Tibetan PHD2, an allele with loss-of-function properties

Abstract

Tibetans have adapted to the chronic hypoxia of high altitude and display a distinctive suite of physiologic adaptations, including augmented hypoxic ventilatory response and resistance to pulmonary hypertension. Genome-wide studies have consistently identified compelling genetic signatures of natural selection in two genes of the Hypoxia Inducible Factor pathway, PHD2 and HIF2A The product of the former induces the degradation of the product of the latter. Key issues regarding Tibetan PHD2 are whether it is a gain-of-function or loss-of-function allele, and how it might contribute to high-altitude adaptation. Tibetan PHD2 possesses two amino acid changes, D4E and C127S. We previously showed that in vitro, Tibetan PHD2 is defective in its interaction with p23, a cochaperone of the HSP90 pathway, and we proposed that Tibetan PHD2 is a loss-of-function allele. Here, we report that additional PHD2 mutations at or near Asp-4 or Cys-127 impair interaction with p23 in vitro. We find that mice with the Tibetan Phd2 allele display augmented hypoxic ventilatory response, supporting this loss-of-function proposal. This is phenocopied by mice with a mutation in p23 that abrogates the PHD2:p23 interaction. Hif2a haploinsufficiency, but not the Tibetan Phd2 allele, ameliorates hypoxia-induced increases in right ventricular systolic pressure. The Tibetan Phd2 allele is not associated with hemoglobin levels in mice. We propose that Tibetans possess genetic alterations that both activate and inhibit selective outputs of the HIF pathway to facilitate successful adaptation to the chronic hypoxia of high altitude.

Keywords: EGLN1; EPAS1; HIF; PHD2; high-altitude adaptation.

Fig. 1.

Effect of mutations in vicinity of PHD2 Asp-4 and Cys-127 on the PHD2:p23 interaction. (A) The HIF pathway. See text for details. VHL, von Hippel Lindau protein. For simplicity, Aryl Hydrocarbon Nuclear Receptor Translocator has been omitted. (B) Diagram of PHD2, showing locations of zinc finger (ZF) domain, prolyl hydroxylase (PH) domain, Asp-4 (diamond), and Cys-127 (triangle). Sequence of residues 1 to 7 and 121 to 133 are shown at the bottom, with Asp-4 and Cys-127 as indicated. (C) The HSP90 pathway. See text for details. (D) Model for zinc finger-dependent recruitment of PHD2 to the HSP90 pathway to facilitate HIF-α hydroxylation. PLXE motifs in p23 and FKBP38 are shown, as are Asp-4 and Cys-127 in PHD2. (E, F, and H) HEK293 FT cells were transfected with expression plasmids for the indicated proteins. The cells were lysed and subjected to immunoprecipitation with anti-Flag antibodies, and then the immunoprecipitates and aliquots of lysate were examined by Western blotting, as indicated. In H, the D4E and C127S substitutions refer to the native amino acid sequence. (G) Diagram of internal deletion mutants of PHD2.

Fig. 2.

Phd2 ^Tib gene targeting strategy. (A) Numbers directly above exons denote exon number. Italicized numbers either above or below exons indicate percentage identity of amino acid sequence encoded by exon with that of the corresponding Tibetan exon. (B) Hepa 1-6 cells were transfected with expression plasmids for the indicated proteins. hmPHD2 denotes a chimeric protein consisting of human PHD2 (1 to 297) and mouse Phd2 (275 to 400). The amino acid sequence of this humanized WT Phd2 is provided in SI Appendix, Fig. S2. The cells were lysed and subjected to immunoprecipitation with anti-Flag antibodies, and then the immunoprecipitates and aliquots of lysate were examined by Western blotting, as indicated. (C) DNA sequencing chromatograms of tail DNA confirming D4E and C127S substitutions. The sequence is from 3ʹ to 5ʹ (reverse complement). Amino acids 4 and 127 are as indicated. (D) Immortalized mouse embryonic fibroblasts with the indicated genotypes were lysed and incubated with either control mAb or anti-PHD2 mAb 6.9, and immunoprecipitations were performed with protein G-agarose. The immunoprecipitates and aliquots of the lysate were then examined by western blotting with anti-p23 antibodies.

