Integrative analysis of HIF binding and transactivation reveals its role in maintaining histone methylation homeostasis Xiaobo Xiaa, Madeleine E. Lemieuxa, Wei Lib,c,1, Jason S. Carrolld,2, Myles Brownd, X. Shirley Liub,c, and Andrew L. Kunga,e,3 Departments of aPediatric Oncology, bBiostatistics and Computational Biology, and dMedical Oncology, Dana–Farber Cancer Institute, 44 Binney Street, and Harvard Medical School, Boston, MA 02115; cHarvard School of Public Health, 677 Huntington Avenue, Boston, MA 02115; and eDivision of Pediatric Hematology/Oncology, Children’s Hospital Boston, 300 Longwood Avenue, Boston, MA 02115 Edited by Steven L. McKnight, University of Texas Southwestern Medical Center, Dallas, TX, and approved January 23, 2009 (received for review October 9, 2008) Adaptation to hypoxia is mediated through a coordinated transcrip- promoter or enhancers of Ϸ50 genes (10, 11). Alignment of the tional response driven largely by hypoxia-inducible factor 1 (HIF-1). sequences encompassing these well-characterized functional HREs We used ChIP-chip and gene expression profiling to identify direct (transcriptionally activated by hypoxia) has revealed a consensus HIF- targets of HIF-1 transactivation on a genome-wide scale. Several 1-binding motif (the core HRE) of 5Ј-RCGTG-3Ј (R ϭ AorG). hundred direct HIF-1 targets were identified and, as expected, were Because the core HRE is too promiscuous to accurately predict highly enriched for proteins that facilitate metabolic adaptation to binding a priori, we used ChIP-chip to define HIF-1 chromatin hypoxia. Surprisingly, there was also striking enrichment for the binding on a genome-wide level. We integrated these results with family of 2-oxoglutarate dioxygenases, including the jumonji-domain gene expression profiling to interrogate mechanisms regulating histone demethylases. We demonstrate that these histone demethy- hypoxia-induced gene expression and to more comprehensively lases are direct HIF targets, and their up-regulation helps maintain identify direct targets of HIF-1 transactivation. We found that the epigenetic homeostasis under hypoxic conditions. These results sug- family of 2-oxoglutarate-dependent dioxygenases are coordinately gest that the coordinated increase in expression of several oxygen- targeted by HIF, and up-regulated expression helps maintain global CELL BIOLOGY dependent enzymes by HIF may help compensate for decreased levels levels of histone methylation under hypoxic conditions. of oxygen under conditions of cellular hypoxia. Results ChIP-chip ͉ hypoxia ͉ jumonji protein ͉ dioxygenase ͉ epigenetics Identification of HIF-1-Binding Sites by ChIP-chip. To identify HIF-1- binding sites across the genome, HepG2 cells were grown under dequate oxygenation is essential for normal physiology and both normoxic (ambient) and hypoxic (0.5% O2) conditions, and Afunctioning of all cells. In response to decreased oxygen ChIP was performed by using a HIF-1␣ polyclonal antibody without tension, a coordinated transcriptional response is activated to appreciable cross-reactivity to HIF-2␣ (Fig. S1A). ChIP and input maintain cellular homeostasis. This transcriptional program is DNA were hybridized to Affymetrix GeneChip Human Tiling 2.0R mediated, at least in part, by activation of the heterodimeric Array Sets, consisting of probes covering the entire nonrepetitive transcription factors hypoxia-inducible factor 1 and 2 (HIF-1 and human genome at 35-bp resolution. The model-based analysis for HIF-2). The HIFs are essential for several physiological processes, tiling arrays (MAT) algorithm (12) was used to identify probe signal including normal development, erythropoietin (EPO) production, peaks comparing triplicate biological replicates of HIF-1 ChIP and wound healing. Unfortunately, HIF signaling also contributes DNA to their matched inputs by using an initial P value cutoff of to the pathophysiology of tumors by facilitating metabolic adapta- 1EϪ5. Consistent with the fact that HIF-1␣ protein levels increase tion and by promoting angiogenesis, invasion, and metastasis (1). dramatically in hypoxic compared with normoxic samples (Fig. The activity of HIF-1 and HIF-2 are controlled at multiple levels. S1B), 91% of the putative HIF-1-binding sites were characterized The primary point of regulation is at the level of abundance of the by a positive peak call only under hypoxic conditions (e.g., ENO1 ␣-subunits. Under normal oxygen (normoxic) conditions, HIF-1␣ and INSIG2;Fig.1A). However, because HIF-1␣ is not entirely and HIF-2␣ are hydroxylated by the HIF prolyl hydroxylases absent in normoxia, peaks in which probe intensities were signifi- EGLN1–3, which target the proteins for binding to the von Hippel– cantly increased under hypoxic conditions by comparison with Lindau (vHL) ubiquitin E3 ligase complex and rapid proteosomal normoxia were also retained (e.g., JMJD1A and ZNF292;Fig.1A). degradation (2–6). As oxygen levels drop, prolyl-hydroxylation A minority of peaks (7%) had peak intensities that did