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. 2010 Jan 22;285(4):2601-9.
doi: 10.1074/jbc.M109.021824. Epub 2009 Oct 30.

Complex regulation of the transactivation function of hypoxia-inducible factor-1 alpha by direct interaction with two distinct domains of the CREB-binding protein/p300

Jorge L Ruas  1 , Utta Berchner-Pfannschmidt, Sohail Malik, Katarina Gradin, Joachim Fandrey, Robert G Roeder, Teresa Pereira, Lorenz Poellinger
Affiliations

Complex regulation of the transactivation function of hypoxia-inducible factor-1 alpha by direct interaction with two distinct domains of the CREB-binding protein/p300

Jorge L Ruas et al. J Biol Chem. .

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 alpha. The C-terminal activation domain of HIF-1 alpha 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 alpha interacts with endogenous CBP and that this interaction facilitates its transactivation function. Our results show that interaction of HIF-1 alpha 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 alpha than of p53. Chromatin immunoprecipitation demonstrated that at 1% O(2) CBP is recruited to a HIF-1 alpha 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 alpha N-TAD and mediating the transactivation function of this domain. Thus, we suggest that in hypoxia HIF-1 alpha 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 alpha.

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Figures

FIGURE 1.
FIGURE 1.
HIF-1α N-TAD interacts with endogenous CBP. A, schematic representation of HIF-1α and CBP/p300 domains. HIF-1α contains an N-terminal DNA-binding domain followed by a helix-loop-helix dimerization interface (bHLH) and the PAS domains (blue boxes labeled A and B). The N- (N) and C-terminal (C) transactivation domains are located in the C-terminal portion of the protein. CBP/p300 contains several domains that mediate interaction with other proteins, such as the NR domain (interaction with nuclear receptors), CH1 and CH3, and the C-terminal glutamine-rich domain (Q). B, HIF-1α N-TAD interacts with endogenous CBP. Nuclear extracts from HeLa cells kept at normoxia (N) or treated with 2,2′-dipyridyl (Dp) were incubated with GST-fused VP16, C-TAD, or N-TAD. Precipitated proteins were separated by SDS-PAGE, and CBP was detected by immunoblot analysis using an anti-CBP antibody (α-CBP). C, Coomassie staining of bacterially expressed GST-fused proteins. D, N-TAD transactivation activity is inhibited by expression of E1A. HEK 293 cells were transfected with 500 ng of GAL4-driven luciferase reporter gene and 10 ng of plasmids encoding FLAG-GAL-C-TAD (left panel) or FLAG-GAL4-N-TAD (right panel) in the absence or presence of increasing concentrations of an E1A expression plasmid (10, 20, or 50 ng). The data are presented as luciferase activity relative to cells transfected with pFLAG-GAL4 and incubated at normoxia. The values represent the means ± S.E. of three independent experiments performed in duplicate. E, full-length HIF-1α mutant with a nonfunctional C-TAD is responsive to CBP. HEK 293 cells were transfected with a HRE-driven luciferase reporter plasmid and plasmids encoding FLAG-HIF-1α or FLAG-HIF-1α(L808A/L809A) (F-HIF-1α(LL808AA)) in the presence or absence of a CBP expressing plasmid. The data are presented as luciferase activity relative to cells transfected with pCMX and incubated at normoxia. The values represent the means ± S.E. of three independent experiments performed in duplicate.
FIGURE 2.
FIGURE 2.
The N-terminal transactivation domain of HIF-1α interacts with the CH3 region of CBP. A, HIF-1α N-TAD is the major contributor for the interaction of full-length HIF-1α with the CH3 domain of CBP. In vitro translated [35S]methionine-labeled wild-type or mutant HIF-1α proteins were incubated with GST-fused CBP domains, as indicated. Precipitated proteins were separated by SDS-PAGE and detected by autoradiography. B, Coomassie staining of bacterially expressed GST-fused proteins. Arrowheads indicate the bands with the correct molecular sizes. C, HIF-1α N-TAD interacts directly with the CH3 domain of CBP. Purified baculovirus-expressed FLAG-CH3 domain was precipitated by bacterially expressed GST-N-TAD. After separation by SDS-PAGE, the proteins were analyzed by Western blotting using an anti-FLAG (α-Flag) antibody.
