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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

Distribution of HIF-1α- and HIF-2α-binding sites. A, distribution of high stringency (MA score >4) HIF-1α- and HIF-2α-binding sites referred to the nearest transcriptional start site. Number of HIF-binding peaks expressed per 500-bp bin is shown. B, frequency distribution of RCGTG motifs at gene loci that bind HIF-1α or HIF-2α. The number of RCGTG motifs expressed per 500-bp bin is shown.
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Figure 2: Distribution of HIF-1α- and HIF-2α-binding sites. A, distribution of high stringency (MA score >4) HIF-1α- and HIF-2α-binding sites referred to the nearest transcriptional start site. Number of HIF-binding peaks expressed per 500-bp bin is shown. B, frequency distribution of RCGTG motifs at gene loci that bind HIF-1α or HIF-2α. The number of RCGTG motifs expressed per 500-bp bin is shown.

Mentions: Third, we analyzed the distribution of the captured sequences and associated RCGTG motifs in relation to the transcriptional start site of the closest gene. Clearly the design of the array (intended to represent sequences from −7.5 to +2.5 kb of the transcription start site) places limits on this analysis, although a proportion of the immunoprecipitated sequences lay outside these regions, most likely representing changes in gene annotation between that on which the array was designed and the (latest) annotation that we used for locus assignment. Several interesting features were observed irrespective of whether the precise position of the peak of enrichment profile or the associated HRE was used in the analysis. The distribution of immunoprecipitated sequences was closely similar for both HIF-1α and HIF-2α with the greatest density of HIF-binding sites being observed within 2 kb of the transcriptional start. This region comprised two distinct frequency peaks. One is a sharp peak centered 500 bp upstream of the transcriptional start site, similar to that observed for other transcription factors (44). The other is a broader peak centered 1000 bp downstream of the transcriptional start site. This distribution contrasted markedly with the distribution of all RCGTG motifs at the same set of promoters, which revealed a single peak centered at the transcriptional start site (Fig. 2).


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)

Distribution of HIF-1α- and HIF-2α-binding sites. A, distribution of high stringency (MA score >4) HIF-1α- and HIF-2α-binding sites referred to the nearest transcriptional start site. Number of HIF-binding peaks expressed per 500-bp bin is shown. B, frequency distribution of RCGTG motifs at gene loci that bind HIF-1α or HIF-2α. The number of RCGTG motifs expressed per 500-bp bin is shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Distribution of HIF-1α- and HIF-2α-binding sites. A, distribution of high stringency (MA score >4) HIF-1α- and HIF-2α-binding sites referred to the nearest transcriptional start site. Number of HIF-binding peaks expressed per 500-bp bin is shown. B, frequency distribution of RCGTG motifs at gene loci that bind HIF-1α or HIF-2α. The number of RCGTG motifs expressed per 500-bp bin is shown.
Mentions: Third, we analyzed the distribution of the captured sequences and associated RCGTG motifs in relation to the transcriptional start site of the closest gene. Clearly the design of the array (intended to represent sequences from −7.5 to +2.5 kb of the transcription start site) places limits on this analysis, although a proportion of the immunoprecipitated sequences lay outside these regions, most likely representing changes in gene annotation between that on which the array was designed and the (latest) annotation that we used for locus assignment. Several interesting features were observed irrespective of whether the precise position of the peak of enrichment profile or the associated HRE was used in the analysis. The distribution of immunoprecipitated sequences was closely similar for both HIF-1α and HIF-2α with the greatest density of HIF-binding sites being observed within 2 kb of the transcriptional start. This region comprised two distinct frequency peaks. One is a sharp peak centered 500 bp upstream of the transcriptional start site, similar to that observed for other transcription factors (44). The other is a broader peak centered 1000 bp downstream of the transcriptional start site. This distribution contrasted markedly with the distribution of all RCGTG motifs at the same set of promoters, which revealed a single peak centered at the transcriptional start site (Fig. 2).

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