AB6002 Sigma-AldrichAnti-MMP-1 Antibody, hinge region

The appropriate expression of the roughly 30,000 human genes requires multiple layers of control. The oncoprotein MYC, a transcriptional regulator, contributes to many of the identified control mechanisms, including the regulation of chromatin, RNA polymerases, and RNA processing. Moreover, MYC recruits core histone-modifying enzymes to DNA. We identified an additional transcriptional cofactor complex that interacts with MYC and that is important for gene transcription. We found that the trithorax protein ASH2L and MYC interact directly in vitro and co-localize in cells and on chromatin. ASH2L is a core subunit of KMT2 methyltransferase complexes that target histone H3 lysine 4 (H3K4), a mark associated with open chromatin. Indeed, MYC associates with H3K4 methyltransferase activity, dependent on the presence of ASH2L. MYC does not regulate this methyltransferase activity but stimulates demethylation and subsequently acetylation of H3K27. KMT2 complexes have been reported to associate with histone H3K27-specific demethylases, while CBP/p300, which interact with MYC, acetylate H3K27. Finally WDR5, another core subunit of KMT2 complexes, also binds directly to MYC and in genome-wide analyses MYC and WDR5 are associated with transcribed promoters. Thus, our findings suggest that MYC and ASH2L-KMT2 complexes cooperate in gene transcription by controlling H3K27 modifications and thereby regulate bivalent chromatin.

We previously reported on the A. thaliana gene EDM2, which is required for several developmental processes and race-specific immunity. Although EDM2 encodes a nuclear protein with features commonly observed in epigenetic factors, its role in chromatin silencing remains unknown. Here we demonstrate that silencing states of several transposons in edm2 mutants are altered. Levels of their transcripts anti-correlate with those of the repressive epigenetic marks H3K27me1, H3K9me2, and DNA-methylation at CHG sites. In addition, double mutant analysis revealed epistasis between EDM2 and the major histone H3K9-methyltransferase gene KRYPTONITE/SUVH4 in the control of H3K9me2 and CHG methylation. Moreover, we found that the expressivity of several mutant edm2 phenotypes exhibits stochastic variation reminiscent of mutants of known epigenetic modifiers. We propose that EDM2 affects the expression of transposons and developmentally important genes by modulating levels of repressive chromatin marks in a locus dependent manner.

Jarid2/Jumonji, the founding member of the Jmj factor family, critically regulates various developmental processes, including cardiovascular development. The Jmj family was identified as histone demethylases, indicating epigenetic regulation by Jmj proteins. Deletion of Jarid2 in mice resulted in cardiac malformation and increased endocardial Notch1 expression during development. Although Jarid2 has been shown to occupy the Notch1 locus in the developing heart, the precise molecular role of Jarid2 remains unknown. Here we show that deletion of Jarid2 results in reduced methylation of lysine 9 on histone H3 (H3K9) at the Notch1 genomic locus in embryonic hearts. Interestingly, SETDB1, a histone H3K9 methyltransferase, was identified as a putative cofactor of Jarid2 by yeast two-hybrid screening, and the physical interaction between Jarid2 and SETDB1 was confirmed by coimmunoprecipitation experiments. Concurrently, accumulation of SETDB1 at the site of Jarid2 occupancy was significantly reduced in Jarid2 knock out (KO) hearts. Employing genome-wide approaches, putative Jarid2 target genes regulated by SETDB1 via H3K9 methylation were identified in the developing heart by ChIP-chip. These targets are involved in biological processes that, when dysregulated, could manifest in the phenotypic defects observed in Jarid2 KO mice. Our data demonstrate that Jarid2 functions as a transcriptional repressor of target genes, including Notch1, through a novel process involving the modification of H3K9 methylation via specific interaction with SETDB1 during heart development. Therefore, our study provides new mechanistic insights into epigenetic regulation by Jarid2, which will enhance our understanding of the molecular basis of other organ development and biological processes.

