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Destruction of a Distal Hypoxia Response Element Abolishes Trans-Activation of the PAG1 Gene Mediated by HIF-Independent Chromatin Looping

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Destruction of a Distal Hypoxia Response Element Abolishes Trans-Activation of the PAG1 Gene Mediated by HIF-Independent Chromatin Looping 5810–5823 Nucleic Acids Research, 2015, Vol. 43, No. 12 Published online 24 May 2015 doi: 10.1093/nar/gkv506 Destruction of a distal hypoxia response element abolishes trans-activation of the PAG1 gene mediated by HIF-independent chromatin looping 1,† 1,† 2 3 Alexandra Schorg¨ , Sara Santambrogio , James L. Platt , Johannes Schodel¨ ,Maja Downloaded from T. Lindenmeyer1, Clemens D. Cohen1,4,KatrinSchrodter¨ 5, David R. Mole2, Roland H. Wenger1,4,* and David Hoogewijs1,4,5,* 1Institute of Physiology and Zurich¨ Center for Integrative Human Physiology ZIHP, University of Zurich,¨ CH-8057 Zurich,¨ Switzerland, 2Henry Wellcome Building for Molecular Physiology, University of Oxford, Ox3 7BN, UK, http://nar.oxfordjournals.org/ 3Department of Nephrology and Hypertension, Friedrich-Alexander-University Erlangen-Nuremberg, D-91054 Erlangen, Germany, 4National Center of Competence in Research "Kidney.CH", Switzerland and 5Institute of Physiology, University of Duisburg-Essen, D-45122 Essen, Germany Received December 18, 2014; Revised April 18, 2015; Accepted May 02, 2015 at Universitaet Erlangen-Nuernberg, Wirtschafts- und Sozialwissenschaftliche Z on August 15, 2016 ABSTRACT INTRODUCTION A crucial step in the cellular adaptation to oxygen Hypoxia, defined as mismatch between oxygen supply and deficiency is the binding of hypoxia-inducible fac- consumption, plays a crucial role in many physiological tors (HIFs) to hypoxia response elements (HREs) and pathophysiological conditions, such as embryonic de- of oxygen-regulated genes. Genome-wide HIF- velopment, adaptation to high-altitude, wound healing, in- 1␣/2␣/␤ DNA-binding studies revealed that the ma- flammation, cardiovascular diseases and cancer. Hypoxia- inducible factors (HIFs) are the master regulators of cel- jority of HREs reside distant to the promoter re- lular adaptation to hypoxia (1). HIFs are heterodimeric gions, but the function of these distal HREs has transcription factors consisting of a labile oxygen-regulated only been marginally studied in the genomic con- ␣-subunit and a constitutively expressed ␤-subunit. Un- text. We used chromatin immunoprecipitation (ChIP), der normoxic conditions, HIF␣ subunits are hydroxylated gene editing (TALEN) and chromosome conforma- by the oxygen-sensing prolyl-4-hydroxylase domain (PHD) tion capture (3C) to localize and functionally char- enzymes and the factor inhibiting HIF on two prolyl acterize a 82 kb upstream HRE that solely drives and one asparagine residue, respectively. HIF␣ prolyl-4- oxygen-regulated expression of the newly identified hydroxylation leads to the binding of the E3 ubiquitin lig- HIF target gene PAG1. PAG1, a transmembrane adap- ase von Hippel–Lindau protein (pVHL) followed by polyu- biquitination and proteasomal destruction (2,3). Under hy- tor protein involved in Src signalling, was hypoxically ␣ induced in various cell lines and mouse tissues. ChIP poxic conditions HIF subunits remain stable, translo- cate to the nucleus, heterodimerize with HIF␤ and tran- and reporter gene assays demonstrated that the −82 scriptionally activate numerous target genes involved in the kb HRE regulates PAG1, but not an equally distant adaptation to hypoxia. Therefore, HIF complexes interact gene further upstream, by direct interaction with HIF. directly with the consensus core motif 5-RCGTG-3, the ac- Ablation of the consensus HRE motif abolished the tual HIF-binding site (HBS) within the hypoxia response hypoxic induction of PAG1 but not general oxygen element (HRE) (4). signalling. 3C assays revealed that the −82 kb HRE Pan-genomic studies combining techniques to assess physically associates with the PAG1 promoter region, transcriptional activity and protein–DNA interaction func- independent of HIF-DNA interaction. These results tionally identified HREs within the promoter regions as demonstrate a constitutive interaction between the well as HREs far away from transcriptional start sites − (TSSs) (5–10). Interestingly, 60% of all HIF-1␣ and 80% 82 kb HRE and the PAG1 promoter, suggesting a ␣ physiologically important rapid response to hypoxia. of all HIF-2 interactions with HREs locate to distal sites >2.5 kb outside of the TSS (7). This finding contrasts with *To whom correspondence should be addressed. Tel: +49 201723 4623; Fax: +49 201723 4648; Email: [email protected] Correspondence may also be addressed to Roland H. Wenger. Tel: +41 44 635 50 65; Fax: +41 44 635 68 14; Email: [email