E-GEOD-3196 - Transcription profiling of mouse embryonic fibroblasts C57Bl/6x129/Sv F2 e14.5 embryos vs controls grown in hypoxic or normoxic conditions
Submitted on 24 August 2005, released on 15 December 2007, last updated on 27 March 2012
The CH1 protein interaction domain of the transcriptional coactivators p300 and CBP is thought to interact with HIF-1alpha and this interaction is thought to be critical to the expression of HIF-1alpha target genes in response to hypoxia. To test the requirement of the CH1 domain for gene expression in response to hypoxia, rimary mouse embryonic fibroblasts (MEFs) were generated from C57Bl/6x129/Sv F2 e14.5 embryos that contain a deletion in the CH1 domain of three of four alleles of CBP and p300. The remaining allele of p300 or CBP was a conditional knock out allele. Control MEFs with only a single conditional knockout allele of p300 or CBP were also generated. At passage 3 MEFs were infected with Cre Adenovirus and grown until they had expanded at least 100 fold. Subconfluent MEFs were treated with 21% oxygen (normoxia) or 0.1% oxygen (hypoxia) with 5% carbon dioxide at 37 C in a humid chamber for 6hrs. At the start of treatment, medium was removed and replaced with medium (DMEM+10% FBS+pen-strep+ l-glu) that had been preequilibrated overnight in normoxia or hypoxia as appropriate. Immediately after treatment, cells were lysed in Trizol for RNA extraction. 12 samples; 4 genotypes [CBP+/flox (flox1), p300 +/flox (flox2), CBP CH1/flox;p300 CH1/CH1 (triCH1flox1),CBP CH1/CH1;p300 CH1/flox (triCH1flox2)] , 2 treatments (normoxia and hypoxia).
transcription profiling by array, growth condition, individual genetic characteristics, stimulus or stress
Two transactivation mechanisms cooperate for the bulk of HIF-1-responsive gene expression. Lawryn H Kasper, Fayçal Boussouar, Kelli Boyd, Wu Xu, Michelle Biesen, Jerold Rehg, Troy A Baudino, John L Cleveland, Paul K Brindle. EMBO J 24(22):3846-58 (2005)