Monthly Archives: February 2012

Chip-seq Gene expression Illumina Immunoprecipitation TF binding

THE TRANSCRIPTION FACTOR ETS-1 REGULATES ANGIOTENSIN II STIMULATED FIBRONECTIN PRODUCTION IN MESANGIAL CELLS.

THE TRANSCRIPTION FACTOR ETS-1 REGULATES ANGIOTENSIN II STIMULATED FIBRONECTIN PRODUCTION IN MESANGIAL CELLS.

Am J Physiol Renal Physiol. 2012 Feb 22;

Authors: Hua P, Feng W, Rezonzew G, Chumley P, Jaimes EA

Abstract
Angiotensin II (Ang II) produced as result of activation of the renin angiotensin system (RAS) plays a critical role in the pathogenesis of chronic kidney disease via its hemodynamic effects on the renal microcirculation as well as by its non-hemodynamic actions including the production of extracellular matrix proteins such as fibronectin, a multifunctional extracellular matrix protein that plays a major role in cell adhesion and migration as well as in the development of glomerulosclerosis. ETS-1 is an important transcription factor essential for normal kidney development and glomerular integrity. We have previously shown that Ang II increases ETS-1 expression and is required for fibronectin production in mesangial cells. In these studies we determined that Ang II induces phosphorylation of ETS-1 via activation of the type 1 Ang II receptor and that Erk1/2 and Akt/PKB phosphorylation are required for these effects. In addition, we characterized the role of ETS-1 on the transcriptional activation of fibronectin production in mesangial cells. We determined that ETS-1 directly activates the fibronectin promoter and by utilizing gel shift assays and chromatin immunoprecipitation assays identified two different ETS-1 binding sites that promote the transcriptional activation of fibronectin in response to Ang II. In addition we identified the essential role of CREB and its coactivator p300 on the transcriptional activation of fibronectin by ETS-1. These studies unveil novel mechanisms involved in RAS induced production of the extracellular matrix protein fibronectin in mesangial cells and establish the role of the transcription factor ETS-1 as a direct mediators of these effects.

PMID: 22357921 [PubMed - as supplied by publisher]

Chip-seq Gene expression Illumina Immunoprecipitation TF binding

Identification of Transcription Factors Regulating CTNNAL1 Expression in Human Bronchial Epithelial Cells.

Identification of Transcription Factors Regulating CTNNAL1 Expression in Human Bronchial Epithelial Cells.

PLoS One. 2012;7(2):e31158

Authors: Xiang Y, Qin XQ, Liu HJ, Tan YR, Liu C, Liu CX

Abstract
Adhesion molecules play important roles in airway hyperresponsiveness or airway inflammation. Our previous study indicated catenin alpha-like 1 (CTNNAL1), an alpha-catenin-related protein, was downregulated in asthma patients and animal model. In this study, we observed that the expression of CTNNAL1 was increased in lung tissue of the ozone-stressed Balb/c mice model and in acute ozone stressed human bronchial epithelial cells (HBEC). In order to identify the possible DNA-binding proteins regulating the transcription of CTNNAL1 gene in HBEC, we designed 8 oligo- nucleotide probes corresponding to various regions of the CTNNAL1 promoter in electrophoretic mobility shift assays (EMSA). We detected 5 putative transcription factors binding sites within CTNNAL1 promoter region that can recruit LEF-1, AP-2α and CREB respectively by EMSA and antibody supershift assay. Chromatin immunoprecipitation (ChIP) assay verified that AP-2 α and LEF-1 could be recruited to the CTNNAL1 promoter. Therefore we further analyzed the functions of putative AP-2 and LEF-1 sites within CTNNAL1 promoter by site-directed mutagenesis of those sites within pGL3/FR/luc. We observed a reduction in human CTNNAL1 promoter activity of mutants of both AP-2α and LEF-1 sites. Pre-treatment with ASOs targeting LEF-1and AP-2α yielded significant reduction of ozone-stress-induced CTNNAL1 expression. The activation of AP-2α and LEF-1, followed by CTNNAL1 expression, showed a correlation during a 16-hour time course. Our data suggest that a robust transcriptional CTNNAL1 up-regulation occurs during acute ozone-induced stress and is mediated at least in part by ozone-induced recruitments of LEF-1 and AP-2α to the human CTNNAL1 promoter.