protected] †These authors contributed equally to the paper as first authors. C The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Nucleic Acids Research, 2015, Vol. 43, No. 12 5811 the majority of published single-gene studies which show cultured in DMEM/F12 (Sigma) supplemented with 10% a certain bias (∼76% HREs are within 2.5 kb upstream of FCS, 100 IU/ml penicillin, 100 ␮g/ml streptomycin, 36 the TSSs) toward proximal HREs (4). Furthermore, these ng/ml hydrocortisone and ITS solution (5 ␮g/ml insulin, 5 pan-genomic analyses also revealed a considerable variabil- ␮g/ml transferrin, 5 ng/ml selenium; Roche, Basel, Switzer- ity between various cell types, especially for distal HREs land). Hypoxia experiments were performed at 0.2% or (11). Only a few distant HREs have been investigated so 0.5% O2 and5%CO2 in a gas-controlled workstation far, including those regulating the genes IGFBP3 (−57 kb (InvivO2 400; Baker Ruskinn, Bridgend, South Wales, UK). upstream of the TSS) (12), CCND1 (-220 kb) (8), SLC2A3 Cells were transfected using polyethylenimine (Polysciences, (−35 kb) (9) and the putative 5 kidney-inducible element of Warrington, PA, USA) as described previously (19). Downloaded from the EPO gene (−9.2 kb) (13). How these distant HREs in- teract with the promoter regions, whether this interaction is RNA isolation and analysis dependent on the presence of HIFs (or even the HBS itself) and how this promoter–enhancer interaction varies between Total RNA was isolated from cultured cells using the guani- different cell types remains generally unknown. dine isothiocyanate method as described before (17). RNA http://nar.oxfordjournals.org/ By using gene arrays to assess the transcriptional profile from mice exposed to inspiratory hypoxia was obtained as of HeLa cells under normoxic and hypoxic conditions, we described elsewhere (20). Specific mRNAs were quantified identified the lipid raft phosphoprotein associated with gly- by reverse transcription followed by quantitative PCR (RT- cosphingolipid enriched microdomains (PAG1) as a novel qPCR) using a MX3000P light cycler (Agilent, Santa Clara, hypoxia-inducible gene. PAG1 is a ubiquitously expressed CA, USA) as described previously (17). For gene array anal- transmembrane adaptor protein that binds the protein ty- ysis, total RNA was extracted from HeLa cells cultured un- rosine kinase csk and is hence also known as csk-binding der normoxic or hypoxic (16 h, 0.2% O2) conditions with protein (14,15). We identified a distal HRE located −82 kb RNeasy (Qiagen, Venlo, The Netherlands). RNA integrity upstream of the PAG1 TSS and used the TALEN (tran- was evaluated using the Agilent 2100 Bioanalyzer. Two- at Universitaet Erlangen-Nuernberg, Wirtschafts- und Sozialwissenschaftliche Z on August 15, 2016 scription activator-like effector-based nuclease) technique colour labelled samples were hybridized to an Agilent whole to specifically target the HBS of this remote HRE, result- human genome 4×44K oligonucleotide microarray slide. ing in functional ablation of the hypoxia inducibility of For each condition two biological replicates were used. PAG1 gene transcription. Chromosome conformation cap- ture (3C) assays were employed to assess the interaction be- Analysis of human renal biopsies tween this HRE and the PAG1 promoter region under nor- moxic, hypoxic, HIF-depleted and HBS-destructed condi- Human renal biopsy specimens and Affymetrix microar- tions. ray expression data (HG-U133 Plus2.0 Array) were pro- cured within the framework of the European Renal cDNA MATERIALS AND METHODS Bank–Kroner-Fresenius¨ Biopsy Bank (21). Diagnostic re- nal biopsies were obtained from patients after informed Plasmid constructs consent and with approval of the local ethics committees. A reporter gene plasmid containing 1014 bp of the PAG1 Following renal biopsy, the tissue was transferred to RNase promoter was obtained from BioCat (Heidelberg, Ger- inhibitor and microdissected into glomerular (Glom) and many). The PAG1 −82 kb HRE region was amplified tubulointerstitial (Tub) compartments. The microarray ex- by Phusion polymerase-based polymerase chain reaction pression data used in this study came from individual pa- (PCR) (Thermo Fisher Scientific, Waltham, MA, USA) us- tients with diabetic nephropathy (DN, Glom (n = 7), Tub (n ing HeLa or MDA-MB-231 genomic DNA and the primers = 7), focal segmental glomerulosclerosis (FSGS, Glom (n (Microsynth, Balgach, Switzerland) listed in Supplemen- = 16), Tub (n = 7)), rapidly progressive glomerulonephri- tary Table S1. Following restriction digestion with BglII tis (RPGN, Glom (n = 23), Tub (n = 21))aswellaspre- (Thermo Fisher Scientific) fragments of 317 bp and 2kb transplant biopsies from living renal allograft donors as were cloned into the
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