07-436 Sigma-AldrichAnti-monomethyl-Histone H3 (Lys4) Antibody

Role of epigenetic mechanisms towards regulation of the complex life cycle/pathogenesis of Plasmodium falciparum, the causative agent of malaria, has been poorly understood. To elucidate stage-specific epigenetic regulation, we performed genome-wide mapping of multiple histone modifications of P. falciparum. Further to understand the differences in transcription regulation in P. falciparum and its host, human, we compared their histone modification profiles.Our comprehensive comparative analysis suggests distinct mode of transcriptional regulation in malaria parasite by virtue of poised genes and differential histone modifications. Furthermore, analysis of histone modification profiles predicted 562 genes producing anti-sense RNAs and 335 genes having bidirectional promoter activity, which raises the intriguing possibility of RNA-mediated regulation of transcription in P. falciparum. Interestingly, we found that H3K36me2 acts as a global repressive mark and gene regulation is fine tuned by the ratio of activation marks to H3K36me2 in P. falciparum. This novel mechanism of gene regulation is supported by the fact that knockout of SET genes (responsible for H3K36 methylation) leads to up-regulation of genes with highest occupancy of H3K36me2 in wild-type P. falciparum. Moreover, virulence (var) genes are mostly poised and marked by a unique set of activation (H4ac) and repression (H3K9me3) marks, which are mutually exclusive to other Plasmodium housekeeping genes.Our study reveals unique plasticity in the epigenetic regulation in P. falciparum which can influence parasite virulence and pathogenicity. The observed differences in the histone code and transcriptional regulation in P. falciparum and its host will open new avenues for epigenetic drug development against malaria parasite.

Histones are the main structural components of the nucleosome, hence targets of many regulatory proteins that mediate processes involving changes in chromatin. The functional outcome of many pathways is "written" in the histones in the form of post-translational modifications that determine the final gene expression readout. As a result, modifications, alone or in combination, are important determinants of chromatin states. Histone modifications are accomplished by the addition of different chemical groups such as methyl, acetyl and phosphate. Thus, identifying and characterizing these modifications and the proteins related to them is the initial step to understanding the mechanisms of gene regulation and in the future may even provide tools for breeding programs. Several studies over the past years have contributed to increase our knowledge of epigenetic gene regulation in model organisms like Arabidopsis, yet this field remains relatively unexplored in crops. In this study we identified and initially characterized histones H3 and H4 in the monocot crop sugarcane. We discovered a number of histone genes by searching the sugarcane ESTs database. The proteins encoded correspond to canonical histones, and their variants. We also purified bulk histones and used them to map post-translational modifications in the histones H3 and H4 using mass spectrometry. Several modifications conserved in other plants, and also novel modified residues, were identified. In particular, we report O-acetylation of serine, threonine and tyrosine, a recently identified modification conserved in several eukaryotes. Additionally, the sub-nuclear localization of some well-studied modifications (i.e., H3K4me3, H3K9me2, H3K27me3, H3K9ac, H3T3ph) is described and compared to other plant species. To our knowledge, this is the first report of histones H3 and H4 as well as their post-translational modifications in sugarcane, and will provide a starting point for the study of chromatin regulation in this crop.

The role of cellular metabolism in regulating cell proliferation and differentiation remains poorly understood. For example, most mammalian cells cannot proliferate without exogenous glutamine supplementation even though glutamine is a non-essential amino acid. Here we show that mouse embryonic stem (ES) cells grown under conditions that maintain naive pluripotency are capable of proliferation in the absence of exogenous glutamine. Despite this, ES cells consume high levels of exogenous glutamine when the metabolite is available. In comparison to more differentiated cells, naive ES cells utilize both glucose and glutamine catabolism to maintain a high level of intracellular α-ketoglutarate (αKG). Consequently, naive ES cells exhibit an elevated αKG to succinate ratio that promotes histone/DNA demethylation and maintains pluripotency. Direct manipulation of the intracellular αKG/succinate ratio is sufficient to regulate multiple chromatin modifications, including H3K27me3 and ten-eleven translocation (Tet)-dependent DNA demethylation, which contribute to the regulation of pluripotency-associated gene expression. In vitro, supplementation with cell-permeable αKG directly supports ES-cell self-renewal while cell-permeable succinate promotes differentiation. This work reveals that intracellular αKG/succinate levels can contribute to the maintenance of cellular identity and have a mechanistic role in the transcriptional and epigenetic state of stem cells.