PMID: 22359570 [PubMed - in process]

Chip-seq Gene expression Illumina Immunoprecipitation TF binding

The Orphan Nuclear Receptor LRH-1 and ERα Activate GREB1 Expression to Induce Breast Cancer Cell Proliferation.

The Orphan Nuclear Receptor LRH-1 and ERα Activate GREB1 Expression to Induce Breast Cancer Cell Proliferation.

PLoS One. 2012;7(2):e31593

Authors: Chand AL, Wijayakumara DD, Knower KC, Herridge KA, Howard TL, Lazarus KA, Clyne CD

Abstract
BACKGROUND: Liver Receptor Homolog 1 (LRH-1, NR5A2) is an orphan nuclear receptor that is over-expressed in cancers in tissues such as the breast, colon and pancreas. LRH-1 plays important roles in embryonic development, steroidogenesis and cholesterol homeostasis. In tumor cells, LRH-1 induces proliferation and cell cycle progression. High LRH-1 expression is demonstrated in breast cancers, positively correlating with ERα status and aromatase activity. LRH-1 dependent cellular mechanisms in breast cancer epithelial cells are poorly defined. Hence in the present study we investigated the actions of LRH-1 in estrogen receptor α (ERα) positive breast cancer cells.
RESULTS: The study aimed to investigate LRH-1 dependent mechanisms that promote breast cancer proliferation. We identified that LRH-1 regulated the expression of Growth Regulation by Estrogen in Breast Cancer 1 (GREB1) in MCF-7 and MDA-MB-231 cells. Over-expression of LRH-1 increased GREB1 mRNA levels while knockdown of LRH-1 reduced its expression. GREB1 is a well characterised ERα target gene, with three estrogen response elements (ERE) located on its promoter. Chromatin immunoprecipitation studies provided evidence of the co-localisation of LRH-1 and ERα at all three EREs. With electrophoretic mobility shift assays, we demonstrated direct binding of LRH-1 to EREs located on GREB1 and Trefoil Factor 1 (TFF1, pS2) promoters. LRH-1 and ERα co-operatively activated transcription of ERE luciferase reporter constructs suggesting an overlap in regulation of target genes in breast cancer cells. Over-expression of LRH-1 resulted in an increase in cell proliferation. This effect was more pronounced with estradiol treatment. In the presence of ICI 182,780, an ERα antagonist, LRH-1 still induced proliferation.
CONCLUSIONS: We conclude that in ER-positive breast cancer cells, LRH-1 promotes cell proliferation by enhancing ERα mediated transcription of target genes such as GREB-1. Collectively these findings indicate the importance of LRH-1 in the progression of hormone-dependent breast cancer and implicate LRH-1 as a potential avenue for drug development.

PMID: 22359603 [PubMed - in process]

Chip-seq Gene expression Illumina Immunoprecipitation TF binding

A differentially methylated single CpG-site is correlated with estrogen receptor alpha transcription.

A differentially methylated single CpG-site is correlated with estrogen receptor alpha transcription.

J Steroid Biochem Mol Biol. 2012 Feb 10;

Authors: Fürst RW, Kliem H, Meyer HH, Ulbrich SE

Abstract
DNA methylation of the promoter region of estrogen receptor alpha (ESR1) is recognized as an epigenetic mechanism that regulates its mRNA abundance. We questioned whether tissues in male growing piglets were influenced in terms of DNA methylation by the developmentally occurring distinct plasma estradiol-17β (E2) concentrations. Additionally, we aimed at broadening the currently limited understanding of the epigenetic regulation of ESR1 in physiological settings. Three distinct genetic regions of ESR1 were analyzed using a combination of methylation-sensitive high resolution melting (MS-HRM) and pyrosequencing. Unexpectedly, major E2 concentration differences were only marginally associated with minor variations in DNA methylation and mRNA abundance. However, by analyzing two tissues showing the greatest differences in transcript abundance, we were able to find one single CpG site in the +1kb intragenic region of ESR1 strikingly differently methylated between heart vs. epididymis. Interestingly, this single CpG-site was identified as a putative binding site for the transcriptional repressor TG-interacting factor 1 (TGIF) which can recruit histone deacetylase 1 (HDAC1) leading to chromatin condensation. Indeed, chromatin immunoprecipitation confirmed a reduced histone H3 presence at the specific ESR1 location in case of higher DNA methylation. We therefore hypothesize that ESR1 expression may be manifested by a single-CpG-site based methylation difference impairing transcription factor binding.

