High-altitude deer mouse hypoxia-inducible factor-2α shows defective interaction with CREB-binding protein

Abstract

Numerous mammalian species have adapted to the chronic hypoxia of high altitude. Recent genomic studies have identified evidence for natural selection of genes and associated genetic changes in these species. A major gap in our knowledge is an understanding of the functional significance, if any, of these changes. Deer mice (Peromyscus maniculatus) live at both low and high altitudes in North America, providing an opportunity to identify functionally important genetic changes. High-altitude deer mice show evidence of natural selection on the Epas1 gene, which encodes for hypoxia-inducible factor-2α (Hif-2α), a central transcription factor of the hypoxia-inducible factor pathway. An SNP encoding for a T755M change in the Hif-2α protein is highly enriched in high-altitude deer mice, but its functional significance is unknown. Here, using coimmunoprecipitation and transcriptional activity assays, we show that the T755M mutation produces a defect in the interaction of Hif-2α with the transcriptional coactivator CREB-binding protein. This results in a loss of function because of decreased transcriptional activity. Intriguingly, the effect of this mutation depends on the amino acid context. Interchanges between methionine and threonine at the corresponding position in house mouse (Mus musculus) Hif-2α are without effects on CREB-binding protein binding. Furthermore, transfer of a set of deer mouse–specific Hif-2α amino acids to house mouse Hif-2α is sufficient to confer sensitivity of house mouse Hif-2α to the T755M substitution. These findings provide insight into high-altitude adaptation in deer mice and evolution at the Epas1 locus. Numerous mammalian species have adapted to the chronic hypoxia of high altitude. Recent genomic studies have identified evidence for natural selection of genes and associated genetic changes in these species. A major gap in our knowledge is an understanding of the functional significance, if any, of these changes. Deer mice (Peromyscus maniculatus) live at both low and high altitudes in North America, providing an opportunity to identify functionally important genetic changes. High-altitude deer mice show evidence of natural selection on the Epas1 gene, which encodes for hypoxia-inducible factor-2α (Hif-2α), a central transcription factor of the hypoxia-inducible factor pathway. An SNP encoding for a T755M change in the Hif-2α protein is highly enriched in high-altitude deer mice, but its functional significance is unknown. Here, using coimmunoprecipitation and transcriptional activity assays, we show that the T755M mutation produces a defect in the interaction of Hif-2α with the transcriptional coactivator CREB-binding protein. This results in a loss of function because of decreased transcriptional activity. Intriguingly, the effect of this mutation depends on the amino acid context. Interchanges between methionine and threonine at the corresponding position in house mouse (Mus musculus) Hif-2α are without effects on CREB-binding protein binding. Furthermore, transfer of a set of deer mouse–specific Hif-2α amino acids to house mouse Hif-2α is sufficient to confer sensitivity of house mouse Hif-2α to the T755M substitution. These findings provide insight into high-altitude adaptation in deer mice and evolution at the Epas1 locus. The chronic hypoxia of high altitude presents a substantial challenge to metazoans residing in this environment. Recent studies have revealed evidence of genetic adaptation to high altitude in multiple mammalian species. These include humans who reside on the Tibetan plateau, the Andean Altiplano, and Ethiopian Simien Mountains (1Bigham A.W. Lee F.S. Human high-altitude adaptation: Forward genetics meets the HIF pathway.Genes Dev. 2014; 28: 2189-2204Crossref PubMed Scopus (153) Google Scholar), as well as Tibetan dogs, yaks, sheep, and horses (2Witt K.E. Huerta-Sanchez E. Convergent evolution in human and domesticate adaptation to high-altitude environments.Philos. Trans. R. Soc. Lond. B Biol. Sci. 2019; 374: 20180235Crossref PubMed Scopus (34) Google Scholar, 3Pamenter M.E. Hall J.E. Tanabe Y. Simonson T.S. Cross-species insights into genomic adaptations to hypoxia.Front. Genet. 