Two transactivation mechanisms cooperate for the bulk of HIF-1-responsive gene expression

Abstract

The C-terminal activation domain (C-TAD) of the hypoxia-inducible transcription factors HIF-1alpha and HIF-2alpha 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 (DeltaCH1) 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 approximately 35-50% of global HIF-1-responsive gene expression, whereas another HIF transactivation mechanism that is sensitive to the histone deacetylase inhibitor trichostatin A (TSA(S)) accounts for approximately 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.

Figure 1

The CH1 domain of CBP is conserved and required for normal development. (A) CH1 domains of CBP and p300 span two exons and are conserved in mouse (m), human (h), Caenorhabditis elegans (Ce), and Drosophila (Dm). ΔCH1 deletion mutation, exon boundary, conserved residues, and relative amino-acid positions are indicated. (B) Grossly, CBP^ΔCH1/ΔCH1 E18.5 lungs are small compared to WT and p300^ΔCH1/ΔCH1. (C–E) Microscopically, lungs from CBP^ΔCH1/ΔCH1 E18.5 embryos have thicker interstitial septa and decreased alveolar airspace. (F, G) Some CBP^ΔCH1/ΔCH1 E18.5 embryos have cleft palate (arrows).

Figure 2

ΔCH1 mutation does not affect other domains or functions of CBP and p300. (A) Western blot of CBP and p300 showing normal protein levels in CBP^ΔCH1/ΔCH1 and p300^ΔCH1/ΔCH1 MEFs. (B, C) Immunoprecipitation/HAT assay showing that HAT activity in CBP^ΔCH1/ΔCH1 (B) and p300^ΔCH1/ΔCH1 (C) MEFs is comparable to WT MEFs (mean±s.e.m., N=2). (D–F) Transient transfection assays showing that Gal-HIF-1α function is reduced in MEFs with multiple ΔCH1 alleles (mean±s.e.m., N=3) (D), and can be rescued by WT CBP (E, F) or p300 (F), but not by CBPΔCH1 (E) (mean±s.e.m., N=2–4). (G, H) Transactivation by factors utilizing other domains of p300 and CBP or other coactivators is unimpaired (mean±s.d., N=4).

Figure 3

ΔCH1 mutation attenuates the expression of many endogenous hypoxia-inducible genes, but not Eμ-Myc-induced B-cell lymphomagenesis. (A, B) Affymetrix microarray analysis of hypoxia-inducible genes (A) (⩾3-fold induced by hypoxia in WT MEFs, probe sets scored as present in WT hypoxia sample) and non-hypoxia-responsive control genes (B) (±1% hypoxia/normoxia signal ratio in WT MEFs; probe sets scored as present in WT hypoxia and normoxia samples) in WT and CBP^+/ΔCH1;p300^ΔCH1/ΔCH1 MEFs. Each symbol represents hypoxia-dependent expression level for an Affymetrix probe set; note degree of data scatter and slope of the best-fit line. (C–E) qRT–PCR analysis of physiologically important hypoxia-inducible genes Pgf (C), Vegfa (D), and Slc2a1 (Glut1) (E) in WT, CBP^ΔCH1/ΔCH1;p300^+/ΔCH1, and CBP^+/ΔCH1;p300^ΔCH1/ΔCH1 triple-ΔCH1 MEFs, normalized to β-actin mRNA (mean±s.e.m., N=6–34, data from 2–6 independent MEF lines for each genotype). Survival curves for C57BL/6 × 129 F1 hybrid (F) and C57BL/6 Eμ-Myc mice (G) with or without ΔCH1 mutant alleles (indicated) are shown.

Figure 4

The CH1 domain is absolutely required for C-TAD transactivation function but is less essential for HIF target genes. (A, B) Deletion of CBP^flox and (A) p300^flox (B) in MEFs following infection with Cre-expressing adenovirus. MEF genotypes, days post infection, and allele-specific products derived from semiquantitative PCR of genomic DNA are indicated. (C, D) Comparable growth curves for tri-ΔCH1/flox and flox MEFs with or without Cre-adenovirus (Ad Cre) infection. (E, F) Normalized activity of Gal-HIF-1α, Gal-HIF-2α, and Gal-Myb in transiently transfected Δflox and tri-ΔCH1/Δflox MEFs (mean±s.e.m., N=3). (G, H) WT CBP contributes marginally to residual hypoxia-inducible gene expression in triple-ΔCH1 MEFs. qRT–PCR analysis of control flox and tri-ΔCH1/flox MEFs±Cre-adenovirus infection. Slc2a1 (G) and Pfkfb3 (H), tested under normoxia and hypoxia, normalized to β-actin mRNA (mean±s.e.m., N=2–3).

Figure 5

Markedly reduced recruitment of CBPΔCH1 and p300ΔCH1 to HIF-binding sites does not strongly correlate with HIF-responsive transcription. (A–C) Quantitative ChIP assays of Slc2a1, Pfkfb3, and Hig1, using WT, CBP^ΔCH1/ΔCH1, and p300^ΔCH1/ΔCH1 MEFs treated for 2 h with ethanol vehicle (EtOH) or DP/MG132/ALLN (DP) (mean±s.e.m., N=3 independent experiments). Control (NRS) and specific (anti-CBP, anti-p300) immunoprecipitation antisera are indicated. DP-dependent ChIP signal was determined by subtracting the EtOH signal from the DP signal after normalizing to the input DNA signal. (D–F) qRT–PCR analysis of HIF-target gene expression in Δflox #2 and tri-ΔCH1/Δflox #2 MEFs after 6 h DP/MG132/ALLN, normalized to β-actin mRNA (mean±s.e.m., N=3).

Figure 6

TSA and the ΔCH1 mutation have distinct but overlapping effects on the transcription of HIF-responsive genes. (A, B) qRT–PCR analysis of HIF-target gene expression in Δflox and tri-ΔCH1/Δflox MEFs treated with EtOH or TSA, followed by 3 h treatment with EtOH vehicle (EtOH) or DP, normalized to β-actin mRNA (Δflox #1 and tri-ΔCH1/Δflox #1, N=1; Δflox #2 and tri-ΔCH1/Δflox #2, mean±s.e.m., N=2). (C, D) qRT–PCR analysis of cDNA reverse transcribed from primary unspliced RNA transcripts, normalized to β-actin primary unspliced RNA, using PCR primer pairs that span an exon–intron boundary. MEFs treated with EtOH or TSA, and then for 3 h with EtOH or DP are shown. RNA was treated with DNase before reverse transcription. As a check for genomic DNA contamination, samples with no reverse transcriptase added (No RT) were analyzed. (E) Hierarchical clustering analysis of HIF-target gene probe sets in Affymetrix microarrays that showed at least a 1.5-fold induction by DP treatment and were scored as present in both Δflox MEFs treated with DP alone. MEFs of indicated genotypes were treated with EtOH or TSA, followed by a 3 h treatment with EtOH or DP. Microarray signals were normalized by Z-score transformation; red indicates induction by DP and green noninduced levels of expression. Probe sets representing genes of interest are indicated. (F) Average effect on global HIF-responsive transcription of the ΔCH1 mutation and TSA treatment alone and in combination (TSA+ΔCH1) on DP-induced signals (calculated by subtracting the appropriate control signal from the DP-treated signal). Data as in panel E (mean±s.d.).