PMID: 22342840 [PubMed - as supplied by publisher]

Chip-seq Gene expression Illumina Immunoprecipitation TF binding

The SET Domain Protein, Set3p, Promotes the Reliable Execution of Cytokinesis in Schizosaccharomyces pombe.

The SET Domain Protein, Set3p, Promotes the Reliable Execution of Cytokinesis in Schizosaccharomyces pombe.

PLoS One. 2012;7(2):e31224

Authors: Rentas S, Saberianfar R, Grewal C, Kanippayoor R, Mishra M, McCollum D, Karagiannis J

Abstract
In response to perturbation of the cell division machinery fission yeast cells activate regulatory networks that ensure the faithful completion of cytokinesis. For instance, when cells are treated with drugs that impede constriction of the actomyosin ring (low doses of Latrunculin A, for example) these networks ensure that cytokinesis is complete before progression into the subsequent mitosis. Here, we identify three previously uncharacterized genes, hif2, set3, and snt1, whose deletion results in hyper-sensitivity to LatA treatment and in increased rates of cytokinesis failure. Interestingly, these genes are orthologous to TBL1X, MLL5, and NCOR2, human genes that encode components of a histone deacetylase complex with a known role in cytokinesis. Through co-immunoprecipitation experiments, localization studies, and phenotypic analysis of gene deletion mutants, we provide evidence for an orthologous complex in fission yeast. Furthermore, in light of the putative role of the complex in chromatin modification, together with our results demonstrating an increase in Set3p levels upon Latrunculin A treatment, global gene expression profiles were generated. While this analysis demonstrated that the expression of cytokinesis genes was not significantly affected in set3Δ backgrounds, it did reveal defects in the ability of the mutant to regulate genes with roles in the cellular response to stress. Taken together, these findings support the existence of a conserved, multi-protein complex with a role in promoting the successful completion of cytokinesis.

PMID: 22347452 [PubMed - in process]

Chip-seq Gene expression Illumina Immunoprecipitation TF binding

Retinoic acid receptor agonists regulate expression of ATP-binding cassette transporter G1 in macrophages.

Retinoic acid receptor agonists regulate expression of ATP-binding cassette transporter G1 in macrophages.

Biochim Biophys Acta. 2012 Feb 14;

Authors: Ayaori M, Yakushiji E, Ogura M, Nakaya K, Hisada T, Uto-Kondo H, Takiguchi S, Terao Y, Sasaki M, Komatsu T, Iizuka M, Yogo M, Uehara Y, Kagechika H, Nakanishi T, Ikewaki K

Abstract
ABC transporter G1 (ABCG1) plays a pivotal role in HDL-mediated cholesterol efflux and atherogenesis. We investigated whether, and how, retinoic acid receptors (RARs) regulate ABCG1 expression in macrophages. All-trans retinoic acid (ATRA), an RAR ligand, increased ABCG1 protein levels and apoA-I/HDL-mediated cholesterol efflux from the macrophages. Both ATRA and other RAR agonists, TTNPB and Am580, increased major transcripts driven by promoter B upstream of exon 5, though minor transcripts driven by promoter A upstream of exon 1 were only increased by ATRA. The stimulatory effects of ATRA on ABCG1 expression were completely abolished in the presence of RAR/RXR antagonists but were only partially canceled in the presence of an LXR antagonist. Adenovirus with overexpressed oxysterol sulfotransferase abolished the LXR pathway, as previously reported, and ATRA-responsiveness in ABCA1/ABCG1 expressions were respectively attenuated by 38 and 22% compared to the control virus. Promoter assays revealed that ABCG1 levels were regulated more by promoter B than promoter A, and ATRA activated promoter B in a liver X receptor-responsive element (LXRE)-dependent manner. Further, LXRE-B in intron 7, but not LXRE-A in intron 5, enhanced ATRA responsiveness under overexpression of all RAR isoforms-RARα/β/γ. In contrast, the activation of promoter B by TTNPB depended on LXRE-B and RARα, but not on RARβ/γ. Finally, chromatin immunoprecipitation and gel-shift assays revealed a specific and direct repeat 4-dependent binding of RARα to LXRE-B. In conclusion, RAR ligands increase ABCA1/G1 expression and apoA-I/HDL-mediated cholesterol efflux from macrophages, and modulate ABCG1 promoter activity via LXRE-dependent mechanisms.

