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Two transactivation mechanisms are responsible for the bulk of HIF-1alpha-responsive gene expression

ABSTRACT: The C-terminal activation domain (C-TAD) of the hypoxia-inducible transcription factors HIF-1? and HIF-2? binds the CH1 domains of the related transcriptional coactivators CREB-binding protein (CBP) and p300, an oxygen-regulated interaction thought to be highly essential for hypoxia-responsive transcription. The role of the CH1 domain in vivo is unknown, however. We created mutant mice bearing deletions in the CH1 domains (?CH1) of CBP and p300 that abrogate their interactions with the C-TAD, revealing that the CH1 domains of CBP and p300 are genetically non-redundant and indispensable for C-TAD transactivation function. Surprisingly, the CH1 domain was only required for an average of ~35-50% of global HIF-1?-responsive gene expression, whereas another HIF-transactivation mechanism that is sensitive to the histone deacetylase inhibitor trichostatin A (TSAS) accounts for ~70%. Both pathways are required for greater than 90% of the response for some target genes. Our findings suggest that a novel functional interaction between the protein acetylases CBP and p300, and deacetylases, is essential for nearly all HIF-responsive transcription. Keywords: genetic modification, dose response

ORGANISM(S): Mus musculus

PROVIDER: GSE3318 | GEO | 2005/10/24

SECONDARY ACCESSION(S): PRJNA93353

REPOSITORIES: GEO

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Transcription profiling of mouse embryonic fibroblasts from embryos with ?CH1 mutation in p300 and/or CBP atreated with hypoxia or dipyridyl (a hypoxia mimetic) and trichostatin A (a histone deacetylase inhibitor) reveals two transactivation mechanisms are responsible for the bulk of HIF-1alpha-responsive gene expression

Project description:The C-terminal activation domain (C-TAD) of the hypoxia-inducible transcription factors HIF-1? and HIF-2? binds the CH1 domains of the related transcriptional coactivators CREB-binding protein (CBP) and p300, an oxygen-regulated interaction thought to be highly essential for hypoxia-responsive transcription. The role of the CH1 domain in vivo is unknown, however. We created mutant mice bearing deletions in the CH1 domains (?CH1) of CBP and p300 that abrogate their interactions with the C-TAD, revealing that the CH1 domains of CBP and p300 are genetically non-redundant and indispensable for C-TAD transactivation function. Surprisingly, the CH1 domain was only required for an average of ~35-50% of global HIF-1?-responsive gene expression, whereas another HIF-transactivation mechanism that is sensitive to the histone deacetylase inhibitor trichostatin A (TSAS) accounts for ~70%. Both pathways are required for greater than 90% of the response for some target genes. Our findings suggest that a novel functional interaction between the protein acetylases CBP and p300, and deacetylases, is essential for nearly all HIF-responsive transcription. Experiment Overall Design: Three separate affymetrix experiments using mouse embryonic fibroblasts derived from embryos bearing the ?CH1 mutation in p300 and/or CBP and treated with hypoxia or combinations of dipyridyl (a hypoxia mimetic) and trichostatin A (a histone deacetylase inhibitor) are described (GSE3195, GSE3196 and GSE3296). Samples are not directly comparable between experiments because of differences in experiment design and Affymetrix chips used.

2008-06-12 | E-GEOD-3318 | biostudies-arrayexpress

Transcription profiling of mouse embryonic fibroblasts normoxia/hypoxia treatments with mutations in the CH1 domains of p300 and CBP

Project description:The CH1 (TAZ) domain of the transcriptional coactivators p300 and CBP has been reported to interact with the transcription factor HIF-1alpha and this interaction is thought to be critical for HIF-1alpha target gene expression in response to hypoxia. To determine the requirement for the CH1 domain in hypoxia-responsive gene expression, primary mouse embryonic fibroblasts (MEFs) were generated from e14.5 C57B/6x129/Sv F2 embryos that were either wildtype or bore deletion mutations in the CH1 protein binding domains of both alleles of p300 and one allele of CBP (tri_CH1). 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. Experiment Overall Design: 4 samples: two genotypes (1 control, 1 mutant); two treatments (normoxia, hypoxia).

2008-06-12 | E-GEOD-3195 | biostudies-arrayexpress

Transcription profiling of mouse embryonic fibroblasts C57Bl/6x129/Sv F2 e14.5 embryos vs controls grown in hypoxic or normoxic conditions

Project description: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).

2007-12-15 | E-GEOD-3196 | biostudies-arrayexpress

Gene expression under normoxia/hypoxia in mouse embryonic fibroblasts with mutations in the CH1 domains of p300 and CBP

2005-10-24 | GSE3195 | GEO

Transcription profiling of primary mouse embryonic fibroblasts (MEFs) 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

Project description: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. Trichostatin A (TSA), an inhibitor of histone deacetylases, has been reported to repress the expression of HIF-1alpha target genes. To test the requirement of the CH1 domain and TSA for gene expression in response to dipyridyl (a hypoxia mimetic), primary 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 ethanol vehicle or 100ng/ml TSA with 5% carbon dioxide at 37 C in a humid chamber for 30 min., followed by ethanol vehicle or 100 umdipyridyl (DP) for an additional 3hrs. Immediately after treatment, cells were lysed in Trizol for RNA extraction.

