Molecular Basis of Hypoxia-Induced Excessive Erythrocytosis: Critical Role of SENP1 gene Regulation

Since polycythemia is a predominant trait in some high altitude dwellers (Chronic Mountain Sickness, CMS, or Monge's disease) but not others living at the same altitude in the Andes, we took advantage of this human "experiment in nature" and studied both populations (with CMS and without, non-CMS). Although polycythemia could be advantageous at high altitude because it increases O₂-carrying capacity, this adaptive pattern to high altitude has deleterious effects since blood increases its viscosity and induces serious morbidities, such as myocardial infarction and stroke in young adults. In order to understand the molecular basis for polycythemia of high altitude, we generated a disease in-the dish-model by re-programming fibroblasts from CMS and non-CMS subjects. Furthermore, we have obtained candidate genes mined from whole genome sequencing of subjects from this region (CMS and non-CMS) we have already performed. In this study, by manipulating expression of some of these genes in our in-vitro model system, we delineate the functional basis and relevance of such candidate genes for a better molecular understanding of this extreme phenotype. As compared to sea level controls and non-CMS subjects who responded to hypoxia by increasing their RBCs modestly or not at all, CMS cells increased theirs remarkably (up to 60 fold) with a dose-dependent response to graded hypoxia (1.5, 5, 10% O₂). When we knocked down SENP1 (a desumoylase) in CMS iPS cells (using lentiviral constructs) we observed a striking reduction (>90%) of the CMS excessive erythropoietic response to low O₂with an elimination of this extreme phenotype. And, by over-expressing SENP1 in non-CMS iPS cells, the hypoxic response in these subjects increased enormously. By further analyzing RBC differentiation in hypoxia, we found that VEGF, GATA1 and Bcl-xL increased their gene expression in the CMS erythroid cells, in contrast to their minimal expression in the other two populations. We demonstrate also that, by utilizing a SUMO-GATA1 fused construct, GATA1 desumoylation, a target of SENP1, is critical for the CMS phenotype. Unlike GATA1, the over-expression of the anti-apoptotic gene Bcl-xL (a GATA1 target), only partially rescued the blunted erythroid response to hypoxia in non-CMS cells. Furthermore, by using bioinformatics tools, we have identified the potentially likely causal SNP(s) (out of the 66 differential SNPs) that are responsible for upregulation of SENP1 in the CMS cells under hypoxia. Only 7 out of these 95 SNPs had CADD-score with p values of less than 0.1. These 7 SNPs are rs60629297, rs7959755, rs17122612, rs72644843, rs11609399, rs11613781, rs10783232 (Fig. 8). The only missense variant in this list is rs11609399. It is interesting to note that differential SNP rs726354 coincides with binding sites of transcriptional factors such as E2F1, POLR2A, GABPA, TAF1, ELF1, GATA1. Indeed, when we compared the transcriptional response of CMS to that of non-CMS cells in hypoxia, we found that CMS cells up-regulated SENP1, suggesting that this up-regulation could underlie the excessive erythrocytosis present in the CMS population (Zhou et al., 2013). We conclude that the increased erythropoietic sensitivity to hypoxia in CMS subjects is genetic in nature and depends on an increase in SENP1 expression and its desumoylation mediated activation of GATA1 under hypoxia.By combining the iPS technology with this unique Andean population that has adapted (or mal-adapted) to chronic hypoxia over thousands of years, we have built an important in-vitro model. We believe that the utility of this model and findings lie not only for studying hypoxia-induced polycythemia at high altitude but also for other hypoxia-driven diseases experienced at sea level.