QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 22 December 2004
doi: 10.1242/jcs.01617

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Ruas, J. L. Articles by Pereira, T. Search for Related Content PubMed PubMed Citation Articles by Ruas, J. L. Articles by Pereira, T. Social Bookmarking                         What's this?

Role of CBP in regulating HIF-1-mediated activation of transcription

Department of Cell and Molecular Biology, Karolinska Institute, 171 77 Stockholm, Sweden

View larger version (48K): [in a new window]   Fig. 1. CBP mediates the interaction of SRC-1 with HIF-1. (A) Interaction of HIF-1 with endogenous CBP is dependent on the integrity of both HIF-1 transactivation domains. Cells were transfected with 1 µg pFlag-mHIF-1 (Flag-HIF-1) or pFlag-mHIF1(532583)(L808A/L809A) [Flag-HIF-1(532583)LL808AA] and kept at normoxia (N) or treated with 100 µM CoCl2 (C) for 3 hours. 3 mg protein from HEK 293 whole cell extracts was incubated with anti-CBP antibody (-CBP), and precipitated complexes were separated by SDS-PAGE followed by immunoblotting using anti-CBP and anti-Flag (-Flag) antibodies. (B) CBP immunodepletion abolishes interaction between HIF-1 and SRC-1. HEK 293 cells were transfected with 1 µg of pFlag-mHIF-1 and kept at normoxia (N) or treated with 100 µM CoCl2 for 3 hours (C). CBP immunodepletion (CBP ID) was performed by incubating whole cell extracts with either IgG (-) or anti-CBP antibodies (+) for 2 hours at room temperature. Following immunodepletion whole cell extracts were recovered and incubated with anti-SRC-1 antibody (-SRC-1) for an additional 2 hours. Precipitated complexes were separated by SDS-PAGE and analyzed by immunoblotting using anti-Flag, anti-CBP and anti-SRC-1 antibodies. *Non-specific band. Input gels were loaded with 50 µg protein of each whole cell extract.

View larger version (42K): [in a new window]   Fig. 2. CBP mediates in vivo interaction between HIF-1 and SRC-1. (A) Subcellular and intranuclear distribution of YFP-CBP, CFP-HIF-1 and YFP-SRC-1. HEK 293 cells were transfected with 400 ng of pYFP-CBP (YFP-CBP), pCFP-mHIF-1 (CFP-HIF-1) or pYFP-SRC-1 (YFP-SRC-1) and observed using fluorescence microscopy. The YFP-SRC-1-fluorescent signal is pseudocolored red. Bars, 3 µm. (B) SRC-1 colocalizes with CBP but not with HIF-1 in accumulation foci. Cells were transfected with 400 ng of expression plasmids for CFP-SRC-1 or YFP-SRC-1 and YFP-CBP or CFP-HIF-1, respectively. CFP- and YFP-SRC-1-fluorescent signals are pseudocolored red. Bars, 3 µm. (C) Redistribution of CFP-HIF-1 by YFP-SRC-1 is dependent on CBP. HEK 293 cells were transfected with 400 ng of pCFP-mHIF-1 (CFP-HIF-1), pYFP-SRC-1 (YFP-SRC-1) and pRc/RSV-CBP-HA (CBP). The bar chart shows the percentage of cells in which colocalization foci were observed at normoxia (N) or upon treatment with CoCl2 (C). Bars, 3 µm. (D) YFP-SRC-1 enhances the hypoxia-dependent transactivation function of CFP-HIF-1. HEK 293 cells were transfected with 500 ng HRE-responsive reporter plasmid, 50 ng of the CFP or CFP-HIF-1 expression plasmids and 400 ng of YFP-SRC-1 expression plasmid. Total amount of DNA was kept at 1 µg using pFlag-CMV-2. After transfection, cells were kept at normoxia (21% O2) or hypoxia (1% O2) for 36 hours, harvested and luciferase activity was measured. Data are presented as fold induction over the luciferase activity obtained following transfection of cells using pECFP-C1 and incubation of the cells under normoxic conditions. Relative luciferase values obtained with expression of CFP-HIF-1 at normoxia (^) or hypoxia (*) in the presence or absence of expressed YFP-SRC-1 are significantly different (P<0.05). Values represent the mean ± s.e.m. of three independent experiments performed in duplicate. (E) Expression levels of the different CFP- and YFP-fusion proteins. HEK 293 cells were transfected with 400 ng of each expression plasmid (as indicated). 36 hours after transfection medium was changed for treatment in the presence of absence of 100 µM CoCl2 for 2 hours. 50 µg whole cell extracts were submitted to SDS-PAGE followed by immunoblotting using anti-GFP (-GFP) antibodies.

