Genome-wide association of hypoxia-inducible factor (HIF)-1alpha and HIF-2alpha DNA binding with expression profiling of hypoxia-inducible transcripts.
Hypoxia-inducible factor (HIF) controls an extensive range of adaptive responses to hypoxia. To better understand this transcriptional cascade we performed genome-wide chromatin immunoprecipitation using antibodies to two major HIF-alpha subunits, and correlated the results with genome-wide transcript profiling. Within a tiled promoter array we identified 546 and 143 sequences that bound, respectively, to HIF-1alpha or HIF-2alpha at high stringency. Analysis of these sequences confirmed an identical core binding motif for HIF-1alpha and HIF-2alpha (RCGTG) but demonstrated that binding to this motif was highly selective, with binding enriched at distinct regions both upstream and downstream of the transcriptional start. Comparison of HIF-promoter binding data with bidirectional HIF-dependent changes in transcript expression indicated that whereas a substantial proportion of positive responses (>20% across all significantly regulated genes) are direct, HIF-dependent gene suppression is almost entirely indirect. Comparison of HIF-1alpha- versus HIF-2alpha-binding sites revealed that whereas some loci bound HIF-1alpha in isolation, many bound both isoforms with similar affinity. Despite high-affinity binding to multiple promoters, HIF-2alpha contributed to few, if any, of the transcriptional responses to acute hypoxia at these loci. Given emerging evidence for biologically distinct functions of HIF-1alpha versus HIF-2alpha understanding the mechanisms restricting HIF-2alpha activity will be of interest.
Mole
David R
DR
From the Henry Wellcome Building of Molecular Physiology, Oxford OX3 7BN, United Kingdom. Electronic address: drmole@well.ox.ac.uk.
Blancher
Christine
C
The Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford OX3 7BN, United Kingdom.
Copley
Richard R
RR
The Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford OX3 7BN, United Kingdom.
Pollard
Patrick J
PJ
From the Henry Wellcome Building of Molecular Physiology, Oxford OX3 7BN, United Kingdom.
Gleadle
Jonathan M
JM
Renal Unit, Level 6, Flinders Medical Centre, Bedford Park, South Australia 5042, Australia.
Ragoussis
Jiannis
J
The Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford OX3 7BN, United Kingdom.
Ratcliffe
Peter J
PJ
From the Henry Wellcome Building of Molecular Physiology, Oxford OX3 7BN, United Kingdom.
D001483:Base Sequence; D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics* / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D015687:Cell Hypoxia / Q000235:genetics* / Q000502:physiology*; D045744:Cell Line, Tumor; D047369:Chromatin Immunoprecipitation; D004273:DNA, Neoplasm / Q000235:genetics* / Q000378:metabolism*; D005260:Female; D020869:Gene Expression Profiling; D055106:Genome-Wide Association Study; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics* / Q000378:metabolism*; D020411:Oligonucleotide Array Sequence Analysis; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D000099162:Endothelial PAS Domain-Containing Protein 1
D051792:Basic Helix-Loop-Helix Proteins; D004273:DNA, Neoplasm; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D000099162:Endothelial PAS Domain-Containing Protein 1; C497442:HIF1A protein, human