2020; 11: 743Crossref PubMed Scopus (11) Google Scholar). In many of these species, there is evidence for natural selection acting on genes of the hypoxia-inducible factor (HIF) pathway, the main transcriptional pathway by which cells respond to hypoxia (4Kaelin Jr., W.G. Ratcliffe P.J. Oxygen sensing by metazoans: The central role of the HIF hydroxylase pathway.Mol. 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HIF-α dimerizes with aryl hydrocarbon receptor nuclear translocator through their basic Helix Loop Helix-Period Arnt Sim domains to form a transcription factor complex. The other critical oxygen-dependent enzyme in this pathway is factor inhibiting HIF (FIH), which catalyzes asparaginyl hydroxylation in the C-terminal transactivation domain (CTAD) of HIF-α (10Cockman M.E. Webb J.D. Ratcliffe P.J. FIH-dependent asparaginyl hydroxylation of ankyrin repeat domain-containing proteins.Ann. N. Y. Acad. Sci. 2009; 1177: 9-18Crossref PubMed Scopus (62) Google Scholar, 11Strowitzki M.J. Cummins E.P. Taylor C.T. Protein hydroxylation by hypoxia-inducible factor (HIF) hydroxylases: Unique or ubiquitous?.Cells. 2019; 8: 384Crossref PubMed Google Scholar, 12Lando D. Peet D.J. Whelan D.A. Gorman J.J. Whitelaw M.L. Asparagine hydroxylation of the HIF transactivation domain a hypoxic switch.Science. 2002; 295: 858-861Crossref PubMed Scopus (1225) Google Scholar). This hydroxylation blocks the interaction of the CTAD with CREB-binding protein (CBP), a transcriptional coactivator with lysine acetyltransferase activity. CBP is a component of the transcription preinitiation complex, and it acetylates lysines 27 and 18 on histone H3, marks characteristic of active chromatin (13Attar N. Kurdistani S.K. Exploitation of EP300 and CREBBP lysine acetyltransferases by Cancer.Cold Spring Harb. Perspect. Med. 2017; 7a026534Crossref PubMed Scopus (82) Google Scholar). Under hypoxia, asparaginyl hydroxylation is arrested, leading to the binding of HIF-α to CBP and activation of the CTAD. HIF activates hundreds of genes involved in cellular and systemic responses to hypoxia (14Schodel J. Mole D.R. Ratcliffe P.J. Pan-genomic binding of hypoxia-inducible transcription factors.Biol. Chem. 2013; 394: 507-517Crossref PubMed Scopus (54) Google Scholar). HIF-1α, which is ubiquitously expressed, upregulates the genes of glycolysis and thereby promotes a shift from oxidative phosphorylation to anaerobic glycolysis. HIF-2α, with a more restricted expression that is tissue and cell type specific, plays a critical role in other aspects of the hypoxic response (15Lappin T.R. Lee F.S. Update on mutations in the HIF: EPO pathway and their role in erythrocytosis.Blood Rev. 2019; 37: 100590Crossref PubMed Scopus (28) Google Scholar, 16Wenger R.H. Hoogewijs D. Regulated oxygen sensing by protein hydroxylation in renal erythropoietin-producing cells.Am. J. Physiol. Renal Physiol. 2010; 298: F1287-F1296Crossref PubMed Scopus (63) Google Scholar, 17Keith B. Johnson R.S. Simon M.C. HIF1alpha and HIF2alpha: Sibling rivalry in hypoxic tumour growth and progression.Nat. Rev. Cancer. 2012; 12: 9-22Crossref Scopus (1117) Google Scholar, 18Tan Q. Kerestes H. Percy M.J. Pietrofesa R. Chen L. Khurana T.S. Christofidou-Solomidou M. Lappin T.R. Lee F.S. Erythrocytosis and pulmonary hypertension in a mouse model of human HIF2A gain of function mutation.J. Biol. Chem. 2013; 288: 17134-17144Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). North American deer mice (Peromyscus maniculatus) reside at both low and high altitudes, providing an opportunity to examine genes that might facilitate hypoxic adaption (19Storz J.F. Cheviron Z.A. McClelland G.B. Scott G.R. Evolution of physiological performance capacities and environmental adaptation: Insights from high-elevation deer mice (Peromyscus maniculatus).J. Mammal. 