochromatin in Schizosaccharomyces pombe. These results were confirmed by equilibrium peptide binding studies and pulldown experiments with native histones and purified native nucleosomes. The PWWP-H3K36me3 interaction is important for the subnuclear localization of enhanced yellow fluorescent protein-fused Dnmt3a. Furthermore, the PWWP-H3K36me3 interaction increases the activity of Dnmt3a for methylation of nucleosomal DNA as observed using native nucleosomes isolated from human cells after demethylation of the DNA with 5-aza-2′-deoxycytidine as substrate for methylation with Dnmt3a. These data suggest that the interaction of the PWWP domain with H3K36me3 is involved in targeting of Dnmt3a to chromatin carrying that mark, a model that is in agreement with several studies on the genome-wide distribution of DNA methylation and H3K36me3.     

ARABIDOPSIS TRITHORAX-RELATED7 Is Required for Methylation of Lysine 4 of Histone H3 and for Transcriptional Activation of FLOWERING LOCUS C

In the winter-annual accessions of Arabidopsis thaliana, presence of an active allele of FRIGIDA (FRI) elevates expression of FLOWERING LOCUS C (FLC), a repressor of flowering, and thus confers a vernalization requirement. FLC activation by FRI involves methylation of Lys 4 of histone H3 (H3K4) at FLC chromatin. Many multicellular organisms that have been examined contain two classes of H3K4 methylases, a yeast (Saccharomyces cerevisiae) Set1 class and a class related to Drosophila melanogaster Trithorax. In this work, we demonstrate that ARABIDOPSIS TRITHORAX-RELATED7 (ATXR7), a putative Set1 class H3K4 methylase, is required for proper FLC expression. The atxr7 mutation partially suppresses the delayed flowering of a FRI-containing line. The rapid flowering of atxr7 is associated with reduced FLC expression and is accompanied by decreased H3K4 methylation and increased H3K27 methylation at FLC. Thus, ATXR7 is required for the proper levels of these histone modifications that set the level of FLC expression to create a vernalization requirement in winter-annual accessions. Previously, it has been reported that lesions in ATX1, which encodes a Trithorax class H3K4 methylase, partially suppress the delayed flowering of winter-annual Arabidopsis. We show that the flowering phenotype of atx1 atxr7 double mutants is additive relative to those of single mutants. Therefore, both classes of H3K4 methylases appear to be required for proper regulation of FLC expression.     

Characterising the Binding Specificities of the Subunits Associated with the KMT2/Set1 Histone Lysine Methyltransferase

KMT2/Set1 is the catalytic subunit of the complex of proteins associated with Set1 (COMPASS) that is responsible for the methylation of lysine 4 of histone H3 (H3K4) in Saccharomyces cerevisiae. Whereas monomethylated H3K4 (H3K4me1) is found throughout the genome, di- (H3K4me2) and tri- (H3K4me3) methylated H3K4 are enriched at specific loci, which correlates with the promoter and 5′-ends of actively transcribed genes in the case of H3K4me3. The COMPASS subunits contain a number of domains that are conserved in homologous complexes in higher eukaryotes and are reported to interact with modified histones. However, the exact organization of these subunits and their role within the complex have not been elucidated. In this study we showed that: (1) subunits Swd1 and Swd3 form a stable heterodimer that dissociates upon binding to a modified H3K4me2 tail peptide, suggesting a regulatory role in COMPASS; (2) the affinity of the subunit Spp1 for modified histone H3 substrates is much higher than that of Swd1 and Swd3; (3) Spp1 has a preference for H3K4me2/3 methylation state; and (4) Spp1 contains a high-affinity DNA-binding domain in the previously uncharacterised C-terminal region. These data allow us to suggest a mechanism for the regulation of COMPASS activity at an actively transcribed gene.