View larger version (30K): [in a new window]   Fig. 3. F2101P/K2103P mutations in full-length CBP disrupt protein-protein interaction and colocalization with SRC-1. (A) Subcellular localization of YFP-CBP(F2101P/K2103P) (YFP-CBP/H3P). HEK 293 cells were transfected with 400 ng of pYFP-CBP/H3P and treated for 2 hours with 100 µM CoCl2. Bar, 3 µm. (B) YFP-CBP/H3P shows diffuse intranuclear distribution and does not colocalize with SRC-1. HEK 293 cells were transfected with 400 ng of expression plasmids encoding CFP-SRC-1, CFP-HIF-1 and/or YFP-CBP/H3P as described in Fig. 1A. Bar, 3 µm. (C) YFP-CBP/H3P fails to coprecipitate with endogenous SRC-1 in HEK 293 cells. Cells were transfected with 1 µg of either pYFP-CBP or pYFP-CBP/H3P and kept at normoxia (N) or treated with 100 µM CoCl2 (C) for 3 hours. Whole cell extracts corresponding to a total protein concentration of 3 mg were incubated with anti-SRC-1 antibody (-SRC-1) for 2 hours at room temperature. Immunoprecipitated protein complexes were separated as before and immunoblotted using anti-GFP (-GFP) and -SRC-1 antibodies. *Non-specific band. 50 µg of whole cell extract protein was loaded on input gels.

View larger version (15K): [in a new window]   Fig. 4. SRC-1 interacts with HIF-1 via CBP. (A) Enhancement of HIF-1 C-TAD-mediated transcription by SRC-1 is dependent on interaction with CBP. Cells were transfected with 500 ng GAL4-driven reporter gene, 10 ng GAL4 (G4) or GAL4-C-TAD expression plasmid (C-TAD) and 200 ng (+) or 400 ng of expression plasmids (++) encoding CBP, CBP/H3P or SRC-1 and carrier DNA, pFLAG-CMV-2 to keep a constant total DNA concentration of 1 µg. No significant difference was observed in the luciferase values obtained with expression of the C-TAD (*) in the presence or absence of coexpressed CBP/H3P (*) (P<0.05). (B) CBP/H3P does not potentiate HIF-1-mediated transactivation. Cells were transfected with 500 ng HRE-driven reporter gene, 50 ng Flag (Flag) or mHIF-1 (HIF-1) expression plasmids and 400 ng (+) or 800 ng (++) of expression plasmids encoding CBP, CBP/H3P, or SRC-1. Total DNA concentration was kept at 1.35 µg using pFlag-CMV-2. Data are presented as fold induction over the luciferase activity obtained following transfection of cells using GAL4 expression plasmid (A) or pFlag-CMV-2 (B) and incubation of the cells under normoxic conditions. No significant difference was observed in the luciferase values obtained with expression of HIF-1 (*) in the presence or absence of coexpressed CBP/H3P (*) (P<0.05). Values represent the mean ± s.e.m. of three independent experiments performed in duplicate.

View larger version (39K): [in a new window]   Fig. 5. In vivo analysis of colocalization of Arnt, CBP and SRC-1 using CFP and YFP chimeric proteins. (A) CFP- and YFP-Arnt localize in discrete nuclear accumulation foci. Cells were transfected with CFP- and YFP-Arnt expression plasmids and analyzed as described in the legend of Fig. 1A. (B) CFP-Arnt colocalizes with YFP-CBP and YFP-SRC-1 but not with YFP-CBP/H3P. Cells were transfected with 400 ng expression plasmids encoding CFP-Arnt and YFP-CBP or YFP-SRC-1 or YFP-CBP/H3P and analyzed as described in Fig. 1A. Bars, 3 µm.

