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Responsiveness of genes to manipulation of transcription factors in ES cells is associated with histone modifications and tissue specificity (2 of 2)

ABSTRACT: BACKGROUND: In addition to determining static states of gene expression (high vs. low), it is important to characterize their dynamic status. For example, genes with H3K27me3 chromatin marks are not only suppressed but also poised for activation. However, the responsiveness of genes to perturbations has never been studied systematically. To distinguish gene responses to specific factors from responsiveness in general, it is necessary to analyze gene expression profiles of cells responding to a large variety of disturbances, and such databases did not exist before. RESULTS: We estimated the responsiveness of all genes in mouse ES cells using our recently published database on expression change after controlled induction of 53 transcription factors (TFs) and other genes. Responsive genes (N = 4746), which were readily upregulated or downregulated depending on the kind of perturbation, mostly have regulatory functions and a propensity to become tissue-specific upon differentiation. Tissue-specific expression was evaluated on the basis of published (GNF) and our new data for 15 organs and tissues. Non-responsive genes (N = 9562), which did not change their expression much following any perturbation, were enriched in housekeeping functions. We found that TF-responsiveness in ES cells is the best predictor known for tissue-specificity in gene expression. Among genes with CpG islands, high responsiveness is associated with H3K27me3 chromatin marks, and low responsiveness is associated with H3K36me3 chromatin, stronger tri-methylation of H3K4, binding of E2F1, and GABP binding motifs in promoters. CONCLUSIONS: We thus propose the responsiveness of expression to perturbations as a new way to define the dynamic status of genes, which brings new insights into mechanisms of regulation of gene expression and tissue specificity Inhibitors of cell signaling are known to support the pluripotent state of embryonic stem cells (Ying et al. 2008, Nature 453, 519-523, PMID: 18497825). To characterize the effect of inhibitors on the gene expression we treated B6R(5) mouse ES cells (C57BL/6 strain) with FGFR inhibitor PD173074 (100 uM), MEK inhibitor PD98059 (25 uM),and GSK-3 inhibitor BIO (2 uM) 24 hr after plating. Cells were grown without feeders on gelatin coated, 6-well plates, 100,000 cells/well (10^4 cells/cm2), in complete ES medium at 37 0C; 5% CO2. Medium was changed daily. Inhibitors dissolved in DMSO were added 24 hr after plating and cells were harvested 48 hr after treatment (72 hr after plating). Control cells were treated with DMSO. Keywords: cell type comparison design,reference design

