The role of H3K4me3 and H3K9/14ac in the induction by dexamethasone of Per1 and Sgk1, two glucococorticoid early response genes that mediate the effects of acute stress in mammals

Glucocorticoids are known to induce or repress the expression of a wide variety of genes with roles in various biological processes such as the circadian clock and the stress response. We studied the changes in the levels of two histone H3 post-translational modifications associated with active chromatin, H3 trimethylated at lysine 4 (H3K4me3) and H3 acetylated at lysines 9/14 (H3K9/14ac), that take place in the promoters of two glucocorticoid early response genes, Per1 and Sgk1, during their induction by the synthetic glucocorticoid, dexamethasone. Sgk1 mediates the effects of acute and chronic stress on the prefrontal cortex and other parts of the brain, while Per1 is a core circadian clock gene whose expression is strongly induced by the increased levels of blood-borne glucocorticoids that accompany acute and chronic stress. Here we show that dexamethasone rapidly increases the levels of H3K4me3 and H3K9/14ac in the promoters of both genes. Furthermore, the effect of dexamethasone on these genes, regarding both mRNA levels and the abundance of H3K4me3 and H3K9/14ac in their promoters, can be inhibited by the presence of nicotinamide, a metabolic molecule which has been shown to possess anxiolytic properties.     

Computational characterization of substrate and product specificities,and functionality of S‐adenosylmethionine binding pocket in histone lysine methyltransferases from Arabidopsis,rice and maize

Histone lysine methylation by histone lysine methyltransferases (HKMTs) has been implicated in regulation of gene expression. While significant progress has been made to understand the roles and mechanisms of animal HKMT functions, only a few plant HKMTs are functionally characterized. To unravel histone substrate specificity, degree of methylation and catalytic activity, we analyzed Arabidopsis Trithorax‐like protein (ATX), Su (var)3‐9 h omologs protein (SUVH), Su(var)3‐9 related protein (SUVR), ATXR5, ATXR6, and E(Z) HKMTs of Arabidopsis, maize and rice through sequence and structure comparison. We show that ATXs may exhibit methyltransferase specificity toward histone 3 lysine 4 (H3K4) and might catalyse the trimethylation. Our analyses also indicate that most SUVH proteins of Arabidopsis may bind histone H3 lysine 9 (H3K9). We also predict that SUVH7, SUVH8, SUVR1, SUVR3, ZmSET20 and ZmSET22 catalyse monomethylation or dimethylation of H3K9. Except for SDG728, which may trimethylate H3K9, all SUVH paralogs in rice may catalyse monomethylation or dimethylation. ZmSET11, ZmSET31, SDG713, SDG715, and SDG726 proteins are predicted to be catalytically inactive because of an incomplete S‐adenosylmethionine (SAM) binding pocket and a post‐SET domain. E(Z) homologs can trimethylate H3K27 substrate, which is similar to the Enhancer of Zeste homolog 2 of humans. Our comparative sequence analyses reveal that ATXR5 and ATXR6 lack motifs/domains required for protein‐protein interaction and polycomb repressive complex 2 complex formation. We propose that subtle variations of key residues at substrate or SAM binding pocket, around the catalytic pocket, or presence of pre‐SET and post‐SET domains in HKMTs of the aforementioned plant species lead to variations in class‐specific HKMT functions and further determine their substrate specificity, the degree of methylation and catalytic activity.     

Nicotinamide treatment reduces the levels of histone H3K4 trimethylation in the promoter of the mper1 circadian clock gene and blocks the ability of dexamethasone to induce the acute response

Circadian rhythms, which measure time on a scale of 24 h, are generated by one of the most ubiquitous endogenous mechanisms, the circadian clock. SIRT1, a class III histone deacetylase, and PARP-1, a poly(ADP-ribose) polymerase, are two NAD⁺-dependent enzymes that have been shown to be involved in the regulation of the clock. Here we present evidence that the metabolite nicotinamide, an inhibitor of SIRT1, PARP-1 and mono(ADP-ribosyl) transferases, blocks the ability of dexamethasone to induce the acute response of the circadian clock gene, mper1, while it concomitantly reduces the levels of histone H3 trimethylation of lysine 4 (H3K4me3) in the mper1 promoter. Moreover, application of alternative inhibitors of SIRT1 and ADP-ribosylation did not lead to similar results. Therefore, inhibition of these enzymes does not seem to be the mode by which NAM exerts these effects. These results suggest the presence of a novel mechanism, not previously documented, by which NAM can alter gene expression levels via changes in the histone H3K4 trimethylation state.     

