Complex Regulation of the Transactivation Function of Hypoxia-inducible Factor-1α by Direct Interaction with Two Distinct Domains of the CREB-binding Protein/p300

Abstract

Activation of transcription in response to low oxygen tension is mediated by the hypoxia-inducible factor-1 (HIF-1). HIF-1 is a heterodimer of two proteins: aryl hydrocarbon receptor nuclear translocator and the oxygen-regulated HIF-1␣. The C-terminal activation domain of HIF-1␣ has been shown to interact with cysteine/ histidine-rich region 1 (CH1) of the coactivator CBP/p300 in a hypoxia-dependent manner. However, HIF forms lacking C-terminal activation domain (naturally occurring or genetically engineered) are still able to activate transcription of target genes in hypoxia. Here, we demonstrate that the N-terminal activation domain (N-TAD) of HIF-1␣ interacts with endogenous CBP and that this interaction facilitates its transactivation function. Our results show that interaction of HIF-1␣ N-TAD with CBP/p300 is mediated by the CH3 region of CBP known to interact with, among other factors, p53. Using fluorescence resonance energy transfer experiments, we demonstrate that N-TAD interacts with CH3 in vivo. Coimmunoprecipitation assays using endogenous proteins showed that immunoprecipitation of CBP in hypoxia results in the recovery of a larger fraction of HIF-1␣ than of p53. Chromatin immunoprecipitation demonstrated that at 1% O 2 CBP is recruited to a HIF-1␣ but not to a p53 target gene. Upon activation of both pathways, lower levels of chromatin-associated CBP were detected at either target gene promoter. These results identify CBP as the coactivator directly interacting with HIF-1␣ N-TAD and mediating the transactivation function of this domain. Thus, we suggest that in hypoxia HIF-1␣ is a major CBP-interacting transcription factor that may compete with other CBP-dependent factors, including p53, for limiting amounts of this coactivator, underscoring the complexity in the regulation of gene expression by HIF-1␣.

Upstream Stimulatory Factor 2 and Hypoxia-Inducible Factor 2α (HIF2α) Cooperatively Activate HIF2 Target Genes during Hypoxia

“…However, reduction of ARNT did not alter binding of USF2 and Pol II to the PAI1 promoter. Consistent with a reported role of HIF in recruiting CBP and p300 (5,81), hypoxic Hep3B/ARNT shRNA cells exhibited a noticeable reduction of CBP and p300 binding to the PAI1 promoter (Fig. 7A).…”

“…7A), indicating that USF2 is responsible for hypoxia-mediated increased CBP and p300 recruitment to the PAI1 promoter. HIF's role in activating HIF target genes was thought to be recruitment of CBP and p300 histone acetylases by C-terminal transactivation domains (C-TADs) as well as N-terminal TADs (N-TADs) of HIF1␣ and HIF2␣ (55,81). Thus, we conducted ChIP experiments in normoxic or hypoxic Hep3B/ARNT shRNA cells that have 80% ARNT reduction.…”

HIF-2α Enhances β-Catenin/TCF-Driven Transcription by Interacting with β-Catenin

“…To further investigate the role of HIF-2a in b-catenin activation, we examined the possible involvement of p300 coactivator. It has been reported that the transcriptional activity of b-catenin is promoted by p300 (17) and that p300 is recruited by TADs of HIF-as (18,19). Therefore, we examined p300 recruitment by b-catenin, and found that the b-catenin-p300 binding was enhanced by full-length HIF-2a, but not by the d67 mutant (Fig.…”

Hypoxia-inducible Factor-1α Enhances Haptoglobin Gene Expression by Improving Binding of STAT3 to the Promoter

“…Because of the absence of an expected HIF-1␣-binding element in the region, it appeared that HIF-1␣ binding might be indirect. The transcription coactivator p300/CBP associates with HIF-1␣, as well as STAT3, in the formation of an active transcription complex for the transactivation of several genes (22,23). Therefore, we examined the association of p300/CBP in the complex.…”