Project description:To examine the regulation of HSF1-mediated gene expression in control and heat shock conditions, we performed DNA microarray analysis. mRNA levels in control and heat-shocked MEF cells, which were infected with adenovirus expressing scramble RNA or HSF1 short hairpin RNA, were analyzed by DNA microarray analysis using GeneChip Mouse Gene 1.0 ST Array (Affymetrix).
Project description:To examine the expressions of HSF1 and SSBP1-mediated gene in control and heat shock conditions, we performed DNA microarray analysis.
Project description:To examine the expressions of HSF1 and SSBP1-mediated gene in control and heat shock conditions, we performed DNA microarray analysis. mRNA levels in control and heat-shocked MEF cells, which were infected with adenovirus expressing scramble RNA, or shRNA against HSF1 and SSBP1, were analyzed by DNA microarray analysis using GeneChip Mouse Gene 1.0 ST Array (Affymetrix).
Project description:In somatic cells elevated temperature induces activation of the heat shock transcription factor 1 (HSF1) what leads to heat shock proteins synthesis and cytoprotection. However, in the male germ cells (spermatocytes) upon HSF1 activation, caspase-3 dependent apoptosis is induced and spermatogenic cells are actively eliminated. To find out molecular targets of HSF1 in all promoter regions, and to elucidate a mechanism of such diverse HSF1 activity we carried out genome-wide HSF1 binding analysis in control and heat-shocked cells, either spermatogenic or somatic. As model somatic cells we used hepatocytes that respond to hyperthermia in a classical way by induction of heat shock genes transcription. As spermatogenic cells we used a fraction of cells enriched with spermatocytes, which are the most sensitive to damage in elevated temperatures. Using isolated spermatocytes we avoided the influence of the somatic testicular component on the our final results.
Project description:In somatic cells elevated temperature induces activation of the heat shock transcription factor 1 (HSF1) what leads to heat shock proteins synthesis and cytoprotection. However, in the male germ cells (spermatocytes) upon HSF1 activation, caspase-3 dependent apoptosis is induced and spermatogenic cells are actively eliminated. To find out molecular targets of HSF1 in all promoter regions, and to elucidate a mechanism of such diverse HSF1 activity we carried out genome-wide HSF1 binding analysis in control and heat-shocked cells, either spermatogenic or somatic. As model somatic cells we used hepatocytes that respond to hyperthermia in a classical way by induction of heat shock genes transcription. As spermatogenic cells we used a fraction of cells enriched with spermatocytes, which are the most sensitive to damage in elevated temperatures. Using isolated spermatocytes we avoided the influence of the somatic testicular component on the our final results. On Affymetrix GeneChipM-BM-. Mouse Promoter 1.0R Arrays we analyzed DNA immunoprecipitated (using anty-HSF1 antibody) from spermatocytes or hepatocytes, either untreated (control) or immediately after heat shock performed in vitro for 5-20 minutes. ChIP on chip analyses were done in triplicate.
Project description:In somatic cells elevated temperature induces activation of the heat shock transcription factor 1 (HSF1) what leads to heat shock proteins synthesis and cytoprotection. However, in the male germ cells (spermatocytes) upon HSF1 activation, caspase-3 dependent apoptosis is induced and spermatogenic cells are actively eliminated. To elucidate a mechanism of such diverse HSF1 activity we carried out genome-wide transcriptional analysis in control and heat-shocked cells, either spermatogenic or somatic. As model somatic cells we used hepatocytes that respond to hyperthermia in a classical way by induction of heat shock genes transcription. As spermatogenic cells we used a fraction of cells enriched with spermatocytes, which are the most sensitive to damage in elevated temperatures. Using isolated spermatocytes we avoided the influence of the somatic testicular component on the our final results. Genes that are differently regulated during hyperthermia in both types of cells have been identified.