2019; 100: 910-922Crossref PubMed Scopus (31) Google Scholar). One mechanism by which this occurs is through amino acid substitutions in the α and β chains of hemoglobin that increase binding affinity for oxygen (20Storz J.F. Moriyama H. Mechanisms of hemoglobin adaptation to high altitude hypoxia.High Alt. Med. Biol. 2008; 9: 148-157Crossref PubMed Scopus (124) Google Scholar). It has recently been reported that the Epas1 gene, which encodes for Hif-2α, is under natural selection in high-altitude deer mice (21Schweizer R.M. Velotta J.P. Ivy C.M. Jones M.R. Muir S.M. Bradburd G.S. Storz J.F. Scott G.R. Cheviron Z.A. Physiological and genomic evidence that selection on the transcription factor Epas1 has altered cardiovascular function in high-altitude deer mice.PLoS Genet. 2019; 15e1008420Crossref PubMed Scopus (20) Google Scholar). The Epas1 gene is the target of selection in multiple high-altitude species (2Witt K.E. Huerta-Sanchez E. Convergent evolution in human and domesticate adaptation to high-altitude environments.Philos. Trans. R. Soc. Lond. B Biol. Sci. 2019; 374: 20180235Crossref PubMed Scopus (34) Google Scholar, 3Pamenter M.E. Hall J.E. Tanabe Y. Simonson T.S. Cross-species insights into genomic adaptations to hypoxia.Front. Genet. 2020; 11: 743Crossref PubMed Scopus (11) Google Scholar). However, there is a dearth of knowledge regarding mechanisms by which high-altitude Epas1 alleles might be adaptive. In deer mice, the high-altitude allele is correlated with decreased expression of adrenal catecholamine synthesis genes and an increased heart rate under hypoxic conditions. The highest ranking SNP in the Epas1 gene resides in exon 14 and encodes for a T755M polymorphism. This SNP is not present in deer mice at sea level, and it rises to a frequency of >0.8 in deer mouse populations at high altitude (21Schweizer R.M. Velotta J.P. Ivy C.M. Jones M.R. Muir S.M. Bradburd G.S. Storz J.F. Scott G.R. Cheviron Z.A. Physiological and genomic evidence that selection on the transcription factor Epas1 has altered cardiovascular function in high-altitude deer mice.PLoS Genet. 2019; 15e1008420Crossref PubMed Scopus (20) Google Scholar). In the primary sequence, amino acid 755 resides between and is distant from the ODD domain and CTAD. Therefore, it is not intuitively obvious whether the polymorphism has any functional significance. There are no other Epas1 SNPs under selection that produce a change in the amino acid sequence. Here we provide evidence that high-altitude deer mouse Hif-2α is a loss of function allele that produces a defect in the interaction of Hif-2α with Cbp. This provides a framework for understanding high-altitude adaptation in deer mice and shows a naturally occurring mutation in a mammalian Epas1 gene that affects the function of the CTAD of Hif-2α. More broadly, it provides important information for understanding convergent evolution at the Epas1 locus in mammalian high-altitude species. To test whether the T755M mutation affects protein stability, we transfected HEK293FT cells with constructs for WT and T755M deer mouse Hif-2α, exposed some cells to hypoxia (0.5% O2), and then examined protein levels by Western blotting. We did not observe any significant difference in protein levels between WT and T755M deer mouse Hif-2α under either normoxia or hypoxia (Fig. 1A, lanes 2 and 3, and lanes 5 and 6). The ODD domain of Hif-2α interacts with two proteins—Phd2 when not hydroxylated, and Vhl when hydroxylated. We examined these interactions. We were not able to detect binding between Hif-2α and Phd2 under hypoxia (data not shown). As an alternative, we used dimethyloxalylglycine, an active site inhibitor of 2-oxoglutarate dependent enzymes (which includes Phd2), that allows for the formation of enzyme:substrate complexes (22Cockman M.E. Webb J.D. Kramer H.B. Kessler B.M. Ratcliffe P.J. Proteomics-based identification of novel factor inhibiting hypoxia-inducible factor (FIH) substrates indicates widespread asparaginyl hydroxylation of ankyrin repeat domain-containing proteins.Mol. Cell. Proteomics. 2009; 8: 535-546Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). We transfected cells with WT or T755M deer mouse Hif-2α constructs along with one for Phd2, exposed cells to dimethyloxalylglycine, and examined Phd2 immunoprecipitates for Hif-2α. We do not see any appreciable differences in the interaction of Phd2 with WT and T755M deer mouse Hif-2α (Fig. 1, B and C). To examine the binding of Vhl with hydroxylated Hif-2α, we transfected cells with the Hif-2α constructs along with one encoding for deer mouse Vhl. We treated cells with the proteasome inhibitor MG-132 to stabilize the prolyl hydroxylated from of Hif-2α. We observe no significant differences in the amount of WT and T755M Hif-2α recovered in Vhl immunoprecipitates (Fig. 1, D and E). The CTAD (residues 834–874) of Hif-2α can bind Fih. We fused WT or T755M deer mouse Hif-2α (669–874), which contains the CTAD, to the DNA-binding domain of the GAL4 protein. We transfected cells with these constructs along with one for deer mouse Fih and exposed cells to hypoxia (0.5% O2) to stabilize the nonhydroxylated form of Hif-2α. Hif-2α (669–874) is detectable in Fih immunoprecipitates, and we do not see a difference in the interaction with Fih between WT and T755M deer mouse Hif-2α (669–874) (Fig. 1F, lanes 2 and 4). In its nonhydroxylated form, the CTAD can interact with Cbp through the zinc finger–containing CH1 (also known as TAZ1) domain of the latter (23Dames S.A. Martinez-Yamout M. De Guzman R.N. Dyson H.J. Wright P.E. Structural basis for Hif-1 alpha/CBP recognition in the cellular hypoxic response.Proc. Natl. Acad. Sci. U. S. A. 2002; 99: PubMed Scopus Google Scholar). We transfected cells with the Hif-2α (669–874) constructs along with one for the CH1 domain of deer mouse Cbp (residues fused to We exposed the cells to hypoxia (0.5% O2) to hydroxylation and examined Cbp immunoprecipitates for Hif-2α. In to the with Phd2, and the T755M mutation in Hif-2α the interaction with Cbp (Fig. We the with WT and T755M Hif-2α (Fig. of these Western a Hif-2α in the Cbp Therefore, the mutation is that it produces a but not loss of studies have that an at the site of hydroxylation in deer mouse allows Cbp binding and CTAD activation under normoxia D. Peet D.J. Whelan D.A. Gorman J.J. Whitelaw M.L. Asparagine hydroxylation of the HIF transactivation domain a hypoxic switch.Science. 2002; 295: 858-861Crossref PubMed Scopus (1225) Google Scholar). studies using Hif-2α (669–874) show that this in Hif-2α allows binding under normoxia in a to the T755M mutation (Fig. lanes 2 and 4). We the interaction of these two Hif-2α (669–874) with Cbp in a cellular by mouse cells with the Hif-2α (669–874) constructs and then the Hif-2α. The T755M mutation the interaction with Cbp in this (Fig. lanes 2 and The protein is a of Cbp with a zinc that interacts with the CTAD M.J. Structural basis for of by hypoxia-inducible Natl. Acad. Sci. U. S. A. 2002; 99: PubMed Scopus Google Scholar). We examined the interaction of Hif-2α (669–874) with the CH1 zinc domain of deer mouse (residues In to binding is with the T755M mutation (Fig. lanes 2 and 4). we the that there might be effects on the binding of the T755M to Phd2, or the results that the T755M mutation in deer mouse Hif-2α interaction with Cbp. To transcriptional we transfected cells with constructs for mutations at the prolyl and asparaginyl that to activity under normoxia of Hif-2α activity in the of along with a gene by of hypoxia response from the gene Q. Lee F.S. interaction of hypoxia-inducible with the von 2001; Google Scholar). In the of the the T755M of deer mouse Hif-2α has transcriptional activity WT (Fig. We examined the transcriptional activity of the (669–874) protein the under normoxia and hypoxia with the of a gene by GAL4 binding gene activity from the WT Hif-2α (669–874) protein, and the T755M mutation activity under either normoxia or hypoxia (Fig. The activity of Hif-2α is in these gene assays, with the to loss of interaction with Cbp and the of interaction with We the effect of the T755M mutation on Hif-2α gene targets by cells WT and T755M deer mouse Hif-2α from an locus the (Fig. 3, and These two constructs were in the to under normoxia and from Deer mouse Hif-2α transcriptional activation of the target genes and (Fig. 3, the T755M mutation activation of these The deer mouse mutation changes 755 to an amino that is the amino acid at 755 in house mouse (Mus musculus) Hif-2α We examined the effect of the on the interaction between house mouse Hif-2α with house mouse Cbp the of which is to deer mouse Cbp In to deer mouse Hif-2α, we that both WT and house mouse Hif-2α interact with Cbp (Fig. lanes 2 and 4). Therefore, the between methionine and threonine at this position in house mouse Hif-2α the functional effect that is in deer mouse Hif-2α. amino acid deer mouse Hif-2α (669–874) from house mouse Hif-2α at (Fig. This that the loss of function effect of the T755M mutation in Hif-2α is in the of amino acid substitutions that are present in deer mouse Hif-2α We constructs in which of deer mouse Hif-2α (669–874) were into the corresponding of house mouse Hif-2α (residues to which confer sensitivity to the T755M mutation (Fig. We to that the of between deer mouse and house mouse Hif-2α and and of The two of and to house mouse Hif-2α that the T755M mutation Cbp binding (Fig. In with H3, the T755M mutation binding (Fig. lanes and In in which the deer mouse of into and we that the T755M mutation binding to Cbp in both that the effect is (Fig. This that multiple deer mouse amino acid substitutions in (which includes are to confer sensitivity of Cbp binding in house mouse Hif-2α to the of methionine at Under prolyl and asparaginyl hydroxylation Hif-2α (Fig. under hypoxia, this is (Fig. We that the North American high-altitude deer mouse T755M mutation in Hif-2α produces a loss of function effect via binding to Cbp (Fig. In the corresponding SNP in the Epas1 gene is associated with decreased levels of catecholamine gene expression in the adrenal (21Schweizer R.M. Velotta J.P. Ivy C.M. Jones M.R. Muir S.M. Bradburd G.S. Storz J.F. Scott G.R. Cheviron Z.A. Physiological and genomic evidence that selection on the transcription factor Epas1 has altered cardiovascular function in high-altitude deer mice.PLoS Genet. 2019; 15e1008420Crossref PubMed Scopus (20) Google Scholar). that is for catecholamine synthesis in cells J.M. factor is for the of the of 2009; PubMed Scopus Google Scholar), our results provide a for The might to increased heart which has been (21Schweizer R.M. Velotta J.P. Ivy C.M. Jones M.R. Muir S.M. Bradburd G.S. Storz J.F. Scott G.R. Cheviron Z.A. Physiological and genomic evidence that selection on the transcription factor Epas1 has altered cardiovascular function in high-altitude deer mice.PLoS Genet. 2019; 15e1008420Crossref PubMed Scopus (20) Google Scholar). 755 is in a of deer mouse Hif-2α that resides between the ODD domain and CTAD (Fig. and is not as highly conserved as either the ODD domain or CTAD. We that the differences in the of and at this position are dependent and in deer but not house Hif-2α. This depends on a in deer mouse Hif-2α (residues that includes multiple amino acid This an of J.F. mutations and for protein Biol. PubMed Scopus (31) Google Scholar), to the that has been in high-altitude deer mouse hemoglobin N. A. Moriyama H. Storz J.F. mutations in deer mouse 2013; PubMed Scopus Google Scholar). We the Deer mouse and house mouse Hif-2α from a (Fig. The is by the other amino acids are by deer mouse Hif-2α amino acid substitutions that are by for and for the other amino acid changes (Fig. The in deer mice is under but is at a high altitude. The provide a genetic in which a of to the allows the of a functional change in Hif-2α at high altitude the In a between and is is for the in Hif-2α in which and it is that it might be a of Hif-2α to in human which is in a of between a zinc and the domain of a in in the of the WT protein, is without an appreciable effect in However, in the of a mutation that is to the zinc the results in interaction of with the leading to loss of function D. Q. A.W. Lee F.S. Tibetan binding to high altitude adaptation to altered oxygen Biol. Chem. 2014; Full Text Full Text PDF PubMed Scopus Google Scholar, D. E. L. Percy M.J. J. Khurana T.S. A.W. Lappin T.R. et an allele with Natl. Acad. Sci. U. S. A. 2020; PubMed Scopus (11) Google Scholar). 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Lee F.S. mutations a critical role for C-terminal to the Biol. Chem. 2009; Full Text Full Text PDF PubMed Scopus Google Scholar, A. J. of and C-terminal of HIF1alpha and to their target gene J. Sci. 2020; Scopus Google Scholar). The that have no of with the of this D. S. and S. L. D. S. D. A. and S. L. D. S. and S. L. A. B. and S. L. and A. B. This by S. and S. L. and A. and a from the of The is the of the and not the of the of

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