The term "position effect" is used when the expression of a gene is deleteriously affected by an alteration in its chromosomal environment even though the integrity of the protein coding sequences is maintained. We describe a patient affected by epilepsy and severe neurodevelopment delay carrying a balanced translocation t(15;16)(p11.2;q12.1)dn that we assume caused a position effect as a result of the accidental juxtaposition of heterochromatin in the euchromatic region.FISH mapped the translocation breakpoints (bkps) to 15p11.2 within satellite III and the 16q12.1 euchromatic band within the ITFG1 gene. The expression of the genes located on both sides of the translocation were tested by means of real-time PCR and three, all located on der(16), were found to be variously perturbed: the euchromatic gene NETO2/BTCL2 was silenced, whereas VPS35 and SHCBP1, located within the major heterochromatic block of chromosome 16q11.2, were over-expressed. Pyrosequencing and chromatin immunoprecipitation of NETO2/BTCL2 and VPS35 confirmed the expression findings. Interphase FISH analysis showed that der(16) localised to regions occupied by the beta satellite heterochromatic blocks more frequently than der(15).To the best of our knowledge, this is the first report of a heterochromatic position effect in humans caused by the juxtaposition of euchromatin/heterochromatin as a result of chromosomal rearrangement. The overall results are fully in keeping with the observations in Drosophila and suggest the occurrence of a human heterochromatin position effect associated with the nuclear repositioning of the der(16) and its causative role in the patient's syndromic phenotype.

DNA methylation of promoter regions is a common event in prostate cancer, one of the most common cancers in men worldwide. Because prior reports demonstrating that DNA methylation is important in prostate cancer studied a limited number of genes, we systematically quantified the DNA methylation status of 1505 CpG dinucleotides for 807 genes in 78 paraffin-embedded prostate cancer samples and three normal prostate samples. The ERG gene, commonly repressed in prostate cells in the absence of an oncogenic fusion to the TMPRSS2 gene, was one of the most commonly methylated genes, occurring in 74% of prostate cancer specimens. In an independent group of patient samples, we confirmed that ERG DNA methylation was common, occurring in 57% of specimens, and cancer-specific. The ERG promoter is marked by repressive chromatin marks mediated by polycomb proteins in both normal prostate cells and prostate cancer cells, which may explain ERG's predisposition to DNA methylation and the fact that tumors with ERG DNA methylation were more methylated, in general. These results demonstrate that bead arrays offer a high-throughput method to discover novel genes with promoter DNA methylation such as ERG, whose measurement may improve our ability to more accurately detect prostate cancer.

The PLZF/RARA fusion protein generated by the t(11;17)(q23;q21) translocation in acute promyelocytic leukaemia (APL) is believed to act as an oncogenic transcriptional regulator recruiting epigenetic factors to genes important for its transforming potential. However, molecular mechanisms associated with PLZF/RARA-dependent leukaemogenesis still remain unclear.We searched for specific PLZF/RARA target genes by ChIP-on-chip in the haematopoietic cell line U937 conditionally expressing PLZF/RARA. By comparing bound regions found in U937 cells expressing endogenous PLZF with PLZF/RARA-induced U937 cells, we isolated specific PLZF/RARA target gene promoters. We next analysed gene expression profiles of our identified target genes in PLZF/RARA APL patients and analysed DNA sequences and epigenetic modification at PLZF/RARA binding sites. We identify 413 specific PLZF/RARA target genes including a number encoding transcription factors involved in the regulation of haematopoiesis. Among these genes, 22 were significantly down regulated in primary PLZF/RARA APL cells. In addition, repressed PLZF/RARA target genes were associated with increased levels of H3K27me3 and decreased levels of H3K9K14ac. Finally, sequence analysis of PLZF/RARA bound sequences reveals the presence of both consensus and degenerated RAREs as well as enrichment for tissue-specific transcription factor motifs, highlighting the complexity of targeting fusion protein to chromatin. Our study suggests that PLZF/RARA directly targets genes important for haematopoietic development and supports the notion that PLZF/RARA acts mainly as an epigenetic regulator of its direct target genes.

It is well known that biosynthesis of glycans takes place in organ- and tissue-specific manners and glycan expression is controlled by various factors including glycosyltransferases. The expression mechanism of glycosyltransferases, however, is poorly understood. Here we investigated the expression mechanism of a brain-specific glycosyltransferase, GnT-IX (N-acetylglucosaminyltransferase IX, also designated as GnT-Vb), which synthesizes branched O-mannose glycan. Using an epigenetic approach, we revealed that the genomic region around the transcriptional start site of the GnT-IX gene was highly associated with active chromatin histone marks in a neural cell-specific manner, indicating that brain-specific GnT-IX expression is under control of an epigenetic "histone code." By EMSA and ChIP analyses we identified two regulatory proteins, NeuroD1 and CTCF that bind to and activate the GnT-IX promoter. We also revealed that GnT-IX expression was suppressed in CTCF- and NeuroD1-depleted cells, indicating that a NeuroD1- and CTCF-dependent epigenetic mechanism governs brain-specific GnT-IX expression. Several other neural glycosyltransferase genes are also found to be regulated by epigenetic histone modifications. This is the first report demonstrating a molecular mechanism at the chromatin level underlying tissue-specific glycan expression.

