Hypoxic Induction of the Regulator of G-Protein Signalling 4 Gene Is Mediated by the Hypoxia-Inducible Factor Pathway Sam W. Z. Olechnowicz 1,2¤ , Anthony O. Fedele 3 , Daniel J. Peet 1,2 * 1 School of Molecular and Biomedical Science, University of Adelaide, Adelaide, Australia, 2 ARC Special Research Centre for the Molecular Genetics of Development, University of Adelaide, Adelaide, Australia, 3 Lysosomal Diseases Research Unit (LDRU), SA Pathology, North Adelaide, Australia Abstract The transcriptional response to hypoxia is largely dependent on the Hypoxia Inducible Factors (HIF-1 and HIF-2) in mammalian cells. Many target genes have been characterised for these heterodimeric transcription factors, yet there is evidence that the full range of HIF-regulated genes has not yet been described. We constructed a TetON overexpression system in the rat pheochromocytoma PC-12 cell line to search for novel HIF and hypoxia responsive genes. The Rgs4 gene encodes the Regulator of G-Protein Signalling 4 (RGS4) protein, an inhibitor of signalling from G-protein coupled receptors, and dysregulation of Rgs4 is linked to disease states such as schizophrenia and cardiomyopathy. Rgs4 was found to be responsive to HIF-2a overexpression, hypoxic treatment, and hypoxia mimetic drugs in PC-12 cells. Similar responses were observed in human neuroblastoma cell lines SK-N-SH and SK-N-BE(2)C, but not in endothelial cells, where Rgs4 transcript is readily detected but does not respond to hypoxia. Furthermore, this regulation was found to be dependent on transcription, and occurs in a manner consistent with direct HIF transactivation of Rgs4 transcription. However, no HIF binding site was detectable within 32 kb of the human Rgs4 gene locus, leading to the possibility of regulation by long- distance genomic interactions. Further research into Rgs4 regulation by hypoxia and HIF may result in better understanding of disease states such as schizophrenia, and also shed light on the other roles of HIF yet to be discovered. Citation: Olechnowicz SWZ, Fedele AO, Peet DJ (2012) Hypoxic Induction of the Regulator of G-Protein Signalling 4 Gene Is Mediated by the Hypoxia-Inducible Factor Pathway. PLoS ONE 7(9): e44564. doi:10.1371/journal.pone.0044564 Editor: Kaustubh Datta, University of Nebraska Medical Center, United States of America Received June 20, 2012; Accepted August 8, 2012; Published September 7, 2012 Copyright: ß 2012 Olechnowicz et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was funded by the ARC Special Centre for the Molecular Genetics of Development (CMGD, http://www.cmgd.adelaide.edu.au) and the National Health and Medical Research Council of Australia (NHMRC, http://www.nhmrc.gov.au). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: daniel.peet@adelaide.edu.au ¤ Current address: Nuffield Department of Surgical Sciences, University of Oxford, Oxford, United Kingdom Introduction Oxygen is critical for the survival of metazoan cells, and as such there is an adaptive response to chronic cellular hypoxia, involving activation of the Hypoxia Inducible Factor (HIF) pathway. HIF is a heterodimeric transcription factor which binds at over 100 genomic sites to activate transcription of hypoxia-adaptive genes [1,2]. HIF is comprised of two basic-Helix-Loop-Helix/Per-Arnt- Sim domain (bHLH/PAS) proteins, known as HIF-a and HIF-b [3], which dimerise in the nucleus to activate transcription [4]. HIF-a subunits are hypoxia responsive, while HIF-b (alternatively known as ARNT) is constitutively expressed and unresponsive to hypoxia. There are three genes encoding HIF-a subunits: Hif1a, which encodes HIF-1a protein [5]; Epas1, which encodes HIF-2a protein, also known as HIF-Like Factor (HLF) or Endothelial Per-ARNT-Sim protein-1 (EPAS1) [6–8]; and Hif3a, which encodes various splice variants of HIF-3a protein [9,10]. However, only HIF-1 and HIF-2 are active transcription factors, formed by respective dimerisation between HIF-1a or HIF-2a with HIF-b. HIF-1a and HIF-2a are not redundant, and have differing physiological roles, as established by several independent transgenic and knockout mouse lines [11–15]. In oxygenated cells, HIF-a subunits are rapidly degraded and inactivated by the oxygen-dependent PHD and FIH hydroxylases [16–18], but in hypoxic conditions they are stabilised and transactivate specific genes such as Vegf [19] and Dec1 [20]. Both forms of HIF bind directly to a DNA sequence known as the Hypoxia Response Element (HRE), with a consensus sequence of RCGTG [1,3,21]. Known HREs are most commonly found within 2kb of the target gene transcription start site (TSS) [22], however there are examples of more distant functional HREs, such as that of the Phd3 gene which is found over 10 kb from its TSS [21]. Differences between HIF-1 and HIF-2 target selection are caused by interaction with other transcription factors or chromatin context, rather than through DNA target sequence selection [23,24]. Rgs4 is a member of a large family of genes encoding RGS G- Protein signalling regulators, which bind to GTP-bound G a and increase the rate of GTP hydrolysis, causing reduced signal transduction from the associated G-protein coupled receptors [25– 27]. Rgs4 is expressed predominantly in neural and vascular cells [28,29], and the Rgs4 locus has been linked to disease states relating to both types of cell, notably in schizophrenia [30]. Although the causes of schizophrenia are not yet understood, one PLOS ONE | www.plosone.org 1 September 2012 | Volume 7 | Issue 9 | e44564