The tumor suppressor p53 has been studied extensively as a direct transcriptional activator of protein-coding genes. Recent studies, however, have shed light on novel regulatory functions of p53 within noncoding regions of the genome. Here, we use a systematic approach that integrates transcriptome-wide expression analysis, genome-wide p53 binding profiles and chromatin state maps to characterize the global regulatory roles of p53 in response to DNA damage. Notably, our approach identified conserved features of the p53 network in both human and mouse primary fibroblast models. In addition to known p53 targets, we identify many previously unappreciated mRNAs and long noncoding RNAs that are regulated by p53. Moreover, we find that p53 binding occurs predominantly within enhancers in both human and mouse model systems. The ability to modulate enhancer activity offers an additional layer of complexity to the p53 network and greatly expands the diversity of genomic elements directly regulated by p53.

The quiescent centre (QC) in the Arabidopsis root apical meristem is essential for stem cell organization. Here we show that the loss of REPRESSOR OF WUSCHEL1 (ROW1), a PHD domain-containing protein, leads to QC failure, defects in cell differentiation and ectopic expression of WUSCHEL-RELATED HOMEOBOX 5 (WOX5) in cells that normally express ROW1. The wox5-1/row1-3 double mutants show similar phenotypes to wox5-1 indicating that WOX5 is epistatic to ROW1. ROW1 specifically binds trimethylated histone H3 lysine 4 (H3K4me3) in the WOX5 promoter region to repress its transcription. QC expression of ROW1 results in a wox5-1-like phenotype with undetectable WOX5 transcripts. We propose that ROW1 is essential for QC maintenance and for stem cell niche development through the repression of WOX5 in the proximal meristem.

Noncoding microRNAs inhibit translation and lower the transcript stability of coding mRNA, however miR-369 s, in aberrant silencing genomic regions, stabilizes target proteins under cellular stress. We found that in vitro differentiation of embryonic stem cells led to chromatin methylation of histone H3K4 at the miR-369 region on chromosome 12qF in mice, which is expressed in embryonic cells and is critical for pluripotency. Proteomic analyses revealed that miR-369 stabilized translation of pyruvate kinase (Pkm2) splicing factors such as HNRNPA2B1. Overexpression of miR-369 stimulated Pkm2 splicing and enhanced induction of cellular reprogramming by induced pluripotent stem cell factors, whereas miR-369 knockdown resulted in suppression. Furthermore, immunoprecipitation analysis showed that the Argonaute complex contained the fragile X mental retardation-related protein 1 and HNRNPA2B1 in a miR-369-depedent manner. Our findings demonstrate a unique role of the embryonic miR-369-HNRNPA2B1 axis in controlling metabolic enzyme function, and suggest a novel pathway linking epigenetic, transcriptional, and metabolic control in cell reprogramming.

Recent advances in rice flowering studies have shown that the accurate control of flowering by photoperiod is regulated by key mechanisms that involve the regulation of flowering genes including Heading date1 (Hd1), Early hd1 (Ehd1), Hd3a, and RFT1. The chromatin mechanism involved in the regulation of rice flowering genes is presently not well known. Here we show that the rice enhancer of zeste [E(z)] genes SDG711 and SDG718, which encode the polycomb repressive complex2 (PRC2) key subunit that is required for trimethylation of histone H3 lysine 27 (H3K27me3), are respectively, involved in long day (LD) and short day (SD) regulation of key flowering genes. The expression of SDG711 and SDG718 is induced by LD and SD, respectively. Over-expression and down-regulation of SDG711 respectively, repressed and promoted flowering in LD, but had no effect in SD. By contrast, down-regulation of SDG718 had no effect in LD but delayed flowering in SD. SDG711 and SDG718 repressed OsLF (a repressor of Hd1) respectively in LD and SD, leading to a higher expression of Hd1 thus late flowering in LD and early flowering in SD. SDG711 was also found to be involved in the repression of Ehd1 in LD. SDG711 was shown to directly target to OsLF and Ehd1 loci to mediate H3K27me3 and gene repression. The function of the rice E(z) genes in LD repression and SD promotion of flowering suggests that PRC2-mediated epigenetic repression of gene expression is involved in the accurate photoperiod control of rice flowering.

