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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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Both HIF-1α and HIF-2α colocalize with ETV4 to the nucleus within molecular proximity. U2OS cells were transiently transfected with the indicated CFP or YFP plasmids, and FRET analysis was performed at 20% O2 or 1% O2 24–48 h post-transfection. (A) Microscopic images showing the subcellular localization of the exogenous proteins. Fluorescence intensity of FRET signals is visualized by false colors on a color bar from low (blue) to high (white) intensity. (B) FRET efficiencies for CFP-ETV4 and YFP-HIF-1α (upper panel) or YFP-HIF-2α (lower panel) fusion protein pairs were calculated from 20 to 40 randomly selected cells which displayed various fluorescent acceptor/donor ratios. Scatter plots were fit to a single-site binding model. FRET efficiencies are given as the percentage of transferred energy relative to the energy absorbed by the donor.
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gkr978-F5: Both HIF-1α and HIF-2α colocalize with ETV4 to the nucleus within molecular proximity. U2OS cells were transiently transfected with the indicated CFP or YFP plasmids, and FRET analysis was performed at 20% O2 or 1% O2 24–48 h post-transfection. (A) Microscopic images showing the subcellular localization of the exogenous proteins. Fluorescence intensity of FRET signals is visualized by false colors on a color bar from low (blue) to high (white) intensity. (B) FRET efficiencies for CFP-ETV4 and YFP-HIF-1α (upper panel) or YFP-HIF-2α (lower panel) fusion protein pairs were calculated from 20 to 40 randomly selected cells which displayed various fluorescent acceptor/donor ratios. Scatter plots were fit to a single-site binding model. FRET efficiencies are given as the percentage of transferred energy relative to the energy absorbed by the donor.

Mentions: Fluorescence resonance energy transfer (FRET) analyses of coexpressed ETV4 and HIF-1α marked with cyan or yellow fluorescent protein tags (CFP and YFP, respectively) resulted in a robust energy transfer between both factors. Similar FRET efficiencies were observed when YFP-labeled HIF-2α was used together with CFP-ETV4 (Figure 5A and B). The intracellular distance of the two nuclear proteins was calculated to be 5.6–5.7 nm and did not differ in oxygenated or hypoxic cells, which might be explained by saturation of the HIF-α degradation pathways by exogenous overexpression of the transcription factors. Notably, efficient energy transfer between HIF-1α and p300 at ambient oxygen tensions has been reported previously (32).Figure 5.


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)

Both HIF-1α and HIF-2α colocalize with ETV4 to the nucleus within molecular proximity. U2OS cells were transiently transfected with the indicated CFP or YFP plasmids, and FRET analysis was performed at 20% O2 or 1% O2 24–48 h post-transfection. (A) Microscopic images showing the subcellular localization of the exogenous proteins. Fluorescence intensity of FRET signals is visualized by false colors on a color bar from low (blue) to high (white) intensity. (B) FRET efficiencies for CFP-ETV4 and YFP-HIF-1α (upper panel) or YFP-HIF-2α (lower panel) fusion protein pairs were calculated from 20 to 40 randomly selected cells which displayed various fluorescent acceptor/donor ratios. Scatter plots were fit to a single-site binding model. FRET efficiencies are given as the percentage of transferred energy relative to the energy absorbed by the donor.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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gkr978-F5: Both HIF-1α and HIF-2α colocalize with ETV4 to the nucleus within molecular proximity. U2OS cells were transiently transfected with the indicated CFP or YFP plasmids, and FRET analysis was performed at 20% O2 or 1% O2 24–48 h post-transfection. (A) Microscopic images showing the subcellular localization of the exogenous proteins. Fluorescence intensity of FRET signals is visualized by false colors on a color bar from low (blue) to high (white) intensity. (B) FRET efficiencies for CFP-ETV4 and YFP-HIF-1α (upper panel) or YFP-HIF-2α (lower panel) fusion protein pairs were calculated from 20 to 40 randomly selected cells which displayed various fluorescent acceptor/donor ratios. Scatter plots were fit to a single-site binding model. FRET efficiencies are given as the percentage of transferred energy relative to the energy absorbed by the donor.
Mentions: Fluorescence resonance energy transfer (FRET) analyses of coexpressed ETV4 and HIF-1α marked with cyan or yellow fluorescent protein tags (CFP and YFP, respectively) resulted in a robust energy transfer between both factors. Similar FRET efficiencies were observed when YFP-labeled HIF-2α was used together with CFP-ETV4 (Figure 5A and B). The intracellular distance of the two nuclear proteins was calculated to be 5.6–5.7 nm and did not differ in oxygenated or hypoxic cells, which might be explained by saturation of the HIF-α degradation pathways by exogenous overexpression of the transcription factors. Notably, efficient energy transfer between HIF-1α and p300 at ambient oxygen tensions has been reported previously (32).Figure 5.

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