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  • Coordinated transcriptional regulation of calmegin,a testis-specific molecular chaperon, by histone deacetylase and CpG methyltransferase. 16264275

    Calmegin is a testis-specific molecular chaperon playing a key role in spermatogenesis. However, the transcriptional regulatory mechanisms for calmegin expression are entirely unknown. Herein, we revealed that calmegin is transcriptionally regulated by histone deacetylase (HDAC) and CpG methyltransferase. The cDNA microarray analysis of the human fibrosarcoma cells treated with trichostatin A (TSA) showed an increased level of calmegin mRNA. The induction of calmegin mRNA by TSA was added by the treatment with 5-aza-2'-deoxycytidine (5'Aza-dC), implying that epigenetic alterations are involved in the transcriptional repression of the gene. Moreover, chromatin immunoprecipitation assay using an anti-acetyl-histone H3 antibody exhibited that the proximal region (-152 ~ -31) of the calmegin promoter is responsible for HDAC-mediated transcriptional repression of the gene. These results demonstrate that calmegin expression is regulated by HDAC and CpG methyltransferase in a coordinative way.
    Document Type:
    Reference
    Product Catalog Number:
    06-599
    Product Catalog Name:
    Anti-acetyl-Histone H3 Antibody

  • Repression of the cardiac myosin light chain-2 gene in skeletal muscle requires site-specific association of antithetic regulator, Nished, and HDACs. 19604314

    The transcriptional activation mechanisms that regulate tissue-specific expression of cardiac muscle genes have been extensively investigated, but little is known of the regulatory events involved in repression of cardiac-specific genes in non-cardiac cells. We have previously reported that Nished, a ubiquitous transcription factor, interacts with a positive sequence element, the Intron Regulatory Element (IRE) as well as a negatively acting element, the Cardiac-Specific Sequence (CSS), in myosin light chain-2 (MLC2v) gene to promote activation and repression of the gene in cardiac and skeletal muscle cells respectively. Here, we show that the negative regulation of cardiac MLC2v gene in skeletal muscle cells is mediated via the interaction of Nished with histone deacetylase (HDAC) co-repressor. Treatment of cells with the HDAC inhibitor, Trichostatin A (TSA), alleviates the repressor activity of Nished in a dose-dependent manner. Co-transfection studies in primary muscle cells in culture and in Nished expressing stable skeletal muscle cell line demonstrate that Nished down-regulates the cardiac MLC2 gene expression when its association is restricted to CSS alone. Chromatin immunoprecipitation data suggest that the CSS-mediated repression of cardiac MLC2v gene in skeletal muscle cells excludes the participation of the positive element IRE despite the presence of an identical Nished binding site. Taken together, it appears that the negative control of MLC2v transcription is based on a dual mode of regulations, one that affords inaccessibility of IRE to Nished and second that promotes the formation of the transcription repression complex at the inhibitory CSS site to silence the cardiac gene in skeletal muscle cell.
    Document Type:
    Reference
    Product Catalog Number:
    17-295
    Product Catalog Name:
    Chromatin Immunoprecipitation (ChIP) Assay Kit

  • Hydrogen sulfide attenuates cytokine production through the modulation of chromatin remodeling. 25873160

    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.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Histone deacetylase inhibition attenuates cardiac hypertrophy and fibrosis through acetylation of mineralocorticoid receptor in spontaneously hypertensive rats. 25667225

    Inhibition of histone deacetylases (HDACs) by valproic acid (VPA) attenuates inflammatory, hypertrophic, and fibrotic responses in the hearts of spontaneously hypertensive rats (SHRs); however, the molecular mechanism is still unclear. We hypothesized that HDAC inhibition (HDACi) attenuates cardiac hypertrophy and fibrosis through acetylation of mineralocorticoid receptor (MR) in SHRs. Seven-week-old SHRs and Wistar-Kyoto rats were treated with an HDAC class I inhibitor (0.71% w/v in drinking water; VPA) for 11 weeks. Sections of heart were visualized after trichrome stain as well as H&E stain. Histone modifications, such as acetylation (H3Ac [acetylated histone 3]) and fourth lysine trimethylation (H3K4me3) of histone 3, and recruitment of MR and RNA polymerase II (Pol II) into promoters of target genes were measured by quantitative real-time polymerase chain reaction after chromatin immunoprecipitation assay. MR acetylation was determined by Western blot with anti-acetyl-lysine antibody after immunoprecipitation with anti-MR antibody. Treatment with VPA attenuated cardiac hypertrophy and fibrosis. Although treatment with VPA increased H3Ac and H3K4me3 on promoter regions of MR target genes, expression of MR target genes as well as recruitment of MR and Pol II on promoters of target genes were decreased. Although HDACi did not affect MR expression, it increased MR acetylation. These results indicate that HDACi attenuates cardiac hypertrophy and fibrosis through acetylation of MR in spontaneously hypertensive rats.
    Document Type:
    Reference
    Product Catalog Number:
    17-371
    Product Catalog Name:
    EZ-ChIP™

  • Runx1t1 (Runt-related transcription factor 1; translocated to, 1) epigenetically regulates the proliferation and nitric oxide production of microglia. 24586690