2007-07-07 | E-GEOD-3296 | biostudies-arrayexpress

Gene expression in hypoxic MEFs having only p300 and CBP with deleted CH1 domains

2005-10-24 | GSE3196 | GEO

Gene expression in MEFs in response to treatment with dipyridyl and trichostatin A

Project description: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. Trichostatin A (TSA), an inhibitor of histone deacetylases, has been reported to repress the expression of HIF-1alpha target genes. To test the requirement of the CH1 domain and TSA for gene expression in response to dipyridyl (a hypoxia mimetic), primary 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 ethanol vehicle or 100ng/ml TSA with 5% carbon dioxide at 37 C in a humid chamber for 30 min., followed by ethanol vehicle or 100 umdipyridyl (DP) for an additional 3hrs. Immediately after treatment, cells were lysed in Trizol for RNA extraction. Keywords: genetic modification, dose response

2005-10-24 | GSE3296 | GEO

Expression data from wild type and CBP/p300 null MEFs +/- 3hr treatment with 100uM dipyridyl (DP)

Project description:Genome-wide distribution of histone H3K18 and H3K27 acetyltransferases, Crebbp (CBP) and Ep300 (p300), is used to map enhancers and promoters, but whether these elements functionally require CBP/p300 remains largely uncertain. We investigated this relationship by comparing genomic CBP recruitment with gene expression in wild type and CBP/p300 double-knockout fibroblasts. ChIP-seq revealed nearby CBP recruitment for 20 percent of constitutively expressed genes, but surprisingly, three-quarters of these were unaffected or slightly activated by CBP/p300 deletion. Computationally defined enhancer-promoter-units (EPUs) having a CBP peak within two kilobases of the enhancer-like element provided better predictive value, with CBP/p300 deletion attenuating expression of 40 percent of such EPU assigned constitutively expressed genes. We next examined signaling-responsive (Hypoxia Inducible Factor) gene expression and CBP recruitment, and found that 97 percent of inducible genes were within 50 kilobases of an inducible CBP peak, and 70 percent of these required CBP/p300 for full inducible expression. Unexpectedly however, most inducible CBP peaks occurred near signal-nonresponsive genes. 12 samples, 3 each wild type and CBP/p300 null treated for 3hrs with 100uM dipyridyl orethanol vehicle.

2014-09-29 | E-GEOD-54452 | biostudies-arrayexpress

CREBBP (CBP) ChIP-seq in wild type and CBP null MEFs +/- dipyridyl

Project description:Genome-wide distribution of histone H3K18 and H3K27 acetyltransferases, Crebbp (CBP) and Ep300 (p300), is used to map enhancers and promoters, but whether these elements functionally require CBP/p300 remains largely uncertain. We investigated this relationship by comparing genomic CBP recruitment with gene expression in wild type and CBP/p300 double-knockout fibroblasts. ChIP-seq revealed nearby CBP recruitment for 20 percent of constitutively expressed genes, but surprisingly, three-quarters of these were unaffected or slightly activated by CBP/p300 deletion. Computationally defined enhancer-promoter-units (EPUs) having a CBP peak within two kilobases of the enhancer-like element provided better predictive value, with CBP/p300 deletion attenuating expression of 40 percent of such EPU assigned constitutively expressed genes. We next examined signaling-responsive (Hypoxia Inducible Factor) gene expression and CBP recruitment, and found that 97 percent of inducible genes were within 50 kilobases of an inducible CBP peak, and 70 percent of these required CBP/p300 for full inducible expression. Unexpectedly however, most inducible CBP peaks occurred near signal-nonresponsive genes. eight samples total; Two wild type and two CBP null primary mouse embryonic fibroblast (MEF) lines, each treated with 100uM 2,2-dipyridyl or ethanol vehicle for 2 hours

2014-09-29 | E-GEOD-54453 | biostudies-arrayexpress

Regulation of p300 acetyltransferase activity by the TAZ2 domain [ATAC-seq]

Project description:The E1A binding protein P300 (EP300, also known as p300, lysine acetyltransferase 3B or KAT3B) and its close paralogue CREB-binding protein (CREBBP, aka CBP or KAT3A) possess intrinsic histone acetyltransferase (HAT) activity that can act on both histone and non-histone proteins. P300 and CBP are composed of multiple conserved protein domains, many of which are in proximity to the HAT domain modulating its catalytic activity. Here we show that the TAZ2 domain regulates the intrinsic catalytic activity of p300 in vitro and histone acetylation and chromatin accessibility in cells. Our study extends the knowledge of p300 self-regulation and provides new therapeutic stratagies for human cancers with corresponding p300/CBP mutations.

2023-08-07 | GSE193647 | GEO

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