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• W2080299092 abstract "Article20 October 2005free access Two transactivation mechanisms cooperate for the bulk of HIF-1-responsive gene expression Lawryn H Kasper Lawryn H Kasper Department of Biochemistry, St Jude Children's Research Hospital, Memphis, TN, USA Search for more papers by this author Fayçal Boussouar Fayçal Boussouar Department of Biochemistry, St Jude Children's Research Hospital, Memphis, TN, USA Search for more papers by this author Kelli Boyd Kelli Boyd Department of Pathology, St Jude Children's Research Hospital, Memphis, TN, USA Search for more papers by this author Wu Xu Wu Xu Department of Biochemistry, St Jude Children's Research Hospital, Memphis, TN, USA Search for more papers by this author Michelle Biesen Michelle Biesen Department of Biochemistry, St Jude Children's Research Hospital, Memphis, TN, USA Search for more papers by this author Jerold Rehg Jerold Rehg Department of Pathology, St Jude Children's Research Hospital, Memphis, TN, USA Search for more papers by this author Troy A Baudino Troy A Baudino Department of Biochemistry, St Jude Children's Research Hospital, Memphis, TN, USAPresent address: Department of Cell and Developmental Biology, University of South Carolina, Columbia, SC 29209, USA Search for more papers by this author John L Cleveland John L Cleveland Department of Biochemistry, St Jude Children's Research Hospital, Memphis, TN, USA Search for more papers by this author Paul K Brindle Corresponding Author Paul K Brindle Department of Biochemistry, St Jude Children's Research Hospital, Memphis, TN, USA Search for more papers by this author Lawryn H Kasper Lawryn H Kasper Department of Biochemistry, St Jude Children's Research Hospital, Memphis, TN, USA Search for more papers by this author Fayçal Boussouar Fayçal Boussouar Department of Biochemistry, St Jude Children's Research Hospital, Memphis, TN, USA Search for more papers by this author Kelli Boyd Kelli Boyd Department of Pathology, St Jude Children's Research Hospital, Memphis, TN, USA Search for more papers by this author Wu Xu Wu Xu Department of Biochemistry, St Jude Children's Research Hospital, Memphis, TN, USA Search for more papers by this author Michelle Biesen Michelle Biesen Department of Biochemistry, St Jude Children's Research Hospital, Memphis, TN, USA Search for more papers by this author Jerold Rehg Jerold Rehg Department of Pathology, St Jude Children's Research Hospital, Memphis, TN, USA Search for more papers by this author Troy A Baudino Troy A Baudino Department of Biochemistry, St Jude Children's Research Hospital, Memphis, TN, USAPresent address: Department of Cell and Developmental Biology, University of South Carolina, Columbia, SC 29209, USA Search for more papers by this author John L Cleveland John L Cleveland Department of Biochemistry, St Jude Children's Research Hospital, Memphis, TN, USA Search for more papers by this author Paul K Brindle Corresponding Author Paul K Brindle Department of Biochemistry, St Jude Children's Research Hospital, Memphis, TN, USA Search for more papers by this author Author Information Lawryn H Kasper1,‡, Fayçal Boussouar1,‡, Kelli Boyd2, Wu Xu1, Michelle Biesen1, Jerold Rehg2, Troy A Baudino1, John L Cleveland1 and Paul K Brindle 1 1Department of Biochemistry, St Jude Children's Research Hospital, Memphis, TN, USA 2Department of Pathology, St Jude Children's Research Hospital, Memphis, TN, USA ‡These authors contributed equally to this study *Corresponding author. Department of Biochemistry, St Jude Children's Research Hospital, 332 N Lauderdale, Memphis, TN 38105, USA. Tel.: +1 901 495 2522; Fax: +1 