• Genome-wide association studies identified regulatory elements controlling erythropoietin gene expression • Anemia-associated polymorphisms affect known and novel EPO hypoxia response elements The gene encoding erythropoietin ( EPO ) is induced by hypoxia via multiple hypoxia response elements (HREs) located in distal upstream, proximal promoter and distal downstream regions. Hypoxia-inducible factor (HIF)-2 interacts with these HREs and upregulates Epo production to enhance erythropoiesis under hypoxemic conditions. While the 3' HRE regulating hepatic EPO expression is well established, the functional relevance of the upstream/promoter HREs for renal EPO expression remained controversial. We analyzed three novel single-nucleotide polymorphisms (SNPs) that are strongly associated with decreased red blood cell traits. These SNPs reside within potential HREs and abrogated ectopic reporter gene induction by hypoxia. In the -9.2 kb risk allele, the normal core HRE (TACGTG) was converted to a palindromic HRE (CACGTG) by the SNP. Because palindromic HREs are commonly involved in the regulation of other HIF target genes, we swapped DNA elements between the non-functional -9.2 kb HRE and a functional palindromic HRE in reporter gene experiments. However, even when the hypoxia ancillary sequence (HAS) was considered, the palindromic EPO HRE remained inactive. Following gene editing of Kelly neuroblastoma cells, the HRE-destructive -3.9 kb but not the palindromic -9.2 kb SNP abolished hypoxic induction of endogenous Epo mRNA and protein. This discrepancy between reporter gene and chromatin contexts may be related to the previously reported role of the -9.2 kb HRE in HIF-2 interaction with EPO promoter and 3' HREs. In conclusion, anemia-associated SNPs defined novel functional elements regulating the EPO gene and provided new mechanistic insights into the role of the -9.2 kb HRE.
Burger et al. (Sun,) studied this question.