Fig. 3.

Increased HVR in Tibetan Phd2 mice. (A) Model for Tibetan PHD2. Compared with wild-type PHD2, Tibetan PHD2 has a defect in its interaction with p23. Hence, Tibetan PHD2 hydroxylates HIF-α less efficiently than wild-type PHD2 (as indicated by difference in thickness of the arrow from PHD2 to HIF-α). (B) Minute ventilation under normoxia (21% O₂/3% CO₂) was compared between Phd2 ^WT/WT and Phd2 ^Tib/Tib mice. (C–E) Mice were acutely exposed to 12% O₂/3% CO₂, and HVR (C), tidal volume change (D), and respiratory frequency change (E) were measured. In B–E, mice were 2 to 4 mo of age, with n = 18 to 19 per group. *P < 0.05. P values were as follows: (B) 0.246, (C) 0.020, (D) 0.018, (E) 0.493.

Fig. 4.

Increased HVR in p23 ^AA/AA and Phd2 ^Tib/Tib;Fkbp38 ^AA/AA mice. (A) The recruitment of PHD2 to HSP90 pathway (Left) is impaired by PXLE > PXAA mutations in p23 or Fkbp38 (Right). (B and C) HEK293 FT cells were transfected with expression plasmids for the indicated proteins. The cells were lysed and subjected to immunoprecipitation with anti-Flag antibodies, and then the immunoprecipitates and aliquots of lysate were examined by Western blotting as indicated. (D) DNA sequencing chromatograms of tail DNA from p23 ^AA/AA and Fkbp38 ^AA/AA mice. The sequences are from (Left) 5ʹ to 3ʹ and (Right) 3ʹ to 5ʹ. Mutated amino acids are in bold. (E and F) Mice with the indicated genotypes were maintained in normoxia (21% O₂/3% CO₂) and then acutely exposed to 12% O₂/3% CO₂, and HVR were measured. In E, mice were 2 to 3 mo of age, with n = 14 per group. In F, mice were 2 to 3 mo of age, with n = 25 to 26 per group. *P < 0.05; **P < 0.01. P values were as follows: E 0.007, F 0.030.

Fig. 5.

Hypoxic Hb and RVSP responses in Tibetan Phd2 mice with or without Hif2a haploinsufficiency. (A and B) Mice with indicated genotypes were exposed to 3 wk of hypoxia (12% O₂). Hb (A) or RVSP (B) was then measured. In A, mice were 3 to 9 mo of age, with n = 9 to 10 per group. In B, mice were 6 to 9 mo of age, with n = 7 to 9 per group. *P < 0.05; ns = nonsignificant (P > 0.05). (C) Model for reconfiguration of the HIF pathway in Tibetans. Low-altitude inhabitants at low altitude (normoxia) have high PHD2 activity and low HIF-2α protein levels. At high altitude (hypoxia), PHD2 activity decreases, resulting in high HIF-2α protein levels. Tibetans harbor a hypomorphic PHD2 allele that leads to augmented HVR. Tibetans also possess a hypomorphic HIF-2α allele that blunts selective aspects of the hypoxic response, including the right ventricular pressure and erythropoietic responses. Sizes of PHD2 and HIF-2α icons indicate their relative activity. P values were as follows: (A) Phd2 ^WT/WT vs. Phd2 ^Tib/Tib = 0.999. Phd2 ^WT/WT; Hif2a ^+/− vs. Phd2 ^Tib/Tib; Hif2a ^+/− = 0.932. (B) Phd2 ^WT/WT vs. Phd2 ^Tib/Tib = 0.949. Phd2 ^WT/WT; Hif2a ^+/− vs. Phd2 ^Tib/Tib; Hif2a ^+/− = 0.852. Phd2 ^WT/WT vs. Phd2 ^WT/WT; Hif2a ^+/− = 0.010. Phd2 ^Tib/Tib vs. Phd2 ^Tib/Tib; Hif2a ^+/− = 0.005.