Project description:Transcription factor access to regulatory elements is prevented by the nucleosome. Heat shock factor 1 (HSF1) is a winged helix transcription factor that plays roles in control and stressed conditions by gaining access to target elements, but mechanisms of HSF1 access have not been well known in mammalian cells. We show a physical interaction between the wing motif of human HSF1 and replication protein A (RPA), which is involved in DNA metabolism. Depletion of RPA1 abolishes HSF1 access to the promoter of HSP70 in unstressed conditions, and delays its rapid activation in response to heat shock. The HSF1-RPA complex leads preloading of RNA polymerase II and opens chromatin structure by recruiting a histone chaperone FACT. Furthermore, this interaction is required for melanoma cell proliferation. These results provide a mechanistic basis for constitutive HSF1 access to nucleosomal DNA, which is important for both basal and inducible gene expression. To examine whether the HSF1-RPA complex regulates the expression of genes, mRNA levels in MEFs with/without HSF1- or RPA1-shRNA were analyzed by DNA microarray analysis using GeneChip Mouse Gene 1.0 ST Arrays (Affymetrix).
Project description:Although HSF1 is known to play an important role in regulating the cellular response to proteotoxic stressors, little is known about the structure and function of the HSF1 signaling network under both stressed and unstressed conditions. In this study, we used a combination of chromatin immunoprecipitation (ChIP) microarray analysis and time course gene expression microarray analysis with and without siRNA-mediated inhibition of HSF1 comprehensively identify genes directly and indirectly regulated by HSF1 and examine the structure of the extended HSF1 signaling network. Correlation between promoter binding and gene expression was not significant for all genes bound by HSF1 suggesting that HSF1 binding per se is not sufficient for expression. However, the correlation with promoter binding was significant for genes identified as HSF1-regulated following siRNA knockdown allowing the identification of direct transcriptional targets of HSF1. Among promoters bound by HSF1 following heat shock, a gene ontology (GO) analysis showed significant enrichment only in categories related to protein folding. In contrast, analysis of the extended HSF1 signaling network showed enrichment in a variety of categories related to protein folding, anti-apoptosis, RNA splicing, ubiquitination and others, highlighting a complex transcriptional program directly and indirectly regulated by HSF1.,SUBMITTER_CITATION: Genome-wide analysis of human HSF1 signaling reveals a transcriptional program linked to cellular adaptation and survival Authors: Todd J. Page, Devanjan Sikder, Longlong Yang, Linda Pluta, Russell D. Wolfinger, Thomas Kodadek, and Russell S. Thomas Journal: Molecular Biosystems 2:627-639
Project description:In somatic cells elevated temperature induces activation of the heat shock transcription factor 1 (HSF1) what leads to heat shock proteins synthesis and cytoprotection. However, in the male germ cells (spermatocytes) upon HSF1 activation, caspase-3 dependent apoptosis is induced and spermatogenic cells are actively eliminated. To elucidate a mechanism of such diverse HSF1 activity we carried out genome-wide transcriptional analysis in control and heat-shocked cells, either spermatogenic or somatic. As model somatic cells we used hepatocytes that respond to hyperthermia in a classical way by induction of heat shock genes transcription. As spermatogenic cells we used a fraction of cells enriched with spermatocytes, which are the most sensitive to damage in elevated temperatures. Using isolated spermatocytes we avoided the influence of the somatic testicular component on the our final results. Genes that are differently regulated during hyperthermia in both types of cells have been identified. On Affymetrix gene chip arrays we analyzed RNA isolated from spermatocytes or hepatocytes, either untreated (control) or after heat shock and 2h of recovery at physiological temperature. Analyses were done in triplicate.
Project description:Although HSF1 is known to play an important role in regulating the cellular response to proteotoxic stressors, little is known about the structure and function of the HSF1 signaling network under both stressed and unstressed conditions. In this study, we used a combination of chromatin immunoprecipitation (ChIP) microarray analysis and time course gene expression microarray analysis with and without siRNA-mediated inhibition of HSF1 comprehensively identify genes directly and indirectly regulated by HSF1 and examine the structure of the extended HSF1 signaling network. Correlation between promoter binding and gene expression was not significant for all genes bound by HSF1 suggesting that HSF1 binding per se is not sufficient for expression. However, the correlation with promoter binding was significant for genes identified as HSF1-regulated following siRNA knockdown allowing the identification of direct transcriptional targets of HSF1. Among promoters bound by HSF1 following heat shock, a gene ontology (GO) analysis showed significant enrichment only in categories related to protein folding. In contrast, analysis of the extended HSF1 signaling network showed enrichment in a variety of categories related to protein folding, anti-apoptosis, RNA splicing, ubiquitination and others, highlighting a complex transcriptional program directly and indirectly regulated by HSF1. Keywords: Time Course, Heat Shock, siRNA