17-625 Sigma-AldrichChIPAb+ Trimethyl-Histone H3 (Lys9) - ChIP Validated Antibody and Primer Set

Hydrogen sulfide (H2S) is an endogenous gaseous biological mediator, which regulates, among others, the oxidative balance of cells under normal physiological conditions, as well as in various diseases. Several previous studies have reported that H2S attenuates inflammatory mediator production. In this study, we investigated the role of H2S in chromatin modulation in an in vitro model of lipopolysaccharide (LPS)-induced inflammation and evaluated its effects on inflammatory cytokine production. Tamm-Horsfall protein 1 (THP-1) differentiated macrophages were pre-treated with sodium hydrosulfide (NaHS) (an H2S donor) at 0.01, 0.1, 0.5 or 1 mM for 30 min. To stimulate cytokine production, the cells were challenged with bacterial LPS (1 µg/ml) for 1, 4, 8 or 24 h. Histone H3 acetylation was analyzed by chromatin immunoprecipitation (ChIP), cytokine production was measured by ELISA and histone deacetylase (HDAC) activity was analyzed using a standard biochemical assay. H2S inhibited the production of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in a concentration-dependent manner; it was most effective at the two highest concentrations used. This effect was associated with a decrease in histone H3 acetylation at the IL-6 and TNF-α promoters in the cells exposed to H2S or H2S + LPS. The findings of the present study suggest that H2S suppresses histone acetylation, which, in turn, inhibits chromatin openness, leading to a decrease in the gene transcription of various pro-inflammatory cytokines. Therefore, this mechanism may contribute to the previously demonstrated anti-inflammatory effects of H2S and various H2S donors.

Extrauterine growth restriction (EUGR) plays an important role in the developmental origin of adult cardiovascular diseases. In an EUGR rat model, we reported an elevated pulmonary arterial pressure in adults and genome-wide epigenetic modifications in pulmonary vascular endothelial cells (PVECs). However, the underlying mechanism of the early nutritional insult that results in pulmonary vascular consequences later in life remains unclear.A rat model was used to investigate the physiological and structural effect of EUGR on early pulmonary vasculature by evaluating right ventricular systolic pressure and pulmonary vascular density in male rats. Epigenetic modifications of the Notch1 gene in PVECs were evaluated.EUGR decreased pulmonary vascular density with no significant impact on right ventricular systolic pressure at 3 weeks. Decreased transcription of Notch1 was observed both at 3 and 9 weeks, in association with decreased downstream target gene, Hes-1. Chromatin immunoprecipitation and bisulfite sequencing were performed to analyze the epigenetic modifications of the Notch1 gene promoter in PVECs. EUGR caused a significantly increased H3K27me3 in the proximal Notch1 gene promoter, and increased methylation of single CpG sites in the distal Notch1 gene promoter, both at 3 and 9 weeks.We conclude that EUGR results in decreased pulmonary vascular growth in association with decreased Notch1 in PVECs. This may be mediated by increased CpG methylation and H3K27me3 in the Notch1 gene promoter region.

Tumour-repopulating cells (TRCs) are a self-renewing, tumorigenic subpopulation of cancer cells critical in cancer progression. However, the underlying mechanisms of how TRCs maintain their self-renewing capability remain elusive. Here we show that relatively undifferentiated melanoma TRCs exhibit plasticity in Cdc42-mediated mechanical stiffening, histone 3 lysine residue 9 (H3K9) methylation, Sox2 expression and self-renewal capability. In contrast to differentiated melanoma cells, TRCs have a low level of H3K9 methylation that is unresponsive to matrix stiffness or applied forces. Silencing H3K9 methyltransferase G9a or SUV39h1 elevates the self-renewal capability of differentiated melanoma cells in a Sox2-dependent manner. Mechanistically, H3K9 methylation at the Sox2 promoter region inhibits Sox2 expression that is essential in maintaining self-renewal and tumorigenicity of TRCs both in vitro and in vivo. Taken together, our data suggest that 3D soft-fibrin-matrix-mediated cell softening, H3K9 demethylation and Sox2 gene expression are essential in regulating TRC self-renewal.