not increase decreases, resulting in accumulation of HIF ␣-subunits and het- in hypoxic compared with normoxic samples (e.g., ADI1 and erodimerization with ARNT (HIF-1␤). As a consequence, HIF PHF12;Fig.1A), and these were suspected to be nonspecific. heterodimer levels are nominal under physiologic oxygen levels and increase exponentially with decreasing oxygen tension (7). HIF heterodimers then recruit transcriptional coactivator complexes (8, Author contributions: X.X., M.E.L., J.S.C., M.B., and A.L.K. designed research; X.X. per- formed research; X.X., M.E.L., W.L., J.S.C., X.S.L., and A.L.K. contributed new reagents/ 9), and transactivate target genes containing the cognate hypoxia- analytic tools; X.X., M.E.L., W.L., M.B., X.S.L., and A.L.K. analyzed data; and X.X., M.E.L., and response element (HRE) (1). A.L.K. wrote the paper. The direct transcriptional targets of HIF-1 play important roles in The authors declare no conflict of interest. facilitating both short-term and long-term adaptation to hypoxia (1). This article is a PNAS Direct Submission. Metabolic homeostasis is achieved by shifting from oxidative phos- 1Present address: Division of Biostatistics, Dan L. Duncan Cancer Center, Department of Mo- phorylation to anaerobic glycolysis through increased expression of the lecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030. glycolytic enzymes and glucose transporters, inhibition of the TCA 2Present address: Cancer Research U.K., Cambridge Research Institute, Li Ka Shing Centre, cycle, and induction of pH-regulating systems. A second HIF-1- Robinson Way, Cambridge CB2 0RE, United Kingdom. mediated program increases oxygen delivery by inducing vasodilatation, 3To whom correspondence should be addressed. E-mail: andrew࿝[email protected]. increased vascular permeability, enhanced erythropoiesis and angio- This article contains supporting information online at www.pnas.org/cgi/content/full/ genesis. Specific sites of HIF-1 binding have been validated within the 0810067106/DCSupplemental. www.pnas.org͞cgi͞doi͞10.1073͞pnas.0810067106 PNAS Early Edition ͉ 1of6 Downloaded by guest on October 4, 2021 A B A 1) Hypoxia-unique 2) Hypoxia-enriched 3) Nonspecific HRE+ HRE+ HRE- Hypoxia number of hits Distance from transcription start site (bp) Normoxia ←→→→←← C HRE+ HRE- ENO1 INSIG2 JMJD1A ZNF292 ADI1 PHF12 Norm p<0.001 Norm p=0.894 FDR q<0.001 FDR q=0.936 B 120 60 Enrichment score (ES) Enrichment score 30 20 up hypoxia:normoxia down (ChIP/Input) 10 Fig. 2. Distribution of HIF-1-bound sites and association with transactivation. ChIP-qPCR fold enrichment ChIP-qPCR fold 0 (A) Distribution of HIF-1 ChIP hits relative to the TSS of associated genes. (B) hypoxia/normxia Distribution of core HRE-containing HIF-1 hits (HREϩ) relate to the annotated hypoxia/sh-HIF1α structure of associated genes. (C) GSEA analysis of mRNA expression profiles for hypoxic (0.5%O2, 12 h) vs. normoxic cells. Relative expression was rank-ordered by signal-to-noise (S2N) ratios of triplicate hypoxic samples vs. triplicate normoxic ϩ HK2 samples. Genes associated with HRE HIF-1-binding sites were strongly corre- ID2-LR ENO1 DDIT4 INHA LDHA PGK1 P4HB ADI1 PHF12 RNMT ADFP RAB17 EFNA1 NEDD9 INSIG2 NDRG1 ZNF292 ALDOA IFNAR1 PFKFB4 MIG6-176K MIG6-185K BHLHB2-3' RAPGEF6 ANKRD37 KIAA2013 JMJD2B JARID1B SAMHD1 SERPINE1 JMJD1A JMJD2C-LR MGC45491-LR ANKRD15-23K 10kbupVEGF 5KbUpVEGF JMJD2C-prom lated with the hypoxic phenotype. In contrast, no such enrichment was evident 1) 2) 3) for the ChIP-chip fragments without identifiable core HREs (HREϪ). The color bar indicates up-regulated (red, positive S2N) and down-regulated (blue, negative Fig. 1. HIF-1 ChIP-chip analysis and validation. (A) Representative integrated S2N) genes. genome browser (IGB; Affymetrix) tracks showing peaks of probe intensities (vertical bars) arrayed by chromosomal position. For each locus, tracks are scaled identically. ChIP peaks were identified by using the MAT algorithm and were and P value Ͻ 2.5 ϫ 10Ϫ7) that together resulted in high specificity classified as hypoxia-unique, hypoxia-enriched, or nonspecific. (B) ChIP-chip re- (95.5%) and sensitivity (82.5%) (Fig. S1D). Within a subset of 40 sults were validated by ChIP-qPCR using primer pairs surrounding the putative well-validated HIF-1 targets evaluated by ChIP-qPCR (Table S2), binding sites in the indicated loci. For each locus, the fold enrichment comparing HIF-1 ChIP DNA to input is represented in the bar graph (mean Ϯ SD). To these thresholds produced an empiric sensitivity of 75% and determine specificity for HIF-1, the bottom heat map depicts the fold enrichment specificity of 100%. With these optimized thresholds, a total of 377 comparing hypoxic to normoxic cells and control hypoxic cells to cells in which HIF-1-binding sites were identified across the genome. HIF-1␣ was specifically depleted with a lentiviral shRNA (sh-HIF1␣). Primer pairs The canonical motif that is bound by HIF-1, the core HRE, is locate 5 and 10 kb upstream of the VEGF gene were used as controls.