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Synthetic transactivation screening reveals ETV4 as broad coactivator of hypoxia-inducible factor signaling.

Wollenick K, Hu J, Kristiansen G, Schraml P, Rehrauer H, Berchner-Pfannschmidt U, Fandrey J, Wenger RH, Stiehl DP - Nucleic Acids Res. (2011)

Bottom Line: Among others, the E-twenty six transcription factor ETS translocation variant 4 (ETV4) was found to contribute to PHD2 gene expression particularly under hypoxic conditions.Chromatin immunoprecipitation confirmed ETV4 and HIF-1α corecruitment to the PHD2 promoter.Of 608 hypoxically induced transcripts found by genome-wide expression profiling, 7.7% required ETV4 for efficient hypoxic induction, suggesting a broad role of ETV4 in hypoxic gene regulation.

View Article: PubMed Central - PubMed

Affiliation: Institute of Physiology and Zürich Center for Integrative Human Physiology (ZIHP), University of Zürich, 8057 Zürich, Switzerland.

ABSTRACT
The human prolyl-4-hydroxylase domain (PHD) proteins 1-3 are known as cellular oxygen sensors, acting via the degradation of hypoxia-inducible factor (HIF) α-subunits. PHD2 and PHD3 genes are inducible by HIFs themselves, suggesting a negative feedback loop that involves PHD abundance. To identify novel regulators of the PHD2 gene, an expression array of 704 transcription factors was screened by a method that allows distinguishing between HIF-dependent and HIF-independent promoter regulation. Among others, the E-twenty six transcription factor ETS translocation variant 4 (ETV4) was found to contribute to PHD2 gene expression particularly under hypoxic conditions. Mechanistically, complex formation between ETV4 and HIF-1/2α was observed by mammalian two-hybrid and fluorescence resonance energy transfer analysis. HIF-1α domain mapping, CITED2 overexpression and factor inhibiting HIF depletion experiments provided evidence for cooperation between HIF-1α and p300/CBP in ETV4 binding. Chromatin immunoprecipitation confirmed ETV4 and HIF-1α corecruitment to the PHD2 promoter. Of 608 hypoxically induced transcripts found by genome-wide expression profiling, 7.7% required ETV4 for efficient hypoxic induction, suggesting a broad role of ETV4 in hypoxic gene regulation. Endogenous ETV4 highly correlated with PHD2, HIF-1/2α and several established markers of tissue hypoxia in 282 human breast cancer tissue samples, corroborating a functional interplay between the ETV4 and HIF pathways.

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Transcriptional cooperation between ETV4 and HIF-1 is disrupted by CITED2. (A) Schematic representation of HIF-1α and ETV4 domain structure and fusion constructs used in mammalian two-hybrid assays. PAS, PER-ARNT-SIM; bHLH, basic helix–loop–helix domain; ODD, oxygen-dependent degradation domain; NRR, negative regulatory region; NAD and CAD, amino-carboxy-terminal activation domain and CADs, respectively. A GAL4-DBD was fused to regions encompassing the HIF-1α NAD and CAD. Full-length ETV4 bearing two activation domains (AD, acidic domain; Ct, carboxy-terminal tail) flanking a central ETS domain was fused to a VP16 activation domain (VP16-AD). Numbers indicate the amino acids present in the respective constructs. (B) U2OS cells were cotransfected with a Gal4-responsive reporter plasmid and Gal4-HIF-1α (GH1α) constructs alone or in combination with VP16-ETV4. The GH1α fusion constructs are specified by the aminoterminal starting amino acid of the truncated HIF-1α regions (530, 740 and 786, respectively). Following transfection, cells were evenly split and incubated at 20 or 0.2% O2 before luciferase activities were determined 24 h later. Noninteracting Gal4 DBD-p53 and VP16-AD-CP1 served as negative control (neg. ctrl.), while the interactions between Gal4 DBD-PHD2 and VP16-AD-HIF-2α(ODD) or VP16-AD-FKBP38 were used as positive controls (pos. ctrl. 1 and pos. ctrl. 2, respectively). (C) Scheme of the potential interactions between HIF-1, p300/CBP and ETV4 as assessed by mammalian two-hybrid assays. Both CITED2 and FIH can block the interaction between HIF-1α and p300/CBP. (D) Cotransfection of the indicated amounts of a CITED2 expression construct together with the mammalian two-hybrid expression vectors followed by hypoxic exposure and luciferase activity determination as described for (B). (E) Cotransfection of siRNA directed against p300 together with the mammalian two-hybrid expression vectors followed by hypoxic exposure and luciferase activity determination as described for (B). The p300 knock down efficiency of different siP300 oligonucleotides was analyzed by immunoblotting (upper panel) and siP300#1 was chosen for further experiments. (F) Cotransfection of siRNA directed against FIH together with the mammalian two-hybrid expression vectors followed by hypoxic exposure and luciferase activity determination as described for (B). The efficiency of the siFIH mediated FIH knock down was confirmed by immunoblotting as shown in the inset. (G) ChIP of normoxic or hypoxic PC3 cells using antibodies directed against HIF-1α or ETV4, or control serum. The amount of coprecipitated chromatin derived from the human P2P region (encoded by EGLN1) containing the HBS was determined by PCR followed by agarose gel electrophoresis.
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gkr978-F4: Transcriptional cooperation between ETV4 and HIF-1 is disrupted by CITED2. (A) Schematic representation of HIF-1α and ETV4 domain structure and fusion constructs used in mammalian two-hybrid assays. PAS, PER-ARNT-SIM; bHLH, basic helix–loop–helix domain; ODD, oxygen-dependent degradation domain; NRR, negative regulatory region; NAD and CAD, amino-carboxy-terminal activation domain and CADs, respectively. A GAL4-DBD was fused to regions encompassing the HIF-1α NAD and CAD. Full-length ETV4 bearing two activation domains (AD, acidic domain; Ct, carboxy-terminal tail) flanking a central ETS domain was fused to a VP16 activation domain (VP16-AD). Numbers indicate the amino acids present in the respective constructs. (B) U2OS cells were cotransfected with a Gal4-responsive reporter plasmid and Gal4-HIF-1α (GH1α) constructs alone or in combination with VP16-ETV4. The GH1α fusion constructs are specified by the aminoterminal starting amino acid of the truncated HIF-1α regions (530, 740 and 786, respectively). Following transfection, cells were evenly split and incubated at 20 or 0.2% O2 before luciferase activities were determined 24 h later. Noninteracting Gal4 DBD-p53 and VP16-AD-CP1 served as negative control (neg. ctrl.), while the interactions between Gal4 DBD-PHD2 and VP16-AD-HIF-2α(ODD) or VP16-AD-FKBP38 were used as positive controls (pos. ctrl. 1 and pos. ctrl. 2, respectively). (C) Scheme of the potential interactions between HIF-1, p300/CBP and ETV4 as assessed by mammalian two-hybrid assays. Both CITED2 and FIH can block the interaction between HIF-1α and p300/CBP. (D) Cotransfection of the indicated amounts of a CITED2 expression construct together with the mammalian two-hybrid expression vectors followed by hypoxic exposure and luciferase activity determination as described for (B). (E) Cotransfection of siRNA directed against p300 together with the mammalian two-hybrid expression vectors followed by hypoxic exposure and luciferase activity determination as described for (B). The p300 knock down efficiency of different siP300 oligonucleotides was analyzed by immunoblotting (upper panel) and siP300#1 was chosen for further experiments. (F) Cotransfection of siRNA directed against FIH together with the mammalian two-hybrid expression vectors followed by hypoxic exposure and luciferase activity determination as described for (B). The efficiency of the siFIH mediated FIH knock down was confirmed by immunoblotting as shown in the inset. (G) ChIP of normoxic or hypoxic PC3 cells using antibodies directed against HIF-1α or ETV4, or control serum. The amount of coprecipitated chromatin derived from the human P2P region (encoded by EGLN1) containing the HBS was determined by PCR followed by agarose gel electrophoresis.

Mentions: Intrigued by the HIF-dependent ETV4 effects, we aimed for the characterization of a putative physical interaction between ETV4 and HIF-1α. Using a mammalian two-hybrid system, expression plasmids encoding for ETV4 fused to the activation domain (AD) of viral protein 16 (VP16-ETV4) were cotransfected with HIF-1α oxygen regulatory domains (21,27) fused to a Gal4-DNA binding domain (DBD; Figure 4A). Due to its intrinsic transactivation activity, constructs containing the CAD of HIF-1α [amino acids 775-826 (27)] were sufficient to activate the Gal4-responsive promoter (Figure 4B). Coexpression of ETV4 strikingly superinduced GH1α740-826 and GH1α786-826, particularly under hypoxic conditions, suggesting that HIF-1α CAD and ETV4 cooperate to transactivate target genes (Figure 4B). ETV4 effects on the aminoterminal activation domain (27) (NAD; HIF-1α amino acids 549-582) were negligible. Both, HIF-1α and ETV4 have been demonstrated to interact with the ubiquitous transcriptional coactivators p300/CBP (28,29). To address the question whether the two factors directly interact or whether a ternary complex between HIF-1, p300/CBP and ETV4 is formed (schematically depicted in Figure 4C), binding of HIF-1α CAD to p300 was disrupted by forced overexpression of CBP/p300-interacting transactivator 2 (CITED2), known to negatively regulate HIF function (30). Structural analyses revealed that CITED2 and HIF-1α share an overlapping binding interface in the p300 cysteine–histidine-rich 1 (CH1) domain and competition assays showed a 33-fold higher affinity of CITED2 for binding to p300 CH1 than a corresponding HIF-1α CAD peptide, indicating that CITED2 is a dominant inhibitor of HIF-1α:p300/CBP complex formation (31). HIF-1α CAD:ETV4 interplay was totally abrogated by CITED2 in mammalian two-hybrid experiments (Figure 4D), underscoring the assumption that ETV4 coactivation of HIF-1 requires functional interaction of the latter with p300/CBP.Figure 4.


Synthetic transactivation screening reveals ETV4 as broad coactivator of hypoxia-inducible factor signaling.

Wollenick K, Hu J, Kristiansen G, Schraml P, Rehrauer H, Berchner-Pfannschmidt U, Fandrey J, Wenger RH, Stiehl DP - Nucleic Acids Res. (2011)