ORGANISM(S): Mus musculus

PROVIDER: GSE19814 | GEO | 2011/02/11

SECONDARY ACCESSION(S): PRJNA129755

REPOSITORIES: GEO

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Project description:BACKGROUND: In addition to determining static states of gene expression (high vs. low), it is important to characterize their dynamic status. For example, genes with H3K27me3 chromatin marks are not only suppressed but also poised for activation. However, the responsiveness of genes to perturbations has never been studied systematically. To distinguish gene responses to specific factors from responsiveness in general, it is necessary to analyze gene expression profiles of cells responding to a large variety of disturbances, and such databases did not exist before. RESULTS: We estimated the responsiveness of all genes in mouse ES cells using our recently published database on expression change after controlled induction of 53 transcription factors (TFs) and other genes. Responsive genes (N = 4746), which were readily upregulated or downregulated depending on the kind of perturbation, mostly have regulatory functions and a propensity to become tissue-specific upon differentiation. Tissue-specific expression was evaluated on the basis of published (GNF) and our new data for 15 organs and tissues. Non-responsive genes (N = 9562), which did not change their expression much following any perturbation, were enriched in housekeeping functions. We found that TF-responsiveness in ES cells is the best predictor known for tissue-specificity in gene expression. Among genes with CpG islands, high responsiveness is associated with H3K27me3 chromatin marks, and low responsiveness is associated with H3K36me3 chromatin, stronger tri-methylation of H3K4, binding of E2F1, and GABP binding motifs in promoters. CONCLUSIONS: We thus propose the responsiveness of expression to perturbations as a new way to define the dynamic status of genes, which brings new insights into mechanisms of regulation of gene expression and tissue specificity Inhibitors of cell signaling are known to support the pluripotent state of embryonic stem cells (Ying et al. 2008, Nature 453, 519-523, PMID: 18497825). To characterize the effect of inhibitors on the gene expression we treated B6R(5) mouse ES cells (C57BL/6 strain) with FGFR inhibitor PD173074 (100 uM), MEK inhibitor PD98059 (25 uM),and GSK-3 inhibitor BIO (2 uM) 24 hr after plating. Cells were grown without feeders on gelatin coated, 6-well plates, 100,000 cells/well (10^4 cells/cm2), in complete ES medium at 37 0C; 5% CO2. Medium was changed daily. Inhibitors dissolved in DMSO were added 24 hr after plating and cells were harvested 48 hr after treatment (72 hr after plating). Control cells were treated with DMSO. Keywords: cell type comparison design,reference design Total RNA was isolated by TRIzol (Invitrogen) after 2 days. Cy3-CTP labeled sample targets were prepared with total RNA by Low RNA Input Fluorescent Linear Amplification Kit (Agilent). Cy5-CTP labeled reference target was Stratagene Universal Mouse Reference RNA.

Project description:Background: In addition to determining static states of gene expression (high vs. low), it is important to characterize their dynamic status. For example, genes with H3K27me3 chromatin marks are not only suppressed but also poised for activation. However, the responsiveness of genes to perturbations has never been studied systematically. To distinguish gene responses to specific factors from responsiveness in general, it is necessary to analyze gene expression profiles of cells responding to a large variety of disturbances, and such databases did not exist before. Results: We estimated the responsiveness of all genes in mouse ES cells using our recently published database on expression change after controlled induction of 53 transcription factors (TFs) and other genes. Responsive genes (/N/ = 4746), which were readily upregulated or downregulated depending on the kind of perturbation, mostly have regulatory functions and a propensity to become tissue-specific upon differentiation. Tissue-specific expression was evaluated on the basis of published (GNF) and our new data for 15 organs and tissues. Non-responsive genes (/N/ = 9562), which did not change their expression much following any perturbation, were enriched in housekeeping functions. We found that TF-responsiveness in ES cells is the best predictor known for tissue-specificity in gene expression. Among genes with CpG islands, high responsiveness is strongly associated with H3K27me3 chromatin marks, and low responsiveness is associated with H3K36me3 chromatin, binding of E2F1, and GABP binding motifs in promoters. Conclusions: We thus propose the responsiveness of expression to perturbations as a new way to define the dynamic status of genes, which brings new insights into mechanisms of regulation of gene expression and tissue specificity. This SuperSeries is composed of the following subset Series: GSE19806: Responsiveness of genes to manipulation of transcription factors in ES cells is associated with histone modifications and tissue specificity (1 of 2) GSE19814: Responsiveness of genes to manipulation of transcription factors in ES cells is associated with histone modifications and tissue specificity (2 of 2) Refer to individual Series

Project description:We present a microarray experiment with the same tet-inducible system but with multiple time points within 24 hr to capture early responses to Oct4 suppression. These data were analyzed together with published genome-wide ChIP data. We found that most tentative target genes (TTG) of Oct4 in ES cells were activated by Oct4 and only a few genes were suppressed. The same method was then applied to find target genes of Sox2 and Nanog based mostly on previously published data. Because the interaction between Oct4, Sox2, and Nanog is supported by immunoprecipitation, functional analysis, and co-localization of binding sites, we explored the relationships between their target genes. Keywords: time series design We used ZHBTc4 ES cells with a Tet-inducible Oct4 transgene. Cells were cultured for 2 passages on gelatin-coated plates in order to remove feeder cells and then transferred to gelatin-coated 6-well plates at the density of 1-2x105 cells/well and cultured in complete ES medium. Tetrocycline (TC) was added at 24 hr after cell plating, and then cells were harvested at 0hr (before adding TC), 3 hr, 6 hr, 12 hr, and 24 hr (2 replications each). RNA samples for later time points (24, 48, 72, 96, and 120 hr) were obtained from our earlier experiment with 3 replications. Two Nanog over-expressing clones were tested: (1) integrated transgene in MG1.19 parental cell line and (2) episomal transgene in EBRTcH3 parental line. EBRTcH3 and MG1.19 ESC lines were kind gifts of Dr. Hitoshi Niwa (RIKEN Center for Developmental Biology, Kobe, Japan). Both transgenic and parental cell lines were cultured for 2 passages on gelatin-coated plates and then transferred to gelatin-coated 6-well plates at the density of 1-2x105 cells/well and cultured for 3 days in 3 different conditions: (1) complete ES medium (see above); (2) complete medium without LIF, and (3) complete medium with 1 uM RA. Cells were cultured at 37 0C and 5% CO2 condition and the culture medium was changed daily. Over-expression of Nanog was evaluated by PCR. Total RNAs were extracted using TrizolTM (Invitrogen, USA) and Phase lock gelTM columns (Eppendorf/Brinkman) according to the manufacturer???s protocol. Total RNAs were precipitated with isopropanol, washed with 70% ethanol, and dissolved in DEPC-treated H2O. 2.5 ug of total RNA samples were labeled with Cy3-CTP using a Low RNA Input Fluorescent Linear Amplification Kit (Agilent, USA). A reference target (Cy5-CTP-labeled) was prepared from the Universal Mouse Reference (UMR) RNA (Stratagene, USA).

Project description:BACKGROUND: In addition to determining static states of gene expression (high vs. low), it is important to characterize their dynamic status. For example, genes with H3K27me3 chromatin marks are not only suppressed but also poised for activation. However, the responsiveness of genes to perturbations has never been studied systematically. To distinguish gene responses to specific factors from responsiveness in general, it is necessary to analyze gene expression profiles of cells responding to a large variety of disturbances, and such databases did not exist before. RESULTS: We estimated the responsiveness of all genes in mouse ES cells using our recently published database on expression change after controlled induction of 53 transcription factors (TFs) and other genes. Responsive genes (N = 4746), which were readily upregulated or downregulated depending on the kind of perturbation, mostly have regulatory functions and a propensity to become tissue-specific upon differentiation. Tissue-specific expression was evaluated on the basis of published (GNF) and our new data for 15 organs and tissues. Non-responsive genes (N = 9562), which did not change their expression much following any perturbation, were enriched in housekeeping functions. We found that TF-responsiveness in ES cells is the best predictor known for tissue-specificity in gene expression. Among genes with CpG islands, high responsiveness is associated with H3K27me3 chromatin marks, and low responsiveness is associated with H3K36me3 chromatin, stronger tri-methylation of H3K4, binding of E2F1, and GABP binding motifs in promoters. CONCLUSIONS: We thus propose the responsiveness of expression to perturbations as a new way to define the dynamic status of genes, which brings new insights into mechanisms of regulation of gene expression and tissue specificity. Total RNA was isolated by TRIzol (Invitrogen) after 2 days. Cy3-CTP labeled sample targets were prepared with total RNA by Low RNA Input Fluorescent Linear Amplification Kit (Agilent). Cy5-CTP labeled reference target was produced from mixture of Stratagene Universal Mouse Reference RNA and MC1 cells RNA. Most samples were collected in 2 replications taken from different animals, except bone and fat, which had only one replication.

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Responsiveness of genes to manipulation of transcription factors in ES cells is associated with histone modifications and tissue specificity (2 of 2)

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