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Genome-wide association of hypoxia-inducible factor (HIF)-1alpha and HIF-2alpha DNA binding with expression profiling of hypoxia-inducible transcripts.

Mole DR, Blancher C, Copley RR, Pollard PJ, Gleadle JM, Ragoussis J, Ratcliffe PJ - J. Biol. Chem. (2009)

Bottom Line: 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.Given emerging evidence for biologically distinct functions of HIF-1alpha versus HIF-2alpha understanding the mechanisms restricting HIF-2alpha activity will be of interest.

View Article: PubMed Central - PubMed

Affiliation: Henry Wellcome Building of Molecular Physiology, University of Oxford, Oxford OX3 7BN, United Kingdom. drmole@well.ox.ac.uk

ABSTRACT
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.

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Related in: MedlinePlus

Relationship between amplitude and direction of regulation and chromatin immunoprecipitation by anti-HIF-1α or HIF-2α. Gene loci were assigned to functional groups according to fold up- or down-regulation by DMOG in the Affymetrix expression array. The proportion of gene loci captured by either of the anti-HIF-α immunoprecipitations is given for each of the functional group.
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Figure 5: Relationship between amplitude and direction of regulation and chromatin immunoprecipitation by anti-HIF-1α or HIF-2α. Gene loci were assigned to functional groups according to fold up- or down-regulation by DMOG in the Affymetrix expression array. The proportion of gene loci captured by either of the anti-HIF-α immunoprecipitations is given for each of the functional group.

Mentions: For all matched HIF regions (combining the HIF-1α and HIF-2α sets), six matrices were identified as overrepresented in the HIF-binding loci (Fig. 5). Three are HIF-binding motifs, further validating the currently held HRE consensus; the others being binding motifs for nuclear respiratory factor-1 (47), Myc intron factor (MIF-1) (48), and E2F transcription factors (49). Given the strict definition of statistical significance, it is also likely that a number of other overrepresented matrices are also valid. Indeed, a number of other E2F matrices, as well as those for an array of previously described HIF-interacting transcriptional cofactors, such as STAT, ETS, and MYC (8), were enriched, but failed to reach the statistical threshold (supplemental materials Table S4). When subgroups of HIF-1α and HIF-2α interacting gene loci were analyzed no significant difference was identified between the two sets of genes, although the numbers in this subgroup analysis were smaller.


Genome-wide association of hypoxia-inducible factor (HIF)-1alpha and HIF-2alpha DNA binding with expression profiling of hypoxia-inducible transcripts.

Mole DR, Blancher C, Copley RR, Pollard PJ, Gleadle JM, Ragoussis J, Ratcliffe PJ - J. Biol. Chem. (2009)

Relationship between amplitude and direction of regulation and chromatin immunoprecipitation by anti-HIF-1α or HIF-2α. Gene loci were assigned to functional groups according to fold up- or down-regulation by DMOG in the Affymetrix expression array. The proportion of gene loci captured by either of the anti-HIF-α immunoprecipitations is given for each of the functional group.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2719312&req=5

Figure 5: Relationship between amplitude and direction of regulation and chromatin immunoprecipitation by anti-HIF-1α or HIF-2α. Gene loci were assigned to functional groups according to fold up- or down-regulation by DMOG in the Affymetrix expression array. The proportion of gene loci captured by either of the anti-HIF-α immunoprecipitations is given for each of the functional group.
Mentions: For all matched HIF regions (combining the HIF-1α and HIF-2α sets), six matrices were identified as overrepresented in the HIF-binding loci (Fig. 5). Three are HIF-binding motifs, further validating the currently held HRE consensus; the others being binding motifs for nuclear respiratory factor-1 (47), Myc intron factor (MIF-1) (48), and E2F transcription factors (49). Given the strict definition of statistical significance, it is also likely that a number of other overrepresented matrices are also valid. Indeed, a number of other E2F matrices, as well as those for an array of previously described HIF-interacting transcriptional cofactors, such as STAT, ETS, and MYC (8), were enriched, but failed to reach the statistical threshold (supplemental materials Table S4). When subgroups of HIF-1α and HIF-2α interacting gene loci were analyzed no significant difference was identified between the two sets of genes, although the numbers in this subgroup analysis were smaller.

Bottom Line: 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.Given emerging evidence for biologically distinct functions of HIF-1alpha versus HIF-2alpha understanding the mechanisms restricting HIF-2alpha activity will be of interest.

View Article: PubMed Central - PubMed

Affiliation: Henry Wellcome Building of Molecular Physiology, University of Oxford, Oxford OX3 7BN, United Kingdom. drmole@well.ox.ac.uk

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus