A long hypoxia-inducible factor 3 isoform 2 is a transcription activator that regulates erythropoietin

Hypoxia-inducible factor (HIF), an alpha beta dimer, is the master regulator of oxygen homeostasis with hundreds of hypoxia-inducible target genes. Three HIF isoforms differing in the oxygen-sensitive alpha subunit exist in vertebrates. While HIF-1 and HIF-2 are known transcription activators, HIF-3 has been considered a negative regulator of the hypoxia response pathway. However, the humanHIF3AmRNA is subject to complex alternative splicing. It was recently shown that the long HIF-3 alpha variants can form alpha beta dimers that possess transactivation capacity. Here, we show that overexpression of the long HIF-3 alpha 2 variant induces the expression of a subset of genes, including the erythropoietin (EPO) gene, while simultaneous downregulation of all HIF-3 alpha variants by siRNA targeting a sharedHIF3Aregion leads to downregulation ofEPOand additional genes. EPO mRNA and protein levels correlated withHIF3Asilencing and HIF-3 alpha 2 overexpression. Chromatin immunoprecipitation analyses showed that HIF-3 alpha 2 binding associated with canonical hypoxia response elements in the promoter regions ofEPO. Luciferase reporter assays showed that the identified HIF-3 alpha 2 chromatin-binding regions were sufficient to promote transcription by all three HIF-alpha isoforms. Based on these data, HIF-3 alpha 2 is a transcription activator that directly regulatesEPOexpression.