17-614 Sigma-AldrichChIPAb+ Trimethyl-Histone H3 (Lys4) - ChIP Validated Antibody and Primer Set, rabbit monoclonal

Mesenchymal stem cells (MSCs) are highly plastic cells that are able to transdifferentiate or dedifferentiate under appropriate conditions. In the present study, we reported here that after in vitro induction of osteogenic differentiation, MSCs could be reverted to a primitive stem cell population (dedifferentiated osteogenic MSCs, De-Os-MSCs) with improved cell survival, colony formation, osteogenic potential, migratory capacity and increased expression of Nanog, Oct4 and Sox2. Most importantly, our results showed great superiority of the De-Os-MSCs over untreated MSCs in ectopic bone formation in vivo. Furthermore, Nanog-knockdown in MSCs could reverse these enhanced properties in De-Os-MSCs in vitro, indicating a central role of Nanog in the transcriptional network. In addition, epigenetic regulations including DNA methylation and histone modifications may play important roles in regulating the de-osteogenic differentiation process. And we found decreased methylation and promoter accrual of activating histone marks, such as H3K4me3 and H4ac on both Nanog and Oct4 gene promoters. Taken together, our study demonstrated that epigenetic memory in De-Os-MSCs gained by priming with osteogenic induction medium favored their differentiation along osteoblastic lineage with improved cell survival and migratory abilities, which may have application potential in enhancing their regenerative capacity in mammals.

The fidelity of T cell immunity depends greatly on coupling T cell receptor signaling with specific T cell effector functions. Here, we describe a chromatin-based mechanism that enables integration of TCR specificity into definite T cell lineage commitment. Using natural killer T cells (iNKT cell) as a model of a T cell subset that differentiates in response to specific TCR signaling, we identified a key role of histone H3 lysine 27 trimethylation (H3K27me3) in coupling iNKT cell TCR specificity with the generation of iNKT cells. We found that the Zbtb16/PLZF gene promoter that drives iNKT cell differentiation possesses a bivalent chromatin state characterized by the simultaneous presence of negative and positive H3K27me3 and H3K4me3 modifications. Depletion of H3K27me3 at the Zbtb16/PLZF promoter leads to uncoupling of iNKT cell development from TCR specificity and is associated with accumulation of iNKT-like CD4(+) cells that express a non-iNKT cell specific T cell repertoire. In turn, stabilization of H3K27me3 leads to a drastic reduction of the iNKT cell population. Our data suggest that H3K27me3 levels at the bivalent Zbtb16/PLZF gene define a threshold enabling precise coupling of TCR specificity to lineage commitment.

Cellular antiviral activities are critically controlled by transcriptional activation of interferon-inducible genes, involving interferon regulatory factors (IRFs). Previous data suggested that IRF1 is an activator and IRF2 is a repressor, which functionally antagonize each other in transcriptional regulation. However, it is not clear how these two factors function to regulate cellular antiviral activities.We show that IRF2 is critically required for the induction of the TLR3 and other interferon-inducible genes in a chromatin environment. While both IRF1 and IRF2 directly interact with the BAF chromatin remodeling complex, IRF2 is associated with the TLR3 promoter in the unstimulated state and IRF1 binding to the promoter is strongly induced by stimulation with interferon, suggesting that these two factors may function at different stages of gene induction in the recruitment of the BAF complex. IRF2 acts to maintain the basal level expression, an open chromatin structure, and active histone modification marks (H3K9, K14 acetylation and H3K4 tri-methylation) of the TLR3 promoter in the unstimulated state, while IRF1 serves to rapidly activate the promoter upon stimulation.IRF1 and IRF2 of the IRF family of transcription factors play distinct roles in cellular response to viral infection. IRF2 binds to TLR3 and other IFN-inducible gene promoters and maintains an active chromatin structure in the unstimulated state, which is required for their induction, while IRF1 binding to these promoters activates their transcription upon viral infection. Thus, the division of labor between the IRF transcription factor family members plays a pivotal role in coordinating the transcriptional activation in the cellular antiviral response.