PMID: 22353356 [PubMed - as supplied by publisher]

Chip-seq Gene expression Illumina Immunoprecipitation TF binding

Kinetics of DNA methylation inheritance by the Dnmt1-including complexes during the cell cycle.

Kinetics of DNA methylation inheritance by the Dnmt1-including complexes during the cell cycle.

Cell Div. 2012 Feb 20;7(1):5

Authors: Hervouet E, Nadaradjane A, Gueguen M, Vallette FM, Cartron PF

Abstract
ABSTRACT: BACKGROUND: The clonal transmission of lineage-specific DNA methylation patterns in a mammalian genome during the cellular division is a crucial biological process controlled by the DNA methyltransferase Dnmt1, mainly. To investigate possible dynamic mechanisms of DNA methylation inheritance during the cell cycle, we used a Proximity Ligation In Situ Assay (P-LISA) to analyze the kinetic of formation and DNA recruitment of Dnmt1-including complexes. RESULTS: P-LISA, sequential chromatin immunoprecipitation and quantitative methylation specific PCR revealed that the Dnmt1/PCNA/UHRF1-including complexes are mainly formed and recruited on DNA during the S-phase of cell cycle, while the formation and the DNA recruitment of several Dnmt1/transcription factors-including complexes are not S-phase dependent but are G0/G1 and/or G2/M phases dependent. CONCLUSION: Our data confirm that DNA methylation inheritance occurs in S-phase, and demonstrate that DNA methylation inheritance can also occur in G0/G1 and G2/M phases of the cell cycle.

PMID: 22348533 [PubMed - as supplied by publisher]

Chip-seq Gene expression Illumina Immunoprecipitation TF binding

Kinetics of DNA replication and telomerase reaction at a single-seeded telomere in human cells.

Kinetics of DNA replication and telomerase reaction at a single-seeded telomere in human cells.

Genes Cells. 2012 Mar;17(3):186-204

Authors: Hirai Y, Masutomi K, Ishikawa F

Abstract
In most cancer cells, telomerase is activated to elongate telomere DNA, thereby ensuring numerous rounds of cell divisions. It is thus important to understand how telomerase and the replication fork react with telomeres in human cells. However, the highly polymorphic and repetitive nature of the nucleotide sequences in human subtelomeric regions hampers the precise analysis of sequential events taking place at telomeres in S phase. Here, we have established HeLa cells harboring a single-seeded telomere abutted by a unique subtelomere DNA sequence, which has enabled us to specifically focus on the seeded telomere. We have also developed a modified chromatin immunoprecipitation (ChIP) method that uses restriction digestion instead of sonication to fragment chromatin DNA (RES-ChIP), and a method for immunoprecipitating 5-bromo-2'-deoxyuridine (BrdU)-labeled single-stranded DNA by incubating DNA with anti-BrdU antibody in the nondenaturing condition. We have shown that DNA replication of the seeded telomere takes place during a relatively narrow time window in S phase, and telomerase synthesizes telomere DNA after the replication. Moreover, we have demonstrated that the telomerase catalytic subunit TERT associates with telomeres before telomere DNA replication. These results provide a temporal and spatial framework for understanding DNA replication and telomerase reaction at human telomeres.

PMID: 22353550 [PubMed - in process]

Chip-seq Gene expression Illumina Immunoprecipitation TF binding

Positive regulation of NADPH oxidase 5 by pro-inflammatory-related mechanisms in human aortic smooth muscle cells.

Positive regulation of NADPH oxidase 5 by pro-inflammatory-related mechanisms in human aortic smooth muscle cells.