901 525 8025; E-mail: [email protected] The EMBO Journal (2005)24:3846-3858https://doi.org/10.1038/sj.emboj.7600846 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The C-terminal activation domain (C-TAD) of the hypoxia-inducible transcription factors HIF-1α and HIF-2α 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 (ΔCH1) 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. Introduction The closely related HIF-1α and HIF-2α are crucial for the physiological adaptation to hypoxia that requires the increased expression of genes involved in glucose metabolism, angiogenesis, hematopoiesis, cell survival, invasion, and vascular tone (Giaccia et al, 2003, 2004; Semenza, 2003). The mechanism(s) enabling HIF-dependent stimulation of transcription in vivo is uncertain but is thought to chiefly involve the physical interaction of the C-terminal activation domain (C-TAD) of HIF-1α and HIF-2α with the CH1 (C/H1, TAZ1) domain of CREB-binding protein (CBP; Crebbp) and the closely related p300 (Ep300) (Dames et al, 2002; Freedman et al, 2002; Semenza, 2002). The NMR co-structures of the CH1 domains of CBP and p300 with the C-TAD of HIF-1α have revealed the specificity of this high-affinity (Kd∼7 nM) interaction, including how oxygen-dependent hydroxylation of HIF-1α Asn803 inhibits complex formation with CH1 (Dames et al, 2002; Freedman et al, 2002), which is believed to be important for inhibiting HIF activity under normoxia (Bruick, 2003; Giaccia et al, 2004; Poellinger and Johnson, 2004). CBP and p300 are required for normal development (Tanaka et al, 2000; Alarcon et al, 2004; Kalkhoven, 2004; Kang-Decker et al, 2004; Korzus et al, 2004; Zhou et al, 2004; Wood et al, 2005), consistent with observations that they interact with ∼10% of the ∼2000 mammalian transcriptional regulatory proteins (PB, submitted) (Messina et al, 2004). CBP and p300 possess protein and histone acetyltransferase (PAT, HAT) activities, but it is largely unknown what roles their transcription factor-binding domains play in vivo (Goodman and Smolik, 2000). The CH1 domain is a zinc-containing structure that is highly conserved between CBP and p300, as well as in man, mice, nematodes, and flies (Figure 1A) (Goodman and Smolik, 2000). CH1 has transactivation function when fused to a heterologous DNA-binding domain, consistent with it having a role in the proposed adaptor functions of CBP and p300 (Newton et al, 2000; Zanger et al, 2001), but its main function is thought to involve binding to specific transcription factors in the recruitment of CBP and p300 to promoters. Indeed, 26 of the 37 transcriptional regulators that bind the CH1 region are essential in mice (Supplementary Table S1), but particular interest has focused on HIF-1α and HIF-2α because of the importance of HIF-1 and HIF-2 (a heterodimeric complex of ARNT with HIF-1α or HIF-2α, respectively) in mediating the transcriptional response to hypoxia. 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, CBPΔCH1/ΔCH1 E18.5 lungs are small compared to WT and p300ΔCH1/ΔCH1. (C–E) Microscopically, lungs from CBPΔCH1/ΔCH1 E18.5 embryos have thicker interstitial septa and decreased alveolar airspace. (F, G) Some CBPΔCH1/ΔCH1 E18.5 embryos have cleft palate (arrows). Download figure Download PowerPoint Results Genetically non-redundant roles for the CH1 domains of CBP and p300 in vivo To test the requirements for the CH1 domain in vivo, we introduced the ΔCH1 mutation into one of the two exons encoding CH1 in CBP and p300 by homologous recombination in mouse embryonic stem (ES) cells (Figure 1A and Supplementary Figure S1). The deletions are essentially equivalent in CBP and p300 and remove more