    Microglia, the resident immune cells of the brain, undergo rapid proliferation and produce several proinflammatory molecules and nitric oxide (NO) when activated in neuropathological conditions. Runx1t1 (Runt-related transcription factor 1, translocated to 1) has been implicated in recruiting histone deacetylases (HDACs) for transcriptional repression, thereby regulating cell proliferation. In the present study, Runx1t1 expression was shown to localize in amoeboid microglial cells of the postnatal rat brain, being hardly detectable in ramified microglia of the adult brain. Moreover, a marked expression of Runx1t1was induced and translocated to nuclei in activated microglia in vitro and in vivo. In view of these findings, it was hypothesized that Runx1t1 regulates microglial functions during development and in neuropathological conditions.siRNA-mediated knockdown of Runx1t1 significantly decreased the expression level of cell cycle-related gene, cyclin-dependent kinase 4 (Cdk4) and proliferation index in activated BV2 microglia. It was also shown that HDAC inhibitor (HDACi) treatment mimics the effects of Runx1t1 knockdown on microglial proliferation, confirming that microglial proliferation is associated with Runx1t1 expression and HDACs activity. Further, Runx1t1 and HDACs were shown to promote neurotoxic effect of microglia by repressing expression of LAT2, L-aminoacid transporter-2 (cationic amino acid transporter, y+ system), which normally inhibits NO production. This was confirmed by chromatin immunoprecipitation (ChIP) assay, which revealed that Runx1t1 binds to the promoter region of LAT2 and this binding increased upon microglial activation. However, the enhanced binding of Runx1t1 to the LAT2 promoter could not repress the LAT2 expression when the BV2 microglia cells were treated with HDACi, indicating that Runx1t1 requires HDACs to transcriptionally repress the expression of LAT2.In conclusion, it is suggested that Runx1t1 controls proliferation and the neurotoxic effect of microglia by epigenetically regulating Cdk4 and LAT2 via its interaction with HDACs.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Nuclear corepressor is required for inhibition of phosphoenolpyruvate carboxykinase expression by tumor necrosis factor-alpha. 17456789

    Inhibition of phosphoenolpyruvate carboxykinase (PEPCK) by TNF-alpha contributes to the pathogenesis of hypoglycemia in endotoxin shock. In this study, the molecular mechanism underlying the inhibition was investigated in hepatoma cells (rat H4IIE and human HepG2). PEPCK expression was induced by cAMP, and the induction was reduced by TNF-alpha at protein and mRNA levels in H4IIE cells. The inhibition was observed in the PEPCK gene promoter in a PEPCK-luciferase reporter. Activation of nuclear factor kappaB (NF-kappaB) pathway was required for the transcriptional inhibition of PEPCK gene. Degradation of NF-kappaB inhibitor (IkappaB) and p65 nuclear translocation were involved in the inhibition. An interaction of histone deacetylase 3 (HDAC3) and silencing mediator for retinoic acid receptor and thyroid hormone receptor (SMRT) with the PEPCK gene promoter was induced by TNF-alpha and observed in a chromatin immunoprecipitation assay. The TNF-induced inhibition was blocked by HDAC inhibitor or HDAC3 knockdown. The blocking effect was also observed in knockdown of corepressor SMRT. Point mutation suggests that cAMP response element (CRE) is required for TNF-induced inhibition of the PEPCK gene promoter. Phosphorylation of cAMP response element-binding protein at Ser133 and expression of peroxisome proliferator-activated receptor-gamma coactivator 1alpha were not changed by TNF-alpha in H4IIE cells. The transcriptional activity of CRE-binding protein was inhibited by TNF-alpha in a CRE-luciferase reporter. The data suggests that the nuclear corepressor proteins of HDAC3 and SMRT mediate TNF inhibition of PEPCK transcription. The inhibition mechanism is related to activation of NF-kappaB and inhibition of CRE-binding protein activity by the corepressor. These data suggest a novel activity of nuclear corepressor in the regulation of PEPCK expression by TNF-alpha.
    Document Type:
    Reference
    Product Catalog Number:
    07-353
    Product Catalog Name:
    Anti-acetyl-Histone H3 (Lys14) Antibody

  • HDAC3-dependent reversible lysine acetylation of cardiac myosin heavy chain isoforms modulates their enzymatic and motor activity. 21177250