Free Radic Biol Med. 2012 Feb 17;

Authors: Manea A, Manea SA, Florea IC, Luca CM, Raicu M

Abstract
NADPH oxidase Nox5 subtype expression is significantly increased in vascular smooth muscle cells (SMCs) underlying fibro-lipid atherosclerotic lesions. The mechanisms that up-regulate Nox5 are not understood. Consequently, we characterized the promoter of the human Nox5 gene and investigated the role of various pro-inflammatory transcription factors in the regulation of Nox5 in human aortic SMCs. The Nox5 promoter was cloned in the pGL3 basic reporter vector. Functional analysis was done employing 5' deletion mutants to identify the sequences necessary to effect high levels of expression in SMCs. Transcriptional initiation site was detected by rapid amplification of the 5'-cDNA ends. In silico analysis indicated the existence of typical NF-kB, AP-1, and STAT1/STAT3 sites. Transient overexpression of p65/NF-kB, c-Jun/AP-1 or STAT1/STAT3 increased significantly the Nox5 promoter activity. Chromatin immunoprecipitation demonstrated the physical interaction of c-Jun/AP-1 and STAT1/STAT3 proteins with the Nox5 promoter. Lucigenin-enhanced chemiluminescence, real-time PCR, and Western blot assays showed that pharmacological inhibition and the silencing of p65/NF-kB, c-Jun/AP-1 or STAT1/STAT3, reduced significantly the interferon γ-induced Ca(2+)-dependent Nox activity and Nox5 expression. Up-regulated Nox5 correlated with increases in intracellular Ca(2+), an essential condition for Nox5 activity. NF-kB, AP-1, and STAT1/STAT3 are important regulators of Nox5 in SMCs by either direct or indirect mechanisms. Overexpressed Nox5 may generate free radicals in excess, further contributing to SMCs dysfunction in atherosclerosis.

PMID: 22348975 [PubMed - as supplied by publisher]

Chip-seq Gene expression Illumina Immunoprecipitation TF binding

Homocysteine Upregulates Soluble Epoxide Hydrolase in Vascular Endothelium In Vitro and In Vivo.

Homocysteine Upregulates Soluble Epoxide Hydrolase in Vascular Endothelium In Vitro and In Vivo.

Circ Res. 2012 Feb 21;

Authors: Zhang D, Xie X, Chen Y, Hammock BD, Kong W, Zhu Y

Abstract
RationaleHyperhomocysteinemia is a risk factor of atherogenesis. Soluble epoxide hydrolase (sEH) is a major enzyme that hydrolyzes epoxyeicosatrienoic acids and attenuates their cardiovascular protective effects. Whether homocysteine (Hcy) regulates sEH and the underlying mechanism remains elusive.ObjectiveTo elucidate the mechanism by which Hcy regulates sEH expression and endothelial activation in vitro and in vivo.Methods and ResultsHcy treatment in cultured human endothelial cells dose-dependently and time-dependently upregulated sEH mRNA and protein. Hcy increased the expression of adhesion molecules, which was markedly reversed by inhibiting sEH activity. Hcy-induced sEH upregulation is associated with activation of activating transcription factor-6 (ATF6). Bioinformatics analysis revealed a putative ATF6-binding motif in the promoter region of the sEH gene, which was found at a methylation site. Site-directed mutagenesis and chromatin immunoprecipitation assays demonstrated that Hcy treatment or ATF6 overexpression promoted ATF6 binding to the promoter of sEH and increased its activity. Results of methylation-specific polymerase chain reaction revealed that the ATF6 binding site on the sEH promoter was partially methylated and was demethylated with Hcy. SiRNA knockdown of ATF6α or SP1 blocked and ATF6 overexpression and DNA methyltransferase inhibitor mimicked the effect of homocysteine on sEH upregulation. In vivo, immunofluorescence assay revealed elevated expression of sEH and adhesion molecules in the aortic intima of mice with mild hyperhomocysteinemia, which was attenuated by sEH deletion or inhibition.ConclusionATF6 activation and DNA demethylation may coordinately contribute to Hcy-induced sEH expression and endothelial activation. Inhibition of sEH may be a therapeutic approach for treating Hcy-induced cardiovascular diseases.

PMID: 22354938 [PubMed - as supplied by publisher]