Early postnatal life is considered as a critical time window for the determination of long-term metabolic states and organ functions. Extrauterine growth restriction (EUGR) causes the development of adult-onset chronic diseases, including pulmonary hypertension. However, the effects of nutritional disadvantages during the early postnatal period on pulmonary vascular consequences in later life are not fully understood. Our study was designed to test whether epigenetics dysregulation mediates the cellular memory of this early postnatal event.To test this hypothesis, we isolated pulmonary vascular endothelial cells by magnetic-activated cell sorting from EUGR and control rats. A postnatal insult, nutritional restriction-induced EUGR caused development of an increased pulmonary artery pressure at 9 weeks of age in male Sprague-Dawley rats. Methyl-DNA immune precipitation chip, genome-scale mapping studies to search for differentially methylated loci between control and EUGR rats, revealed significant difference in cytosine methylation between EUGR and control rats. EUGR changes the cytosine methylation at approximately 500 loci in male rats at 9 weeks of age, preceding the development of pulmonary hypertension and these represent the candidate loci for mediating the pathogenesis of pulmonary vascular disease that occurs later in life. Gene ontology analysis on differentially methylated genes showed that hypermethylated genes in EUGR are vascular development-associated genes and hypomethylated genes in EUGR are late-differentiation-associated and signal transduction genes. We validated candidate dysregulated loci with the quantitative assays of cytosine methylation and gene expressions.These results demonstrate that epigenetics dysregulation is a strong mechanism for propagating the cellular memory of early postnatal events, causing changes in the expression of genes and long-term susceptibility to pulmonary hypertension, and further providing a new insight into the prevention and treatment of EUGR-related pulmonary hypertension.

Facioscapulohumeral muscular dystrophy (FSHD) is one of the most common inherited diseases of the skeletal muscle. It is characterized by asymmetric muscle weakness and variable penetrance. FSHD is linked to a reduction in copy number of the D4Z4 3.3 kb macrosatellite repeat, located in 4q35. This causes the epigenetic de-repression of FSHD candidate genes leading to disease. Nevertheless, the molecular mechanism responsible for silencing of FSHD candidate genes in healthy subjects is not fully understood. While a role for DNA methylation has been suggested, so far there is limited information regarding the methylation status of the 325 CpGs contained in each D4Z4 unit. Using a human/rodent monochromosomal hybrid cell line containing a single human chromosome 4, we performed an in depth analysis of DNA methylation for the majority of the CpGs inside D4Z4 at single nucleotide level. We found that D4Z4 is not uniformly methylated and that the level of DNA methylation does not correlate with the density of CpG dinucleotides. Moreover, in several D4Z4 regions characterized by near complete methylation, we found specific unmethylated CpGs. These elements are enriched in transcription factor binding sites that could be involved in muscle-specific D4Z4 activity. Our approach also detected differential methylation among different D4Z4 units, suggesting that the D4Z4 array is a mosaic of euchromatic and heterochromatic domains. Finally, we found that DNA methylation and histone de-acetylation are required to maintain FSHD candidate genes repressed. Taken together, our data underscore new players involved in the epigenetic regulation of the FSHD locus that could be targeted for therapeutic purposes.

Mammalian cells have mechanisms to counteract the effects of metabolic and exogenous stresses, many of that would be mutagenic if ignored. Damage arising during DNA replication is a major source of mutagenesis. The extent of damage dictates whether cells undergo transient cell cycle arrest and damage repair, senescence or apoptosis. Existing dogma defines these alternative fates as distinct choices. Here we show that immortalised breast epithelial cells are able to survive prolonged S phase arrest and subsequently re-enter cycle after many days of being in an arrested, senescence-like state. Prolonged cell cycle inhibition in fibroblasts induced DNA damage response and cell death. However, in immortalised breast epithelia, efficient S phase arrest minimised chromosome damage and protected sufficient chromatin-bound replication licensing complexes to allow cell cycle re-entry. We propose that our observation could have implications for the design of drug therapies for breast cancer.

