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

Jussi-Pekka Tolonen,Hang-Mao Lee,Jorma J Palvimo,Minna Heikkilä,Gong-Hong Wei,Marjo Malinen,Johanna Myllyharju

doi:10.1007/s00018-019-03387-9

Jussi-Pekka Tolonen, Hang-Mao Lee + Show 5 more

Open Access

https://doi.org/10.1007/s00018-019-03387-9

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Abstract

Hypoxia-inducible factor (HIF), an αβ dimer, is the master regulator of oxygen homeostasis with hundreds of hypoxia-inducible 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.

Highlights

Oxygen-dependent organisms have developed elaborate means to maintain appropriate intracellular oxygen levels for the physicochemical reactions that occur within cells
As the microarray data indicate upregulation of EPO by Hypoxia-inducible factor (HIF)-3α2 overexpression (Fig. 1a), and as we have previously shown that siRNA knockdown of all HIF3A splice variants results in downregulation of EPO mRNA by 39–60% and protein by 28–73% in Hep3B cells [12], we carried out further HIF-3α2 overexpression experiments in Hep3B and the EPO-producing SK-N-AS neuroblastoma cells, as well as siHIF3A knockdown experiments in the SK-N-AS cells
We explored HIF-3 target genes by cDNA microarray analysis in Hep3B cells and concluded that eight genes were upregulated by ≥ 2-fold with HIF-3α2 overexpression and 39 genes were downregulated by ≥ 2-fold with siHIF3A treatment suggesting a HIF-3 specific transcriptional program

Summary

Introduction

Oxygen-dependent organisms have developed elaborate means to maintain appropriate intracellular oxygen levels for the physicochemical reactions that occur within cells. Three HIF-α subunit isoforms (HIF-1α, HIF-2, and HIF-3α) have been identified in vertebrates, encoded by three separate genes (HIF1A, EPAS1, and HIF3A), with HIF3A mRNA being subject to diverse alternative splicing [5–10]. HIF-1α, HIF-2α, and some HIF-3α variants contain basic helix–loop–helix–PAS domains, which facilitate heterodimerization with the HIF-β subunit, encoded by the ARNT gene, and binding to DNA [11]. Towards their C-terminus, the HIF-α protein possess an oxygen-dependent degradation domain (ODDD), which accounts for their oxygendependent regulation. HIF-1α and HIF-2α contain two transactivation domains (NTAD and CTAD), whereas the long HIF3A splicing variants contain only the NTAD [12]

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