than 50% of the 88 largely conserved residues of CH1 (aa 329–379 deleted for p300, and aa 342–393 for CBP; an NheI site encoding a flexible Ala–Ser linker was inserted in-frame to facilitate identification of the mutant alleles) (Figure 1A). The ΔCH1 mutation removes critical components of the domain, including two of the four α-helices, five Cys and His residues that bind to two of the three zinc ions in the structure, eight of 14 residues that comprise the conserved hydrophobic core, and much of the binding surface for the HIF-1α C-TAD, including three residues (CBP Asp346, Lys349, Ile353) that contact Asn803 (Dames et al, 2002; Freedman et al, 2002). The structural integrity of CH1 is highly dependent on the hydrophobic core (e.g. p300 Leu344, Leu345), and bound zinc, which strongly indicates that the ΔCH1 mutation will block the interaction with most, if not all, CH1-binding partners (Newton et al, 2000; Gu et al, 2001; Matt et al, 2004). Essentially normal adult mice heterozygous for p300ΔCH1 or CBPΔCH1 were generated at near the expected Mendelian frequency (there was a modest ∼30% decrease in the number of CBP+/ΔCH1 mice), indicating that ΔCH1 is not an overt dominant-negative mutation. Homozygous CBPΔCH1/ΔCH1 mice on a mixed 129 and C57BL/6 strain background typically died shortly after birth (one runted homozygous mutant survived to adulthood out of 651 mice derived from mating CBP+/ΔCH1 mice). In contrast, p300ΔCH1/ΔCH1 adult mice were overtly normal, although they were produced at about 50% of the expected frequency. Analysis of day 0.5 neonates and day 18.5 embryos revealed that CBPΔCH1/ΔCH1 and p300ΔCH1/ΔCH1 mice were present nearer the expected frequency. The rare survival of CBPΔCH1/ΔCH1 mice past the neonatal stage suggested that animals with hybrid vigor would have improved viability. Indeed, F1 hybrid CBPΔCH1/ΔCH1 offspring derived from interbreeding C57BL/6 and 129 congenic CBP+/ΔCH1 mice had markedly enhanced survival to adulthood (∼25% of the expected frequency), but were growth retarded and had craniofacial defects (to be described elsewhere). F1 hybrid CBP+/ΔCH1;p300+/ΔCH1 compound heterozygotes were also smaller than wild-type (WT) littermates and some had craniofacial defects (incomplete penetrance), indicating that the p300 CH1 domain has a role in normal development in the context of the CBPΔCH1 mutation (not shown). Craniofacial abnormalities are a hallmark of Rubinstein–Taybi syndrome, where CBP, and to a lesser degree p300, monoallelic mutations have been identified (Roelfsema et al, 2005); thus, our results suggest that CBP and p300 CH1 domain insufficiency is an important determinant in this human disease. Together, these results demonstrate that the CH1 domains of CBP and p300 are genetically non-redundant, with the CBP CH1 domain being especially important for normal mouse development. Examination of day 18.5 embryos of mixed background revealed that CBPΔCH1/ΔCH1 embryos had lung defects not seen in p300ΔCH1/ΔCH1 embryos. CBPΔCH1/ΔCH1 lungs were smaller than WT and p300ΔCH1/ΔCH1 as a percentage of total body weight (4.1±0.4% for WT, 4.2±0.2 for p300ΔCH1/ΔCH1, and 3.1±0.1 for CBPΔCH1/ΔCH1, N=5–6, P=0.0014, t-test; Figure 1B). CBPΔCH1/ΔCH1 (Figure 1D) lungs had thickened interstitial septa and decreased alveolar air space, compared to WT (Figure 1C) and p300ΔCH1/ΔCH1 (Figure 1E) embryos. Some of the CBP+/ΔCH1 embryos also displayed a similar lung phenotype, possibly explaining the partially penetrant lethality (not shown). Cell proliferation, determined by immunostaining for Ki67, was significantly reduced in CBPΔCH1/ΔCH1 lungs, consistent with a delay in lung maturation, but not in