The SU(VAR)3-9-like histone methyltransferases usually catalyze repressive histone H3K9 methylation and are involved in transcriptional gene silencing in eukaryotic organisms. We identified a putative SU(VAR)3-9-like histone methyltransferase SUVR2 by a forward genetic screen and demonstrated that it is involved in transcriptional gene silencing at genomic loci targeted by RNA-directed DNA methylation (RdDM). We found that SUVR2 has no histone methyltransferase activity and the conserved catalytic sites of SUVR2 are dispensable for the function of SUVR2 in transcriptional silencing. SUVR2 forms a complex with its close homolog SUVR1 and associate with three previously uncharacterized SNF2-related chromatin-remodeling proteins CHR19, CHR27, and CHR28. SUVR2 was previously thought to be a component in the RdDM pathway. We demonstrated that SUVR2 contributes to transcriptional gene silencing not only at a subset of RdDM target loci but also at many RdDM-independent target loci. Our study suggests that the involvement of SUVR2 in transcriptional gene silencing is related to nucleosome positioning mediated by its associated chromatin-remodeling proteins.

DNA sequence variants influence gene expression and cellular phenotypes. In this study, we focused on natural variation in the gene encoding the histone demethylase, KDM4C, which promotes transcriptional activation by removing the repressive histone mark, H3K9me3, from its target genes. We uncovered cis-acting variants that contribute to extensive individual differences in KDM4C expression. We also identified the target genes of KDM4C and demonstrated that variation in KDM4C expression leads to differences in the growth of normal and some cancer cells. Together, our results from genetic mapping and molecular analysis provide an example of how genetic variation affects epigenetic regulation of gene expression and cellular phenotype.

Epigenetic gene silencing by histone modifications and DNA methylation is essential for cancer development. The molecular mechanism that promotes selective epigenetic changes during tumorigenesis is not understood. We report here that the PIAS1 SUMO ligase is involved in the progression of breast tumorigenesis. Elevated PIAS1 expression was observed in breast tumor samples. PIAS1 knockdown in breast cancer cells reduced the subpopulation of tumor-initiating cells, and inhibited breast tumor growth in vivo. PIAS1 acts by delineating histone modifications and DNA methylation to silence the expression of a subset of clinically relevant genes, including breast cancer DNA methylation signature genes such as cyclin D2 and estrogen receptor, and breast tumor suppressor WNT5A. Our studies identify a novel epigenetic mechanism that regulates breast tumorigenesis through selective gene silencing.

Sirtuin 1 (SIRT1) is a nicotinamide adenine dinucleotide (NAD)-dependent deacetylase that is implicated in plethora of biological processes, including metabolism, aging, stress response, and tumorigenesis. Telomerase (TERT) is essential for telomere maintenance. Activation of TERT is considered a crucial step in tumorigenesis, and therefore it is a potential therapeutic target against cancer. We have recently found that SIRT1 expression is highly elevated in hepatocellular carcinoma, and the depletion of SIRT1 leads to substantial reduction in TERT mRNA and protein expression. However, the underlying molecular mechanism of SIRT1-dependent TERT expression remains uncharacterized. Here, we elucidated if SIRT1 regulates TERT expression via transcriptional, epigenetic and post-transcriptional mechanisms. We report that depletion of SIRT1 does not lead to significant change in transcriptional activity and CpG methylation patterns of the TERT promoter, nor does it affect mRNA stability or 3'-UTR regulation of TERT. Intriguingly, depletion of SIRT1 is associated with substantial induction of acetylated histone H3-K9 and reduction of trimethyl H3-K9 at the TERT gene, which are known to be associated with gene activation. Our data revealed that SIRT1 regulates histone acetylation and methylation at the TERT promoter. We postulated that SIRT1 may regulate TERT expression via long-range interaction, or via yet unidentified histone modifications.