Transcriptional cooperation between ETV4 and HIF-1 is disrupted by CITED2. (A) Schematic representation of HIF-1α and ETV4 domain structure and fusion constructs used in mammalian two-hybrid assays. PAS, PER-ARNT-SIM; bHLH, basic helix–loop–helix domain; ODD, oxygen-dependent degradation domain; NRR, negative regulatory region; NAD and CAD, amino-carboxy-terminal activation domain and CADs, respectively. A GAL4-DBD was fused to regions encompassing the HIF-1α NAD and CAD. Full-length ETV4 bearing two activation domains (AD, acidic domain; Ct, carboxy-terminal tail) flanking a central ETS domain was fused to a VP16 activation domain (VP16-AD). Numbers indicate the amino acids present in the respective constructs. (B) U2OS cells were cotransfected with a Gal4-responsive reporter plasmid and Gal4-HIF-1α (GH1α) constructs alone or in combination with VP16-ETV4. The GH1α fusion constructs are specified by the aminoterminal starting amino acid of the truncated HIF-1α regions (530, 740 and 786, respectively). Following transfection, cells were evenly split and incubated at 20 or 0.2% O2 before luciferase activities were determined 24 h later. Noninteracting Gal4 DBD-p53 and VP16-AD-CP1 served as negative control (neg. ctrl.), while the interactions between Gal4 DBD-PHD2 and VP16-AD-HIF-2α(ODD) or VP16-AD-FKBP38 were used as positive controls (pos. ctrl. 1 and pos. ctrl. 2, respectively). (C) Scheme of the potential interactions between HIF-1, p300/CBP and ETV4 as assessed by mammalian two-hybrid assays. Both CITED2 and FIH can block the interaction between HIF-1α and p300/CBP. (D) Cotransfection of the indicated amounts of a CITED2 expression construct together with the mammalian two-hybrid expression vectors followed by hypoxic exposure and luciferase activity determination as described for (B). (E) Cotransfection of siRNA directed against p300 together with the mammalian two-hybrid expression vectors followed by hypoxic exposure and luciferase activity determination as described for (B). The p300 knock down efficiency of different siP300 oligonucleotides was analyzed by immunoblotting (upper panel) and siP300#1 was chosen for further experiments. (F) Cotransfection of siRNA directed against FIH together with the mammalian two-hybrid expression vectors followed by hypoxic exposure and luciferase activity determination as described for (B). The efficiency of the siFIH mediated FIH knock down was confirmed by immunoblotting as shown in the inset. (G) ChIP of normoxic or hypoxic PC3 cells using antibodies directed against HIF-1α or ETV4, or control serum. The amount of coprecipitated chromatin derived from the human P2P region (encoded by EGLN1) containing the HBS was determined by PCR followed by agarose gel electrophoresis.
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gkr978-F4: Transcriptional cooperation between ETV4 and HIF-1 is disrupted by CITED2. (A) Schematic representation of HIF-1α and ETV4 domain structure and fusion constructs used in mammalian two-hybrid assays. PAS, PER-ARNT-SIM; bHLH, basic helix–loop–helix domain; ODD, oxygen-dependent degradation domain; NRR, negative regulatory region; NAD and CAD, amino-carboxy-terminal activation domain and CADs, respectively. A GAL4-DBD was fused to regions encompassing the HIF-1α NAD and CAD. Full-length ETV4 bearing two activation domains (AD, acidic domain; Ct, carboxy-terminal tail) flanking a central ETS domain was fused to a VP16 activation domain (VP16-AD). Numbers indicate the amino acids present in the respective constructs. (B) U2OS cells were cotransfected with a Gal4-responsive reporter plasmid and Gal4-HIF-1α (GH1α) constructs alone or in combination with VP16-ETV4. The GH1α fusion constructs are specified by the aminoterminal starting amino acid of the truncated HIF-1α regions (530, 740 and 786, respectively). Following transfection, cells were evenly split and incubated at 20 or 0.2% O2 before luciferase activities were determined 24 h later. Noninteracting Gal4 DBD-p53 and VP16-AD-CP1 served as negative control (neg. ctrl.), while the interactions between Gal4 DBD-PHD2 and VP16-AD-HIF-2α(ODD) or VP16-AD-FKBP38 were used as positive controls (pos. ctrl. 1 and pos. ctrl. 2, respectively). (C) Scheme of the potential interactions between HIF-1, p300/CBP and ETV4 as assessed by mammalian two-hybrid assays. Both CITED2 and FIH can block the interaction between HIF-1α and p300/CBP. (D) Cotransfection of the indicated amounts of a CITED2 expression construct together with the mammalian two-hybrid expression vectors followed by hypoxic exposure and luciferase activity determination as described for (B). (E) Cotransfection of siRNA directed against p300 together with the mammalian two-hybrid expression vectors followed by hypoxic exposure and luciferase activity determination as described for (B). The p300 knock down efficiency of different siP300 oligonucleotides was analyzed by immunoblotting (upper panel) and siP300#1 was chosen for further experiments. (F) Cotransfection of siRNA directed against FIH together with the mammalian two-hybrid expression vectors followed by hypoxic exposure and luciferase activity determination as described for (B). The efficiency of the siFIH mediated FIH knock down was confirmed by immunoblotting as shown in the inset. (G) ChIP of normoxic or hypoxic PC3 cells using antibodies directed against HIF-1α or ETV4, or control serum. The amount of coprecipitated chromatin derived from the human P2P region (encoded by EGLN1) containing the HBS was determined by PCR followed by agarose gel electrophoresis.
Mentions: Intrigued by the HIF-dependent ETV4 effects, we aimed for the characterization of a putative physical interaction between ETV4 and HIF-1α. Using a mammalian two-hybrid system, expression plasmids encoding for ETV4 fused to the activation domain (AD) of viral protein 16 (VP16-ETV4) were cotransfected with HIF-1α oxygen regulatory domains (21,27) fused to a Gal4-DNA binding domain (DBD; Figure 4A). Due to its intrinsic transactivation activity, constructs containing the CAD of HIF-1α [amino acids 775-826 (27)] were sufficient to activate the Gal4-responsive promoter (Figure 4B). Coexpression of ETV4 strikingly superinduced GH1α740-826 and GH1α786-826, particularly under hypoxic conditions, suggesting that HIF-1α CAD and ETV4 cooperate to transactivate target genes (Figure 4B). ETV4 effects on the aminoterminal activation domain (27) (NAD; HIF-1α amino acids 549-582) were negligible. Both, HIF-1α and ETV4 have been demonstrated to interact with the ubiquitous transcriptional coactivators p300/CBP (28,29). To address the question whether the two factors directly interact or whether a ternary complex between HIF-1, p300/CBP and ETV4 is formed (schematically depicted in Figure 4C), binding of HIF-1α CAD to p300 was disrupted by forced overexpression of CBP/p300-interacting transactivator 2 (CITED2), known to negatively regulate HIF function (30). Structural analyses revealed that CITED2 and HIF-1α share an overlapping binding interface in the p300 cysteine–histidine-rich 1 (CH1) domain and competition assays showed a 33-fold higher affinity of CITED2 for binding to p300 CH1 than a corresponding HIF-1α CAD peptide, indicating that CITED2 is a dominant inhibitor of HIF-1α:p300/CBP complex formation (31). HIF-1α CAD:ETV4 interplay was totally abrogated by CITED2 in mammalian two-hybrid experiments (Figure 4D), underscoring the assumption that ETV4 coactivation of HIF-1 requires functional interaction of the latter with p300/CBP.Figure 4.