Signal transducer and activator of transcription 2 (STAT2), the critical component of type I interferons signaling, is a prototype latent cytoplasmic signal-dependent transcription factor. Activated tyrosine-phosphorylated STAT2 associates with STAT1 and IRF9 to bind the ISRE elements in the promoters of a subset of IFN-inducible genes (ISGs). In addition to activate hundreds of ISGs, IFNα also represses numerous target genes but the mechanistic basis for this dual effect and transcriptional repression is largely unknown. We investigated by ChIP-chip the binding dynamics of STAT2 and "active" phospho(P)-STAT2 on 113 putative IFNα direct target promoters before and after IFNα induction in Huh7 cells and primary human hepatocytes (PHH). STAT2 is already bound to 62% of our target promoters, including most "classical" ISGs, before IFNα treatment. 31% of STAT2 basally bound promoters also show P-STAT2 positivity. By correlating in vivo promoter occupancy with gene expression and changes in histone methylation marks we found that: 1) STAT2 plays a role in regulating ISGs expression, independently from its phosphorylation; 2) P-STAT2 is involved in ISGs repression; 3) "activated" ISGs are marked by H3K4me1 and H3K4me3 before IFNα; 4) "repressed" genes are marked by H3K27me3 and histone methylation plays a dominant role in driving IFNα-mediated ISGs repression.

The purpose of this study was to assess the effects of gadolinium (Gd3+), provided as gadolinium chloride, on fibroblast function.Human dermal fibroblasts in monolayer culture and intact skin in organ culture were exposed to the lanthanide metal (1-20 μM).Increased proliferation was observed, in association with upregulation of matrix metalloproteinase-1 and tissue inhibitor of metalloproteinases-1, without an apparent increase in production of type I procollagen. A platelet-derived growth factor (PDGF) receptor-blocking antibody inhibited fibroblast proliferation in response to Gd3+ as did inhibitors of signaling pathways--that is, mitogen-activated protein kinase and phosphatidylinositol-3 kinase pathways--that are activated by PDGF.The responses to gadolinium chloride are similar to responses previously seen with chelated Gd3+ in clinically used magnetic resonance imaging contrast agents. Fibroblast responses appear to reflect Gd3+-induced PDGF receptor activation and downstream signaling. Increased dermal fibroblast proliferation in conjunction with effects on matrix metalloproteinase-1 and tissue inhibitor of metalloproteinases-1 could contribute to the fibroplastic/fibrotic changes seen in the lesional skin of individuals with nephrogenic systemic fibrosis.

Polycomb (PcG) and Trithorax (TrxG) group proteins act antagonistically to establish tissue-specific patterns of gene expression. The PcG protein Ezh2 facilitates repression by catalysing histone H3-Lys27 trimethylation (H3K27me3). For expression, H3K27me3 marks are removed and replaced by TrxG protein catalysed histone H3-Lys4 trimethylation (H3K4me3). Although H3K27 demethylases have been identified, the mechanism by which these enzymes are targeted to specific genomic regions to remove H3K27me3 marks has not been established. Here, we demonstrate a two-step mechanism for UTX-mediated demethylation at muscle-specific genes during myogenesis. Although the transactivator Six4 initially recruits UTX to the regulatory region of muscle genes, the resulting loss of H3K27me3 marks is limited to the region upstream of the transcriptional start site. Removal of the repressive H3K27me3 mark within the coding region then requires RNA Polymerase II (Pol II) elongation. Interestingly, blocking Pol II elongation on transcribed genes leads to increased H3K27me3 within the coding region, and formation of bivalent (H3K27me3/H3K4me3) chromatin domains. Thus, removal of repressive H3K27me3 marks by UTX occurs through targeted recruitment followed by spreading across the gene.