p300ΔCH1/ΔCH1 lungs (25.0±0.7% for WT, 23.9±1.7 for p300ΔCH1/ΔCH1, 16.9±1.0 for CBPΔCH1/ΔCH1, P=1.5 × 10−7, N=5–6, t-test). HIF-2α has been implicated in lung development in mice, but we did not observe a synergistic genetic interaction in mice doubly heterozygous for an HIF-2α knockout allele and the CBPΔCH1 or p300ΔCH1 mutation (not shown) (Compernolle et al, 2002). Additionally, ∼50% of CBPΔCH1/ΔCH1 newborn mice had cleft palate (Figure 1F and G, arrows), indicative of a role for the CBP CH1 domain in palate morphogenesis. The relative expression of CBP and p300 does not obviously account for the differential effects of the ΔCH1 mutation, as each is expressed ubiquitously and at roughly comparable levels in the embryonic palate and lung (Naltner et al, 2000; Warner et al, 2002). CBPΔCH1/ΔCH1;p300ΔCH1/ΔCH1 compound homozygous mutant mice are not viable, as no such embryos were observed at day 14.5 of gestation (E14.5). However, CBP+/ΔCH1;p300ΔCH1/ΔCH1 and CBPΔCH1/ΔCH1;p300+/ΔCH1 viable embryos that retain one WT CBP or p300 allele, respectively, could be recovered at E14.5. These ‘triple-ΔCH1’ embryos yielded primary mouse embryonic fibroblasts (MEFs) with growth and morphological characteristics comparable to WT MEFs (not shown). CBPΔCH1 and p300ΔCH1 are hypomorphic proteins with specific defects in mediating HIF-dependent transcription The mutant transcripts were correctly spliced, as determined by RT–PCR (Supplementary Figure S1A and B). The biochemical integrity of the CBPΔCH1 and p300ΔCH1 proteins was confirmed by examining their expression and acetyltransferase activities. Western blot established that CBP and p300 protein levels and stability were indistinguishable in WT, CBPΔCH1/ΔCH1, and p300ΔCH1/ΔCH1 MEFs (Figure 2A). HAT activities measured in vitro following immunoprecipitation of CBP and p300 with specific antibodies were comparable between the WT and mutant CBP and p300 (Figure 2B and C). Thus, CBPΔCH1 and p300ΔCH1 are normally expressed hypomorphic proteins, and their HAT domain is intact. 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 CBPΔCH1/ΔCH1 and p300ΔCH1/ΔCH1 MEFs. (B, C) Immunoprecipitation/HAT assay showing that HAT activity in CBPΔCH1/ΔCH1 (B) and p300ΔCH1/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). Download figure Download PowerPoint Transient transfection assays showed that the transactivation function of the HIF-1α C-TAD fused to the Gal4 DNA-binding domain (Gal-HIF-1α) was attenuated about 60–80% in CBPΔCH1/ΔCH1 and p300ΔCH1/ΔCH1 MEFs, and was reduced about 90% in triple-ΔCH1 (CBP+/ΔCH1;p300ΔCH1/ΔCH1) MEFs (Figure 2D). Overexpression of CBP (Figure 2E and F) or p300 (Figure 2F), but not CBPΔCH1, rescued Gal-HIF-1α activity; CBPΔCH1 overexpression did not affect Gal-HIF-1α activity in WT MEFs. Other activation domains fused to Gal4 (Myb, Ets-1, and CREB), which interact with other CBP and p300 domains, or with other coactivators, were not significantly affected by the ΔCH1 mutation (Figure 2G and H). Therefore, HIF-1α C-TAD activity is specifically attenuated by the ΔCH1 mutation, the combined dosage of CBP and p300 CH1 domains is crucial for C-TAD activity, and the CBPΔCH1 protein does not function as a dominant negative. Remarkably, endogenous hypoxia-inducible gene expression was largely unaffected in CBPΔCH1/ΔCH1 and p300ΔCH1/ΔCH1 MEFs (not shown). As the CH1 domain may not be limiting for HIF function in such cells, we also analyzed endogenous gene expression using two types of triple-ΔCH1 mutant MEFs (CBP+/ΔCH1;p300ΔCH1/ΔCH1 and CBPΔCH1/ΔCH1;p300+/ΔCH1). Affymetrix microarrays showed that there was a modest average decrease in the expression levels of 111 hypoxia-inducible