FIGURE 3.
FIGURE 3.
In vivo interaction between HIF-1α transactivation domains and cysteine/histidine-rich regions of CBP. A and B, FRET analysis shows in vivo interaction between CFP-C-TAD/YFP-CH1 and CFP-N-TAD/YFP-CH3. HEK 293 cells were transfected with 600 ng of CFP and 400 ng of YFP-fused expression plasmids. The images show representative cells of donor (D) and acceptor (A) before (bb) and after (ab) selective photobleaching. The cells are shown in false color representing different fluorescence intensities as indicated by the color table shading from black (lowest intensity), through blue, green, yellow, orange, red, and purple to white (highest intensity). C, analysis of FRET efficiency (%) of the corresponding CFP/YFP fusion proteins. HEK 293 cells expressing the different fusion proteins were kept at normoxia or treated with 100 μm CoCl2 for 4 h. The values represent the means ± S.E. of two independent experiments each of 10–20 cells. D, expression of CH3-VP16 protein increases luciferase activity mediated by a HIF-1α mutant bearing a nonfunctional C-TAD. HEK 293 cells were transfected with 300 ng of HRE-driven luciferase reporter gene and 50 ng of HIF-1α or the corresponding mutant expression plasmids in the absence or presence of 400 ng of pFLAG-CH1-VP16 or pFLAG-CH3-VP16. The data are presented as luciferase activity relative to cells transfected with pFLAG and incubated at normoxia. The values represent the means ± S.E. of three independent experiments performed in duplicate.
FIGURE 4.
FIGURE 4.
HIF-1α binding to CBP displays a high affinity interaction. A, endogenous HIF-1α interacts with both the CH1 and the CH3 regions of CBP. HeLa nuclear extracts kept at normoxia (N) or treated with 2,2′-dipyridyl (Dp) were incubated with bacterially expressed GST-fused CH1 and CH3 proteins. Precipitated proteins were separated by SDS-PAGE and detected by Western blot analysis using anti-HIF-1α (α-HIF-1α) and anti-p53 (α-p53) antibodies. B, recruitment of CBP to HIF-1α and p53. Endogenous CBP was immunoprecipitated with an anti-CBP (α-CBP) antibody, and interacting proteins were separated by SDS-PAGE and detected by Western blot analysis using specific antibodies. C and D, recruitment of CBP to target genes promoters is impaired by simultaneously activation of HIF-1α- and p53-regulated pathways. HCT 166 cells were fixed with formaldeyde after 8 h of treatment as described. ChIP was performed using antibodies against IgG, HIF-1α, p53, and CBP. Binding sites of HIF-1α and p53 on the target gene promoters PGK-1 and p21, respectively, were amplified using quantitative reverse transcription-PCR as described under “Experimental Procedures.” The ChIP experiments were repeated three times, and the figure shows the results of a representative experiment.
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References

    1. Ruas J. L., Poellinger L. (2005) Semin. Cell Dev. Biol. 16, 514–522 - PubMed
    1. Lendahl U., Lee K. L., Yang H., Poellinger L. (2009) Nat. Rev. Genet. 10, 821–832 - PubMed
    1. Epstein A. C., Gleadle J. M., McNeill L. A., Hewitson K. S., O'Rourke J., Mole D. R., Mukherji M., Metzen E., Wilson M. I., Dhanda A., Tian Y. M., Masson N., Hamilton D. L., Jaakkola P., Barstead R., Hodgkin J., Maxwell P. H., Pugh C. W., Schofield C. J., Ratcliffe P. J. (2001) Cell 107, 43–54 - PubMed
    1. Bruick R. K., McKnight S. L. (2001) Science 294, 1337–1340 - PubMed
    1. Kamura T., Sato S., Iwai K., Czyzyk-Krzeska M., Conaway R. C., Conaway J. W. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 10430–10435 - PMC - PubMed

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