A long hypoxia-inducible factor 3 isoform 2 is a transcription activator that regulates erythropoietin
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Cellular and Molecular Life Sciences
ORIGINAL ARTICLE
A long hypoxia‑inducible factor 3 isoform 2 is a transcription activator that regulates erythropoietin Jussi‑Pekka Tolonen1,2 · Minna Heikkilä1,2 · Marjo Malinen3 · Hang‑Mao Lee2 · Jorma J. Palvimo4 · Gong‑Hong Wei2 · Johanna Myllyharju1,2 Received: 16 April 2019 / Revised: 12 November 2019 / Accepted: 15 November 2019 © The Author(s) 2019
Abstract Hypoxia-inducible factor (HIF), an αβ dimer, is the master regulator of oxygen homeostasis with hundreds of hypoxiainducible target genes. Three HIF isoforms differing in the oxygen-sensitive α 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 human HIF3A mRNA is subject to complex alternative splicing. It was recently shown that the long HIF-3α variants can form αβ dimers that possess transactivation capacity. Here, we show that overexpression of the long HIF-3α2 variant induces the expression of a subset of genes, including the erythropoietin (EPO) gene, while simultaneous downregulation of all HIF-3α variants by siRNA targeting a shared HIF3A region leads to downregulation of EPO and additional genes. EPO mRNA and protein levels correlated with HIF3A silencing and HIF-3α2 overexpression. Chromatin immunoprecipitation analyses showed that HIF-3α2 binding associated with canonical hypoxia response elements in the promoter regions of EPO. Luciferase reporter assays showed that the identified HIF-3α2 chromatin-binding regions were sufficient to promote transcription by all three HIF-α isoforms. Based on these data, HIF-3α2 is a transcription activator that directly regulates EPO expression. Keywords Hypoxia response · Hypoxia-inducible factor 3 isoform · Transcription activator · Erythropoietin · Chromatin immunoprecipitation · Hypoxia response element
Introduction Oxygen-dependent organisms have developed elaborate means to maintain appropriate intracellular oxygen levels for the physicochemical reactions that occur within cells. The master regulators of oxygen homeostasis are the heterodimeric hypoxia-inducible factors (HIFs), present in some form in all metazoan species studied so far [1, 2]. When intracellular oxygen tension decreases below a typical * Johanna Myllyharju [email protected] 1
Oulu Center for Cell–Matrix Research, University of Oulu, PO Box 5400, 90014 Oulu, Finland
2
Biocenter Oulu and Faculty of Biochemistry and Molecular Medicine, University of Oulu, 90014 Oulu, Finland
3
Department of Environmental and Biological Sciences, University of Eastern Finland, 80100 Joensuu, Finland
4
Institute of Biomedicine, University of Eastern Finland, 70211 Kuopio, Finland
concentration, the HIFs initiate and control graded mechanisms to reduce oxygen consumption and to increase oxygen availability via oxygen-dependent regulation of a genetic hypoxia response pathway [3, 4]. Three HIF-α subunit isoforms (HIF-1α, HIF-2, and HIF-3α) have been identified in
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