Bottom Line: Among others, the E-twenty six transcription factor ETS translocation variant 4 (ETV4) was found to contribute to PHD2 gene expression particularly under hypoxic conditions.Chromatin immunoprecipitation confirmed ETV4 and HIF-1α corecruitment to the PHD2 promoter.Of 608 hypoxically induced transcripts found by genome-wide expression profiling, 7.7% required ETV4 for efficient hypoxic induction, suggesting a broad role of ETV4 in hypoxic gene regulation.

View Article: PubMed Central - PubMed

Affiliation: Institute of Physiology and Zürich Center for Integrative Human Physiology (ZIHP), University of Zürich, 8057 Zürich, Switzerland.

ABSTRACT
The human prolyl-4-hydroxylase domain (PHD) proteins 1-3 are known as cellular oxygen sensors, acting via the degradation of hypoxia-inducible factor (HIF) α-subunits. PHD2 and PHD3 genes are inducible by HIFs themselves, suggesting a negative feedback loop that involves PHD abundance. To identify novel regulators of the PHD2 gene, an expression array of 704 transcription factors was screened by a method that allows distinguishing between HIF-dependent and HIF-independent promoter regulation. Among others, the E-twenty six transcription factor ETS translocation variant 4 (ETV4) was found to contribute to PHD2 gene expression particularly under hypoxic conditions. Mechanistically, complex formation between ETV4 and HIF-1/2α was observed by mammalian two-hybrid and fluorescence resonance energy transfer analysis. HIF-1α domain mapping, CITED2 overexpression and factor inhibiting HIF depletion experiments provided evidence for cooperation between HIF-1α and p300/CBP in ETV4 binding. Chromatin immunoprecipitation confirmed ETV4 and HIF-1α corecruitment to the PHD2 promoter. Of 608 hypoxically induced transcripts found by genome-wide expression profiling, 7.7% required ETV4 for efficient hypoxic induction, suggesting a broad role of ETV4 in hypoxic gene regulation. Endogenous ETV4 highly correlated with PHD2, HIF-1/2α and several established markers of tissue hypoxia in 282 human breast cancer tissue samples, corroborating a functional interplay between the ETV4 and HIF pathways.

Show MeSH
Related in: MedlinePlus