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Chromatin Immunoprecipitation Analysis (chromatin + immunoprecipitation_analysis)
Selected AbstractsJarid2 is among a set of genes differentially regulated by Nkx2.5 during outflow tract morphogenesisDEVELOPMENTAL DYNAMICS, Issue 7 2010Jeremy L. Barth Abstract Nkx2.5, a transcription factor implicated in human congenital heart disease, is required for regulation of second heart field (SHF) progenitors contributing to outflow tract (OFT). Here, we define a set of genes (Lrrn1, Elovl2, Safb, Slc39a6, Khdrbs1, Hoxb4, Fez1, Ccdc117, Jarid2, Nrcam, and Enpp3) expressed in SHF containing pharyngeal arch tissue whose regulation is dependent on Nkx2.5. Further investigation shows that Jarid2, which has been implicated in OFT morphogenesis, is a direct target of Nkx2.5 regulation. Jarid2 expression was up-regulated in SHF mesoderm of Nkx2.5-deficient embryos. Chromatin immunoprecipitation analysis showed Nkx2.5 interaction with consensus binding sites in the Jarid2 promoter in pharyngeal arch cells. Finally, Jarid2 promoter activity and mRNA expression levels were down-regulated by Nkx2.5 overexpression. Given the role of Jarid2 as a regulator of early cardiac proliferation, these findings highlight Jarid2 as one of several potential mediators of the critical role played by Nkx2.5 during OFT morphogenesis. Developmental Dynamics 239:2024,2033, 2010. © 2010 Wiley-Liss, Inc. [source] Human ovarian carcinoma cells: Histone deacetylase inhibitors exhibit antiproliferative activity and potently induce apoptosisCANCER, Issue 12 2004Noriyuki Takai M.D., Ph.D. Abstract BACKGROUND Histone deacetylase inhibitors (HDACIs) can inhibit proliferation, stimulate apoptosis, and induce cell cycle arrest in malignant cells. METHODS The authors investigated the effects of four HDACIs on nine ovarian carcinoma cell lines in vitro and in vivo. Ovarian carcinoma cells were treated with a variety of HDACIs, and their effects on cell growth, the cell cycle, apoptosis, and related events were investigated. The ability of valproic acid (VPA) to inhibit the growth of ovarian tumors in immunodeficient mice was also assessed. RESULTS Clonogenic assays showed that all ovarian carcinoma cell lines were sensitive to the growth-inhibitory effects of the HDACIs. Cell cycle analysis indicated that their exposure to HDACIs decreased the proportion of cells in S phase and increased the proportion of cells in the G0/G1 and/or G2/M phases of the cell cycle. Terminal deoxynucleotidyltransferase-mediated uridine triphosphate end-labeling assays demonstrated that HDACIs induced apoptosis, which occurred in concert with alterations in the expression of genes related to apoptosis, cell growth, and malignant phenotype, including the activation of caspase-9 and caspase-3. Chromatin immunoprecipitation analysis revealed a notable increase in levels of acetylated histones associated with the p21 promoter after treatment with suberoylanilide bishydroxamine. In addition, in experiments involving nude mice, VPA significantly inhibited human ovarian tumor growth without toxic side effects. CONCLUSIONS The results of the current study suggest that HDACIs may be particularly effective in the treatment of ovarian tumors. Cancer 2004. © 2004 American Cancer Society. [source] Transcription regulation of the Saccharomyces cerevisiae PIS1 gene by inositol and the pleiotropic regulator, Ume6pMOLECULAR MICROBIOLOGY, Issue 6 2008Niketa M. Jani Summary In Saccharomyces cerevisiae, transcription of most of the phospholipid biosynthetic genes (e.g. INO1, CHO1, CHO2 and OPI3) is repressed by growth in the presence of inositol and choline and derepressed in their absence. This regulation requires the Ino2p and Ino4p activators and the Opi1p repressor. The PIS1 structural gene is required for the synthesis of the essential lipid phosphatidylinositol. Previous reports show that PIS1 expression is uncoupled from inositol/choline regulation, but is regulated by carbon source, hypoxia and zinc. However, in this study we found that the expression of PIS1 is induced twofold by inositol. This regulation did not require Ino2p and Ino4p, although Ino4p was required for full expression. Ino4p is a basic helix-loop-helix protein that requires a binding partner. Curiously, none of the other basic helix-loop-helix proteins affected PIS1 expression. Inositol induction did require another general regulator of phospholipid biosynthesis, Ume6p. Ume6p was found to be a positive regulator of PIS1 gene expression. Ume6p, and several associated factors, were required for inositol-mediated induction and chromatin immunoprecipitation analysis showed that Ume6p directly regulates PIS1 expression. Thus, we demonstrate novel regulation of the PIS1 gene by Ume6p. [source] Differential mechanism of NF-,B inhibition by two glucocorticoid receptor modulators in rheumatoid arthritis synovial fibroblastsARTHRITIS & RHEUMATISM, Issue 11 2009Valerie Gossye Objective To investigate and compare the molecular mechanisms by which 2 glucocorticoid receptor (GR),activating compounds, dexamethasone (DEX) and Compound A (CpdA), interfere with the NF-,B activation pathway in rheumatoid arthritis (RA) synovial cells. Methods Quantitative polymerase chain reaction was performed to detect the tumor necrosis factor , (TNF,),induced cytokine gene expression of interleukin-1, (IL-1,) and to investigate the effects of DEX and CpdA in RA fibroblast-like synoviocytes (FLS) transfected with small interfering RNA (siRNA) against GR (siGR) compared with nontransfected cells. Immunofluorescence analysis was used to detect the subcellular distribution of NF-,B (p65) under the various treatment conditions, and active DNA-bound p65 was measured using a TransAM assay and by chromatin immunoprecipitation analysis of IL-1,. Signaling pathways were studied via Western blotting of siGR-transfected cells, compared with nontransfected and nontargeting siRNA,transfected control cells, to detect the regulation of phospho-IKK, I,B,, phospho-p38, phospho-ERK, and phospho-JNK. Results Both DEX and CpdA efficiently inhibited IL-1, gene expression in a GR-dependent manner. In addition, CpdA attenuated the TNF,-induced nuclear translocation and DNA binding of p65 in RA FLS, via the attenuation of IKK phosphorylation and subsequent I,B, degradation. CpdA also displayed profound effects on TNF,-induced MAPK activation. The effects of CpdA on TNF,-induced kinase activities occurred independently of the presence of GR. In sharp contrast, DEX did not affect TNF,-induced IKK phosphorylation, I,B, degradation, p65 nuclear translocation, or MAPK activation in RA FLS. Conclusion DEX and CpdA display a dissimilar molecular mechanism of interaction with the NF-,B activation pathway ex vivo. A dual pathway, partially dependent and partially independent of GR (nongenomic), may explain the gene-inhibitory effects of CpdA in RA FLS. [source] |