genes (not necessarily HIF targets; defined by 148 probe sets induced ⩾3-fold in WT MEFs) in CBP+/ΔCH1;p300ΔCH1/ΔCH1 MEFs compared to WT MEFs (best-fit line slope is less than one; Figure 3A). As a control, we examined non-hypoxia-inducible genes represented by 281 probe sets that differed no more than ±1% between normoxia and hypoxia in WT cells, which showed that transcription was not broadly affected in triple-ΔCH1 MEFs (best-fit line slope is close to one with minimal data scatter; Figure 3B). Quantitative real-time RT–PCR (qRT–PCR) analysis of RNA from both types of triple-ΔCH1 MEFs revealed strong to moderate dependence on the CH1 domain for selected hypoxia-inducible genes including placental growth factor (Pgf), vascular endothelial growth factor (Vegf), and glucose transporter-1 (Glut1 or Slc2a1), when normalized to β-actin mRNA (Figure 3C–E, data from 2–6 independent MEF lines for each genotype). Vegf and Slc2a1 are direct HIF targets, but it is unclear if Pgf is a direct or indirect target (Manalo et al, 2005). These three genes play important roles in angiogenesis or glucose metabolism, with Vegf levels being especially critical for angiogenesis and normal development, as Vegf+/− mice die at E11 to E12 (Ferrara et al, 1996). These results suggest that a moderate decrease in the expression of many HIF-target genes could have consequences in both neoplastic and normal cells carrying the ΔCH1 mutation. Interestingly, Vegf deficiency, but apparently not deficiency of the related protein Pgf, leads to defective lung development in mice (Compernolle et al, 2002). 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;p300ΔCH1/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, CBPΔCH1/ΔCH1;p300+/ΔCH1, and CBP+/ΔCH1;p300ΔCH1/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. Download figure Download PowerPoint The ΔCH1 mutation does not significantly attenuate tumorigenesis The interaction of the CH1 domain with HIF is thought to be vital for tumorigenesis and has been proposed as a therapeutic target (Kung et al, 2000; Semenza, 2003). In this regard, the small molecule chetomin has been identified as a pharmacological agent that disrupts the CH1 structure and inhibits hypoxia-inducible transcription and tumor growth in vivo (Kung et al, 2004). The reduced hypoxia-dependent transcriptional response in triple-ΔCH1 MEFs predicts that tumorigenesis would be attenuated by the ΔCH1 mutation. To test this hypothesis, we introduced CBPΔCH1 and p300ΔCH1 alleles into mice carrying an Eμ-Myc transgene, which induces B-cell lymphoma, a highly vascularized solid mass tumor, with high penetrance and short latency. We found that a single CBPΔCH1 (N=8, P=0.014, log-rank test) or p300ΔCH1 (N=20, P=0.006) mutant allele decreased the median survival of C57BL/6 × 129 F1 hybrid Eμ-Myc mice by about 8–10 weeks, when compared to littermates carrying only the Eμ-Myc transgene (N=22; Figure 3F). The survival curves for Eμ-Myc;p300+/ΔCH1 mice on a C57BL/6 background (N=11, P=0.21) were indistinguishable from Eμ-Myc;p300+/+ mice (N=16), showing that strain background also contributes to tumor latency (Figure 3G). Thus, a reduction in the levels of the CH1 domain does not inhibit Myc-induced B-cell lymphomagenesis. We also tested WT and triple-ΔCH1 MEFs (CBP+/ΔCH1;p300ΔCH1/ΔCH1 and CBPΔCH1/ΔCH1;p300+/ΔCH1) transformed with the oncogenes 12S E1A and N61 Ras, for their tumorigenic potential following subcutaneous injection into the flanks of Scid mice. The time for the tumors to reach ∼1 cm3 was not significantly different for four independent WT transformed MEF lines (16.5±1.3 days, mean±s.d.), two CBP+/ΔCH1;p300ΔCH1/ΔCH1 lines (19.9±5.3 days), and three CBPΔCH1/ΔCH1;p300+/ΔCH1 lines (19.5±2.3 days) (ANOVA, P=0.57). Volumes (P=0.54) and weights (P=0.37) of the harvested tumors were also not significantly different between the groups. Similarly, there were no statistically significant differences in the growth rate and size of tumors in nude mice following subcutaneous injection of eight independent lines of WT and ΔCH1 MEFs transformed with retroviruses expressing c-Myc and oncogenic V12 Ras (WT, 31±23 days (N=3); CBPΔCH1/ΔCH1, 12±0 days (N=2); CBPΔCH1/ΔCH1;p300+/ΔCH1, 17±3.8 days (N=3); mean±s.d., P=0.74). Therefore, substantially reducing CH1 domain function also does not significantly affect fibroblastic transformation or tumorigenesis. C-TAD transactivation function absolutely requires the CH1 domain To address if residual CBP or p300 produced from the remaining WT allele in the triple-ΔCH1 cells was sufficient to support hypoxia-inducible transcription, we generated two strains of triple-ΔCH1/flox MEFs that have a Cre/LoxP conditional knockout CBPflox or p300flox allele in place of the WT gene (Kang-Decker et al, 2004) (PB, submitted). Transient expression of Cre recombinase following infection with a Cre-expressing adenovirus resulted in highly efficient recombination of CBPflox to yield a CBPΔflox null allele (i.e. CBPΔCH1/Δflox;p300ΔCH1/ΔCH1 or tri-ΔCH1/Δflox #1 MEFs; Figure 4A), or p300flox to yield a p300Δflox null allele (i.e. CBPΔCH1/ΔCH1;p300ΔCH1/Δflox or tri-ΔCH1/Δflox #2 MEFs; Figure 4B). Tri-ΔCH1/Δflox MEFs had a growth rate and morphology (not shown) comparable to Cre-adenovirus-infected control cells that lacked only a single CBP allele (CBP+/Δflox;p300+/+ or Δflox #1 MEF; Figure 4C) or p300 allele (CBP+/+;p300+/Δflox or Δflox #2 MEF; Figure 4D). Transient transfection assays revealed a dramatic loss of transactivation function for Gal-HIF-1α (>99%) and Gal-HIF-2α (>96%) in both types of tri-ΔCH1/Δflox MEFs, demonstrating that both C-TADs absolutely require the CH1 domain (Figure 4E and F). By contrast, the KIX-domain-dependent activator Gal-Myb functioned normally in tri-ΔCH1/Δflox MEFs (Figure 4E and F). Thus, other coactivators, or other domains of CBP and p300, appear to be unable to mediate C-TAD function. 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 CBPflox and (A) p300flox (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). Download figure Download PowerPoint Endogenous HIF-target gene expression relies on both CH1-dependent and -independent mechanisms We next examined the transcription of hypoxia-inducible genes in triple-ΔCH1/flox and tri-ΔCH1/Δflox MEFs by qRT–PCR. Surprisingly, there was very little difference in the expression of the HIF targets Slc2a1 and Pfkfb3 in both types of tri-ΔCH1/Δflox (infected with Cre-expressing adenovirus) and triple-ΔCH1/flox MEFs (not infected), indicating that WT CBP or p300 was not responsible for residual HIF-target gene expression in triple-ΔCH1 MEFs (Figure 4G and H). The Cre-adenovirus-infected cells were tested at 10, 13, and 16 days post infection, after they had expanded a minimum of 450-fold for tri-ΔCH1/Δflox MEFs (>250-fold for Δflox MEFs), thus greatly diluting any residual WT CBP or p300 protein and mRNA (Figure 4C and D). Comparison of 40 HIF-target genes in three independent lines each of Δflox and tri-ΔCH1/Δflox MEFs using Affymetrix microarrays revealed a 35% average decrease in gene expression (mutant to control signal ratio of 0.65±0.47, mean±s.d.) after 6 h of hypoxia (Supplementary Table S2). Thus, microarray and qRT–PCR analyses revealed that the CH1 domain is vital for a few hypoxia-responsive genes (e.g. Pgf; Figure 3C and Supplementary Table