Brain-derived neurotrophic factor (BDNF) has a crucial role in modulating neural and behavioral plasticity to drugs of abuse. We found a persistent downregulation of exon-specific Bdnf expression in the ventral tegmental area (VTA) in response to chronic opiate exposure, which was mediated by specific epigenetic modifications at the corresponding Bdnf gene promoters. Exposure to chronic morphine increased stalling of RNA polymerase II at these Bdnf promoters in VTA and altered permissive and repressive histone modifications and occupancy of their regulatory proteins at the specific promoters. Furthermore, we found that morphine suppressed binding of phospho-CREB (cAMP response element binding protein) to Bdnf promoters in VTA, which resulted from enrichment of trimethylated H3K27 at the promoters, and that decreased NURR1 (nuclear receptor related-1) expression also contributed to Bdnf repression and associated behavioral plasticity to morphine. Our findings suggest previously unknown epigenetic mechanisms of morphine-induced molecular and behavioral neuroadaptations.

The canonical pathway for IL-1β production requires TLR-mediated NF-κB-dependent Il1b gene induction, followed by caspase-containing inflammasome-mediated processing of pro-IL-1β. Here we show that IL-21 unexpectedly induces IL-1β production in conventional dendritic cells (cDCs) via a STAT3-dependent but NF-κB-independent pathway. IL-21 does not induce Il1b expression in CD4(+) T cells, with differential histone marks present in these cells versus cDCs. IL-21-induced IL-1β processing in cDCs does not require caspase-1 or caspase-8 but depends on IL-21-mediated death and activation of serine protease(s). Moreover, STAT3-dependent IL-1β expression in cDCs at least partially explains the IL-21-mediated pathologic response occurring during infection with pneumonia virus of mice. These results demonstrate lineage-restricted IL-21-induced IL-1β via a non-canonical pathway and provide evidence for its importance in vivo.

Common bean is the most important grain legume in the human diet. Bean improvement efforts have been focused on classical breeding techniques because bean is recalcitrant to both somatic embryogenesis and in vitro regeneration. This study was undertaken to better understand the process of somatic embryogenesis in the common bean. We focused on the mechanisms by which somatic embryogenesis in plants is regulated and the interaction of these mechanisms with plant hormones. Specifically, we examined the role of the gene PvTRX1h, an ortholog of a major known histone lysine methyltransferase in plants, in somatic embryo generation. Given the problems with regeneration and transformation, we chose to develop and use regeneration-competent callus that could be successively transformed. Embryogenic calli of common bean were generated and transformed with the PvTRX1hRiA construction to down-regulate, by RNA interference, expression of the PvTRX1h gene. Plant hormone content was measured by mass spectrometry and gene expression was assessed by q-PCR. Detailed histological analysis was performed on selected transgenic embryogenic calli. It was determined that down-regulation of PvTRX1h gene was accompanied by altered concentrations of plant hormones in the calli. PvTRX1h regulated the expression of genes involved in auxin biosynthesis and embryogenic calli in which PvTRX1h was down-regulated were capable of differentiation into somatic embryos. Also, down-regulation of PvTRX1h showed increased transcript abundance of a gene coding for a second histone lysine methyltransferase, PvASHH2h. Accordingly, the PvTRX1h gene is involved in the synthesis of plant hormones in common bean callus. These results shed light on the crosstalk among histone methyltransferases and plant hormone signaling and on gene regulation during somatic embryo generation.

Metabolic diseases and comorbidities represent an ever-growing epidemic where multiple cell types impact tissue homeostasis. Here, the link between the metabolic and gene regulatory networks was studied through experimental and computational analysis. Integrating gene regulation data with a human metabolic network prompted the establishment of an open-sourced web portal, IDARE (Integrated Data Nodes of Regulation), for visualizing various gene-related data in context of metabolic pathways. Motivated by increasing availability of deep sequencing studies, we obtained ChIP-seq data from widely studied human umbilical vein endothelial cells. Interestingly, we found that association of metabolic genes with multiple transcription factors (TFs) enriched disease-associated genes. To demonstrate further extensions enabled by examining these networks together, constraint-based modeling was applied to data from human preadipocyte differentiation. In parallel, data on gene expression, genome-wide ChIP-seq profiles for peroxisome proliferator-activated receptor (PPAR) γ, CCAAT/enhancer binding protein (CEBP) α, liver X receptor (LXR) and H3K4me3 and microRNA target identification for miR-27a, miR-29a and miR-222 were collected. Disease-relevant key nodes, including mitochondrial glycerol-3-phosphate acyltransferase (GPAM), were exposed from metabolic pathways predicted to change activity by focusing on association with multiple regulators. In both cell types, our analysis reveals the convergence of microRNAs and TFs within the branched chain amino acid (BCAA) metabolic pathway, possibly providing an explanation for its downregulation in obese and diabetic conditions.