Epigenetic factors have been implicated in the regulation of CD4(+) T-cell differentiation. Jmjd3 plays a role in many biological processes, but its in vivo function in T-cell differentiation remains unknown. Here we report that Jmjd3 ablation promotes CD4(+) T-cell differentiation into Th2 and Th17 cells in the small intestine and colon, and inhibits T-cell differentiation into Th1 cells under different cytokine-polarizing conditions and in a Th1-dependent colitis model. Jmjd3 deficiency also restrains the plasticity of the conversion of Th2, Th17 or Treg cells to Th1 cells. The skewing of T-cell differentiation is concomitant with changes in the expression of key transcription factors and cytokines. H3K27me3 and H3K4me3 levels in Jmjd3-deficient cells are correlated with altered gene expression through interactions with specific transcription factors. Our results identify Jmjd3 as an epigenetic factor in T-cell differentiation via changes in histone methylation and target gene expression.

Day-length is important for regulating the transition to reproductive development (flowering) in plants. In the model plant Arabidopsis thaliana, the transcription factor CONSTANS (CO) promotes expression of the florigen FLOWERING LOCUS T (FT), constituting a key flowering pathway under long-day photoperiods. Recent studies have revealed that FT expression is regulated by changes of histone modification marks of the FT chromatin, but the epigenetic regulators that directly interact with the CO protein have not been identified. Here, we show that the Arabidopsis Morf Related Gene (MRG) group proteins MRG1 and MRG2 act as H3K4me3/H3K36me3 readers and physically interact with CO to activate FT expression. In vitro binding analyses indicated that the chromodomains of MRG1 and MRG2 preferentially bind H3K4me3/H3K36me3 peptides. The mrg1 mrg2 double mutant exhibits reduced mRNA levels of FT, but not of CO, and shows a late-flowering phenotype under the long-day but not short-day photoperiod growth conditions. MRG2 associates with the chromatin of FT promoter in a way dependent of both CO and H3K4me3/H3K36me3. Vice versa, loss of MRG1 and MRG2 also impairs CO binding at the FT promoter. Crystal structure analyses of MRG2 bound with H3K4me3/H3K36me3 peptides together with mutagenesis analysis in planta further demonstrated that MRG2 function relies on its H3K4me3/H3K36me3-binding activity. Collectively, our results unravel a novel chromatin regulatory mechanism, linking functions of MRG1 and MRG2 proteins, H3K4/H3K36 methylations, and CO in FT activation in the photoperiodic regulation of flowering time in plants.

Seed development involves a plethora of spatially and temporally synchronised genetic and epigenetic processes. Although it has been shown that epigenetic mechanisms, such as DNA methylation and chromatin remodelling, act on a large number of genes during seed development and germination, to date the global levels of histone modifications have not been studied in a tissue-specific manner in plant embryos. In this study we analysed the distribution of three epigenetic markers, i.e. H4K5ac, H3K4me2 and H3K4me1 in 'matured', 'dry' and 'germinating' embryos of a model grass, Brachypodium distachyon (Brachypodium). Our results indicate that the abundance of these modifications differs considerably in various organs and tissues of the three types of Brachypodium embryos. Embryos from matured seeds were characterised by the highest level of H4K5ac in RAM and epithelial cells of the scutellum, whereas this modification was not observed in the coleorhiza. In this type of embryos H3K4me2 was most evident in epithelial cells of the scutellum. In 'dry' embryos H4K5ac was highest in the coleorhiza but was not present in the nuclei of the scutellum. H3K4me1 was the most elevated in the coleoptile but absent from the coleorhiza, whereas H3K4me2 was the most prominent in leaf primordia and RAM. In embryos from germinating seeds H4K5ac was the most evident in the scutellum but not present in the coleoptile, similarly H3K4me1 was the highest in the scutellum and very low in the coleoptile, while the highest level of H3K4me2 was observed in the coleoptile and the lowest in the coleorhiza. The distinct patterns of epigenetic modifications that were observed may be involved in the switch of the gene expression profiles in specific organs of the developing embryo and may be linked with the physiological changes that accompany seed desiccation, imbibition and germination.