S2), moderately limiting for many (e.g. Slc2a1, Pfkfb3; Figure 4G and H), and mostly dispensable for others (e.g. Hig1; Supplementary Table S2). The ΔCH1 mutation specifically attenuates the recruitment of CBP and p300 to HIF-target genes We next addressed whether the ΔCH1 mutation blocked recruitment of CBP and p300 to endogenous HIF-binding sites. Quantitative real-time PCR chromatin immunoprecipitation (ChIP) assays were performed with the gene-specific signal normalized to the input DNA signal. CBPΔCH1/ΔCH1 and p300ΔCH1/ΔCH1 MEFs showed attenuated HIF-dependent recruitment of CBPΔCH1 or p300ΔCH1 to DNA sequences near the HIF-binding sites of Slc2a1, Pfkfb3, and Hig1 (Figure 5A–C). Recruitment of WT CBP or p300 in the singly homozygous mutant MEFs served as an internal control. The ΔCH1 mutation caused an 80–90% reduction in treatment-dependent CBP or p300 recruitment in CBPΔCH1/ΔCH1 and p300ΔCH1/ΔCH1 MEFs (compared to the WT CBP or p300-dependent signal in the mutant MEFs) treated for 2 h with the hypoxia mimetic dipyridyl (DP) and the proteasome inhibitors MG132 and ALLN, which together induce HIF-1α and HIF-2α (Figure 5A–C). Another independent set of MEF lines confirmed this effect of the ΔCH1 mutation following 4 h of hypoxia (Supplementary Figure S2A–C). Levels of Slc2a1, Pfkfb3, and Hig1 transcripts in tri-ΔCH1/Δflox #2 MEFs compared to Δflox #2 MEFs treated with DP, MG132, and ALLN (Figure 5D–F) were similar to results obtained with hypoxia (Figure 4G and H and Supplementary Table S2). Importantly, control experiments showed that ΔCH1 mutation did not affect recruitment to the Jun/Sp1-binding site of the non-hypoxia-regulated gene vimentin by ChIP (Wu et al, 2003), but that it strongly attenuated the interaction with HIF-1 in a co-immunoprecipitation assay (Supplementary Figure 2D and E). Although the recruitment of CBP and p300 is not completely blocked by the ΔCH1 mutation under conditions that activate HIF-1 and HIF-2, the amount of gene expression remaining in cells that only contain ΔCH1 mutant alleles suggests that a CBP/p300-independent mechanism must account for a large portion of HIF-responsive transcription. 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, CBPΔCH1/ΔCH1, and p300ΔCH1/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). Download figure Download PowerPoint A trichostatin A (TSA)-sensitive pathway cooperates with a CH1-dependent mechanism to mediate the bulk of HIF-responsive transcription Histone deacetylases (HDACs) have been implicated in gene activation dependent on the CBP/p300-interacting transcription factors CREB and HIF (Kim et al, 2001; Brugarolas et al, 2003; Fass et al, 2003). We tested if deacetylase activity is required for the CH1-independent component of HIF-responsive transcription by pretreating MEFs with the specific HDAC inhibitor TSA for 30 min prior to inducing HIF with DP for 3 h (treatment with TSA starting 30 min after DP addition yielded similar results; LH Kasper, data not shown). TSA markedly inhibited the DP-dependent induction of Pfkfb3 and Egln3 in both Δflox controls and tri-ΔCH1/Δflox MEFs to an extent comparable to or greater than that caused by the ΔCH1 mutation alone (Figure 6A and B). TSA treatment in the absence of DP had marginal effects on these three genes over the 3.5 h of treatment, but TSA cooperated with the ΔCH1 mutation to further reduce HIF-responsive gene expression in the presence of DP. In fact, Egln3 DP-dependent expression was reduced nearly 100% by the combination of TSA and the ΔCH1 mutation (when TSA-dependent expression is subtracted from that for DP±TSA)." @default.
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