Both diffusible factors acting in trans and chromatin components acting in cis are implicated in gene regulation, but the extent to which either process causally determines a cell's transcriptional identity is unclear. We recently used cell fusion to define a class of silent genes termed "cis-silenced" (or "occluded") genes, which remain silent even in the presence of trans-acting transcriptional activators. We further showed that occlusion of lineage-inappropriate genes plays a critical role in maintaining the transcriptional identities of somatic cells. Here, we present, for the first time, a comprehensive map of occluded genes in somatic cells. Specifically, we mapped occluded genes in mouse fibroblasts via fusion to a dozen different rat cell types followed by whole-transcriptome profiling. We found that occluded genes are highly prevalent and stable in somatic cells, representing a sizeable fraction of silent genes. Occluded genes are also highly enriched for important developmental regulators of alternative lineages, consistent with the role of occlusion in safeguarding cell identities. Alongside this map, we also present whole-genome maps of DNA methylation and eight other chromatin marks. These maps uncover a complex relationship between chromatin state and occlusion. Furthermore, we found that DNA methylation functions as the memory of occlusion in a subset of occluded genes, while histone deacetylation contributes to the implementation but not memory of occlusion. Our data suggest that the identities of individual cell types are defined largely by the occlusion status of their genomes. The comprehensive reference maps reported here provide the foundation for future studies aimed at understanding the role of occlusion in development and disease.

The histone demethylase JMJD2C, also known as KDM4C/GASC1, has activity against methylated H3K9 and H3K36 and is amplified and/or overexpressed in human cancers. By the generation of Jmjd2c knockout mice, we demonstrate that loss of Jmjd2c is compatible with cellular proliferation, embryonic stem cell (ESC) self-renewal, and embryonic development. Moreover, we report that JMJD2C localizes to H3K4me3-positive transcription start sites in both primary cells and in the human carcinoma KYSE150 cell line containing an amplification of the JMJD2C locus. Binding is dependent on the double Tudor domain of JMJD2C, which recognizes H3K4me3 but not H4K20me2/me3 in vitro, showing a binding specificity different from that of the double Tudor domains of JMJD2A and JMJD2B. Depletion of JMJD2C in KYSE150 cells has a modest effect on H3K9me3 and H3K36me3 levels but impairs proliferation and leads to deregulated expression of a subset of target genes involved in cell cycle progression. Taking these findings together, we show that JMJD2C is targeted to H3K4me3-positive transcription start sites, where it can contribute to transcriptional regulation, and report that the putative oncogene JMJD2C generally is not required for cellular proliferation or embryonic development.

Differentiation of mammary secretory epithelium during pregnancy is characterized by sequential activation of genes over several orders of magnitude. Although the transcription factor STAT5 is key to alveolar development, it is not clear to what extent it controls temporal activation of genetic programs in secretory epithelium. To uncover molecular mechanisms effecting progressive differentiation, we explored genome-wide STAT5 binding and H3K4me3 (i.e., trimethylated histone H3 at K4) marks in mammary tissues at early and midpregnancy and at parturition. STAT5 binding to genes induced during pregnancy was low in immature mammary tissue but increased with epithelial differentiation. Increased STAT5 binding was associated with the establishment of H3K4me3 marks and transcriptional activation. STAT5 binding preceded the formation of H3K4me3 marks in some mammary-specific genes. De novo STAT5 binding was also found at distal sites, indicating enhancers. Furthermore, we established an exhaustive mammary transcriptome. Through integration of RNA-seq and STAT5 and H3K4me4 ChIP-seq data, we discovered novel mammary-specific alternative promoters and genes, including noncoding RNAs. Our findings suggest that STAT5 is an early step in establishing transcription complexes on genes specifically expressed in mammary epithelium. This is the first study in an organ that links progressive chromatin occupancy of STAT5 to the acquisition of H3K4me3 marks and transcription during hormone-induced differentiation.