    Reversible lysine acetylation is a widespread post-translational modification controlling the activity of proteins in different subcellular compartments. We previously demonstrated that a class II histone deacetylase (HDAC), HDAC4, and a histone acetyltransferase, PCAF, associate with cardiac sarcomeres, and a class I and II HDAC inhibitor, trichostatin A, enhances contractile activity of myofilaments. In this study, we show that a class I HDAC, HDAC3, is also present at cardiac sarcomeres. By immunohistochemical and electron microscopic analyses, we found that HDAC3 was localized to the A band of sarcomeres and was capable of deacetylating myosin heavy chain (MHC) isoforms. The motor domains of both cardiac α- and β-MHC isoforms were found to be reversibly acetylated. Biomechanical studies revealed that lysine acetylation significantly decreased the K(m) for the actin-activated ATPase activity of both α- and β-MHC isoforms. By an in vitro motility assay, we found that lysine acetylation increased the actin sliding velocity of α-myosin by 20% and β-myosin by 36%, compared to their respective non-acetylated isoforms. Moreover, myosin acetylation was found to be sensitive to cardiac stress. During induction of hypertrophy, myosin isoform acetylation increased progressively with duration of stress stimuli, independent of isoform shift, suggesting that lysine acetylation of myosin could be an early response of myofilaments to increase contractile performance of the heart. These studies provide the first evidence for localization of HDAC3 at myofilaments and uncover a novel mechanism modulating the motor activity of cardiac MHC isoforms.
    Document Type:
    Reference
    Product Catalog Number:
    06-933
    Product Catalog Name:
    Anti-acetyl-Lysine Antibody

  • Valproate induces widespread epigenetic reprogramming which involves demethylation of specific genes. 17012225

    Valproate (VPA)(1) has been used for decades in the treatment of epilepsy, and is also effective as a mood stabilizer and in migraine therapy. It has been shown that VPA is also a histone deacetylase (HDAC) inhibitor. We have previously shown that VPA could trigger active demethylation of ectopically methylated transiently transfected DNA in HEK 293 cells. We therefore tested whether VPA treatment could bring about stable changes in the epigenome by causing changes in the state of DNA methylation of genomic DNA. Using a microarray gene expression analysis we identified the genes whose expression is induced by VPA treatment in HEK 293 cells. We found that a subset of these genes could also be induced by the classical DNA methylation inhibitor 5-aza-2'-deoxy-cytidine (5-aza-CdR) suggesting that VPA can alter the state of expression of genes, which are stably suppressed by DNA methylation. We mapped the state of methylation of three of these genes, MELANOMA ANTIGEN B2 GENE (MAGEB2), METALLOPROTEINASE 2 (MMP2) and WIF1, which are involved in tumor growth and metastasis. A chromatin immunoprecipitation (ChIP) assay revealed that VPA treatment caused as expected a change in the state of acetylation of these genes. Our data supports the concept that chromatin acetylation and DNA methylation are found in a dynamic interrelation and that the consequences of HDAC inhibitors are not limited to changes in histone acetylation but that they also bring about a change in the state of modification of DNA. The implications of our results on the future therapeutic utilities of VPA in cancer will be discussed.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Histone hyperacetylation induced by histone deacetylase inhibitors is not sufficient to cause growth inhibition in human dermal fibroblasts. 11304533

    Use of specific histone deacetylase inhibitors has revealed critical roles for the histone deacetylases (HDAC) in controlling proliferation. Although many studies have correlated the function of HDAC inhibitors with the hyperacetylation of histones, few studies have specifically addressed whether the accumulation of acetylated histones, caused by HDAC inhibitor treatment, is responsible for growth inhibition. In the present study we show that HDAC inhibitors cause growth inhibition in normal and transformed keratinocytes but not in normal dermal fibroblasts. This was despite the observation that the HDAC inhibitor, suberic bishydroxamate (SBHA), caused a kinetically similar accumulation of hyperacetylated histones. This cell type-specific response to SBHA was not due to the inactivation of SBHA by fibroblasts, nor was it due to differences in the expression of specific HDAC family members. Remarkably, overexpression of HDACs 1, 4, and 6 in normal human fibroblasts resulted in cells that could be growth-inhibited by SBHA. These data suggest that, although histone acetylation is a major target for HDAC inhibitors, the accumulation of hyperacetylated histones is not sufficient to cause growth inhibition in all cell types. This suggests that growth inhibition, caused by HDAC inhibitors, may be the culmination of histone hyperacetylation acting in concert with other growth regulatory pathways.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Histonedeacetylase inhibitor Oxamflatin increase HIV-1 transcription by inducing histone modification in latently infected cells. 21181272

    HIV-1 latency represents a major problem in the eradication of HIV-1 in infected individuals treated with highly active anti-retroviral therapy. Histone deacetylase (HDAC) inhibits HIV-1 gene expression and virus production and may contribute to quiescence of HIV-1 within resting CD4+ T cells. Here, we evaluated the effect of Oxamflatin, a class I HDAC inhibitor, on the epigenetic change at HIV-1 long terminal repeat (LTR) and the induction of the latent viruses in the latency Jurkat T cell line. Flow cytometry assay showed that Oxamflatin activate HIV-1 gene expression in these latently infected cells by 2-17 fold than background levels. Chromatin immunoprecipitation (ChIP) assays further revealed that Oxamflatin increase the acetylation level of histone H3 and histone H4 at the nucleosome 1(nuc-1) site of the HIV-1 LTR compared to mock treatment. We also found that Oxamflatin had a synergization with prostratin, or 5-azacytidine or tumor necrosis factor-α to activate the HIV-1 promoter. Taken together, our results suggest that the histone acetylation plays an important role in regulating HIV-1 LTR gene expression, and Oxamflatin has potential as drug candidates as antilatency therapies.
    Document Type:
    Reference
    Product Catalog Number:
    17-371
    Product Catalog Name:
    EZ-ChIP™