Arabidopsis histone methyltransferase SET DOMAIN GROUP2 is required for regulation of various hormone responsive genes

Histone modifications are known to play important roles in plant development through epigenetic regulation of gene expression. How these modifications regulate downstream targets in response to various environmental cues and developmental stimuli is still largely unknown. Here, we provide evidence that Arabidopsis histone H3K4 methyltransferase SET DOMAIN GROUP2 (SDG2) is required for full activation of hormone responsive genes upon hormone treatment. The pleiotropic phenotypes of sdg2 were closely related to those of auxin deficient mutants and RNA analysis revealed that expression of early hormone responsive genes was significantly reduced in sdg2-5. By ChIP analyses we found that H3K4 tri-methylations on chromatin region of hormone responsive genes such as SAUR27, KIN1 and GASA6 were enriched in WT upon hormone treatments whereas these enrichments were largely abolished in sdg2-5. After hormone treatment, chromatin regions of responsive genes that accumulated H3K4me3 in WT overlapped with those displaying decreased H3K4me3 levels in sdg2-5. Histone H3K4 di-methylation levels on tested genes were increased rather than decreased in sdg2-5, suggesting that SDG2 mediates transition of H3K4me2 to H3K4me3. Taken together, we conclude that the SDG2 activity is required to regulate the expression of hormone responsive genes via histone H3K4 tri-methylation.     

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.     

Regulation of Histone Methylation and Reprogramming of Gene Expression in the Rice Inflorescence Meristem

Rice inflorescence meristem (IM) activity is essential for panicle development and grain production. How chromatin and epigenetic mechanisms regulate IM activity remains unclear. Genome-wide analysis revealed that in addition to genes involved in the vegetative to reproductive transition, many metabolic and protein synthetic genes were activated in IM compared with shoot apical meristem and that a change in the H3K27me3/H3K4me3 ratio was an important factor for the differential expression of many genes. Thousands of genes gained or lost H3K27me3 in IM, and downregulation of the H3K27 methyltransferase gene SET DOMAIN GROUP 711 (SDG711) or mutation of the H3K4 demethylase gene JMJ703 eliminated the increase of H3K27me3 in many genes. SDG711-mediated H3K27me3 repressed several important genes involved in IM activity and many genes that are silent in the IM but activated during floral organogenesis or other developmental stages. SDG711 overexpression augmented IM activity and increased panicle size; suppression of SDG711 by RNA interference had the opposite effect. Double knockdown/knockout of SDG711 and JMJ703 further reduced panicle size. These results suggest that SDG711 and JMJ703 have agonistic functions in reprogramming the H3K27me3/H3K4me3 ratio and modulating gene expression in the IM.     

Structural dynamics and epigenetic modifications of Hoxc loci along the anteroposterior body axis in developing mouse embryos

Hox genes are organized as clusters and specify regional identity along the anteroposterior body axis by sequential expression at a specific time and region during embryogenesis. However, the precise mechanisms underlying the sequential spatio-temporal, collinear expression pattern of Hox genes are not fully understood. Since epigenetic modifications such as chromatin architecture and histone modifications have become crucial mechanisms for highly coordinated gene expressions, we examined such modifications. E14.5 mouse embryos were dissected into three parts along the anteroposterior axis: brain, trunk-anterior, and trunk-posterior. Then, structural changes and epigenetic modifications were analyzed along the Hoxc cluster using chromosome conformation capture and chromatin immunoprecipitation-PCR methods. Hox non-expressing brain tissues had more compact, heterochromatin-like structures together with the strong repressive mark H3K27me3 than trunk tissues. In the trunk, however, a more loose euchromatin-like topology with a reduced amount of H3K27me3 modifications were observed along the whole cluster, regardless of their potency in gene activation. The active mark H3K4me3 was rather closely associated with the collinear expression of Hoxc genes; at trunk-anterior tissues, only 3' anterior Hoxc genes were marked by H3K4me3 upon gene activation, whereas whole Hoxc genes were marked by H3K4me3 and showed expression in trunk-posterior tissues. Altogether, these results indicated that loosening of the chromatin architecture and removing H3K27me3 were not sufficient for, but rather the concomitant acquisition of H3K4me3 drove the collinear expression of Hoxc genes.