View larger version (34K): [in a new window]   Fig. 6. CBP enhances colocalization between CFP-HIF-1 and YFP-Arnt. (A) Cells were transfected with 400 ng of pCFP-mHIF-1 (CFP-HIF-1), pYFP-Arnt (YFP-Arnt) and pRc/RSV-CBP-HA (CBP) or pSG5/hSRC-1 (SRC-1) as indicated and analyzed as described in the legend of Fig. 1A. The bar chart shows the percentage of cells in which colocalization foci were observed at normoxia (N) or upon treatment with CoCl2 (C). Bars, 3 µm. (B) Colocalization between CFP-HIF-1 and YFP-Arnt. HEK 293 cells were transfected with 300 ng pYFP-Arnt (YFP-Arnt) and 700 ng pCFP-mHIF-1 (CFP-HIF-1). The bar chart shows the percentage of cells in which colocalization foci were observed at normoxia (N) or upon treatment with CoCl2 (C). Bars, 3 µm. (C) Protein expression levels of CFP-HIF-1 and YFP-Arnt under the conditions described in B. 36 hours after transfection, medium was changed and cells were given a further 2 hours at normoxia or in the presence of 100 µM CoCl2. 50 µg whole cell extracts were submitted to SDS-PAGE followed by immunoblotting using anti-GFP (-GFP) antibodies. (D) The CFP-HIF-1/YFP-Arnt heterodimer efficiently activates transcription of an HRE-driven reporter gene in a hypoxia-inducible way. HEK 293 cells were transfected with 500 ng HRE-driven reporter gene, 50 ng of the indicated expression plasmid and carrier DNA, pFlag-CMV-2, to keep the total DNA concentration at 1 µg. After transfection cells were kept at normoxia (21% O2) or hypoxia (1% O2) for 36 hours, harvested and luciferase activity was measured. Data are presented as fold induction over the luciferase activity obtained with transfection of the pECFP-C1 and incubated at normoxia. Relative luciferase values obtained with expression of CFP-HIF-1 at normoxia (^) or hypoxia (*) in the presence or absence of expressed YFP-Arnt are significantly different (P<0.05). Values represent the mean ± s.e.m. of three independent experiments performed in duplicate.

View larger version (60K): [in a new window]   Fig. 7. Intranuclear distribution of RNA polymerase IIo and analysis of colocalization with HIF-1, Arnt, CBP and SRC-1. Insets show enlarged examples of the different observed foci in each image. (A) RNA polymerase IIo shows an intranuclear distribution in discrete accumulation foci. Immunocytochemistry for RNA polymerase IIo (RNA pol IIo) was performed using anti-RNA polymerase IIo antibody (CC-3) as described in Materials and Methods. (a) Enlargement of a nuclear accumulation focus of endogenous RNA polymerase IIo. (B) CBP colocalizes with RNA polymerase IIo in a large number of nuclear accumulation foci. Cells were transfected with pYFP-CBP as described in Fig. 1A, and anti-RNA polymerase IIo immunocytochemistry was performed using the CC-3 antibody. An RNA polymerase IIo accumulation focus (a), a YFP-CBP accumulation focus (b) and a focus where RNA polymerase IIo colocalized with YFP-CBP (c) are shown. (C) Colocalization of SRC-1 and RNA polymerase IIo in a few discrete foci. Cells were transfected with pYFP-SRC-1 and immunocytochemistry was performed as indicated above. Enlargement of an RNA polymerase IIo accumulation focus (a), a YFP-SRC-1 accumulation focus (b) and a focus of colocalization between RNA polymerase IIo and YFP-SRC-1 are shown. (D) The intranuclear distribution of Arnt in discrete foci does not overlap with RNA polymerase IIo. Cells were transfected with pYFP-Arnt. Enlarged images of an RNA polymerase IIo accumulation focus (a) and a YFP-Arnt accumulation focus (b) are shown. (E) The intranuclear distribution pattern of HIF-1 and Arnt partially overlaps with that of RNA polymerase IIo. HEK 293 cells were transfected with pCFP-HIF-1 (CFP-HIF-1) and pYFP-Arnt (YFP-Arnt) as indicated in the legend of Fig. 6B. An RNA polymerase IIo accumulation focus (a), a CFP-HIF-1/YFP-Arnt accumulation focus (b) and a focus of colocalization between RNA polymerase IIo and CFP-HIF-1/YFP-Arnt (c) are shown. (F) Distribution of CFP-HIF-1 and YFP-CBP into some discrete foci of colocalization with RNA polymerase IIo. Enlargements of an RNA polymerase IIo accumulation focus (a), a CFP-HIF-1/YFP-CBP accumulation focus (b) and a focus of colocalization between RNA polymerase IIo and CFP-HIF-1/YFP-CBP (c) are shown. All figures are single confocal sections obtained from HEK 293 cells treated with 100 µM CoCl2 for 2 hours. Bar, 3 µm for all images.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter