Project description:Stat5+/- mice were bred into the C57BL/6 background. Stat5+/- mice were intercrossed and mouse embryonic fibroblasts (MEFs) were isolated from 12.5-13.5-day WT or Stat5-/- fetuses. The retroviral-expression vector carrying a wild-type Stat5A gene based on an MSCV-IRES-GFP backbone (gift from Richard Moriggl, Ludwig-Boltzmann Institute, Vienna, Austria) was infected into Stat5-/- MEFs. FACS was used to select GFP+ cells. After 5 hours starvation in serum free medium with 0.1% of BSA, MEFs were treated with growth hormone for 2 hours. Total cellular RNA from each group of the MEFs was extracted with TRIzol reagent (Invitrogen) according to the manufacturer's instructions. Microarray analyses were performed using Affymetrix Mouse Genome 430 2.0 GeneChips (Affymetrix, Santa Clara, CA) (six groups, biological replicates for each group). Expression values were determined with GeneChip Operating Software (GCOS) v1.1.1 software. RMA signals were summarized using GeneSpring GX 10.0.1 (Agilent) and normalized by quantile normalization. All data analysis was performed with GeneSpring software GX 10.01.
Project description:Stat5+/- mice were bred into the C57BL/6 background. Stat5+/- mice were intercrossed and mouse embryonic fibroblasts (MEFs) were isolated from 12.5-13.5-day WT or Stat5-/- fetuses. The retroviral-expression vector carrying a DNA binding domain mutant Stat5A (E437/E438?AA) or tyrosine-phosphorylated Stat5A Y694F mutation based on an MSCV-IRES-GFP backbone (gift from Richard Moriggl, Ludwig-Boltzmann Institute, Vienna, Austria) was infected into Stat5-/- MEFs. FACS was used to select GFP+ cells. After 5 hours starvation in serum free medium with 0.1% of BSA, MEFs were treated with growth hormone for 2 hours. Total cellular RNA from each group of the MEFs was extracted with TRIzol reagent (Invitrogen) according to the manufacturer's instructions. Microarray analyses were performed using Affymetrix Mouse Genome 430 2.0 GeneChips (Affymetrix, Santa Clara, CA) (four groups, biological replicates for each group). Expression values were determined with GeneChip Operating Software (GCOS) v1.1.1 software. RMA signals were summarized using GeneSpring GX 10.0.1 (Agilent) and normalized by quantile normalization. All data analysis was performed with GeneSpring software GX 10.01.
Project description:Histone acetyltransferases (HATs) GCN5/PCAF and CBP/p300 are transcription coactivators. However, how these HATs regulate ligand-induced nuclear receptor target gene expression remains unclear. Here we show in mouse embryonic fibroblasts (MEFs), deletion of GCN5/PCAF specifically eliminates acetylation on H3K9 (H3K9Ac) while deletion of CBP/p300 selectively reduces acetylation on H3K18 and H3K27 (H3K18/27Ac). Treating MEFs with a specific ligand for nuclear receptor PPARdelta induces sequential increases of H3K18/27Ac and H3K9Ac on the promoter of PPARdelta target gene Angptl4, which correlates with a robust ligand-induced Angptl4 expression. Inhibiting transcription elongation blocks ligand-induced H3K9Ac but not H3K18/27Ac on Angptl4 promoter. Finally, we show CBP/p300 and their HAT activities are required, while GCN5/PCAF and H3K9Ac are dispensable, for ligand-induced PPARdelta target gene expression in MEFs. These results highlight the substrate and site specificities of HATs in cells, and suggest that GCN5/PCAF- and CBP/p300-mediated histone acetylations play distinct roles in regulating ligand-induced nuclear receptor target gene expression. PCAF and GCN5 have some redundant function. To identify PCAF/GCN5-regulated genes, immortalized MEFs with PCAF knockout and GCN5 conditional knockout were infected with retroviruses expressing either Cre recombinase or vector alone. We prepared duplicated RNAs from either vector or Cre infected cells (PCAF-/-;GCN5+/- or PCAF-/-;GCN5+/-) and RNAs from either Vector or Cre infected the other independently immortalized cells for 6 affymetrix microarray.
Project description:Stat5+/- mice were bred into the C57BL/6 background. Stat5+/- mice were intercrossed and mouse embryonic fibroblasts (MEFs) were isolated from 12.5-13.5-day WT or Stat5-/- fetuses. The retroviral-expression vector carrying a wild-type Stat5A gene based on an MSCV-IRES-GFP backbone (gift from Richard Moriggl, Ludwig-Boltzmann Institute, Vienna, Austria) was infected into Stat5-/- MEFs. FACS was used to select GFP+ cells. After 5 hours starvation in serum free medium with 0.1% of BSA, MEFs were treated with growth hormone for 2 hours. Total cellular RNA from each group of the MEFs was extracted with TRIzol reagent (Invitrogen) according to the manufacturer's instructions. Microarray analyses were performed using Affymetrix Mouse Genome 430 2.0 GeneChips (Affymetrix, Santa Clara, CA) (six groups, biological replicates for each group). Expression values were determined with GeneChip Operating Software (GCOS) v1.1.1 software. RMA signals were summarized using GeneSpring GX 10.0.1 (Agilent) and normalized by quantile normalization. All data analysis was performed with GeneSpring software GX 10.01. Total 6 groups (S5, S5-GH, WT, WT_GH, KO, KO_GH), biological duplicate replication for each group