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An integrative genomics approach identifies Hypoxia Inducible Factor-1 (HIF-1)-target genes that form the core response to hypoxia.

Benita Y, Kikuchi H, Smith AD, Zhang MQ, Chung DC, Xavier RJ - Nucleic Acids Res. (2009)

Bottom Line: The proximal promoters of these genes were then analyzed for the presence of conserved HIF-1-binding sites.We present experimental validation for ANKRD37 as a novel HIF-1-target gene.Together these analyses demonstrate the potential to recover novel HIF-1-target genes and the discovery of mammalian-regulatory elements operative in the context of microarray data sets.

View Article: PubMed Central - PubMed

Affiliation: Center for Computational and Integrative Biology, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA.

ABSTRACT
The transcription factor Hypoxia-inducible factor 1 (HIF-1) plays a central role in the transcriptional response to oxygen flux. To gain insight into the molecular pathways regulated by HIF-1, it is essential to identify the downstream-target genes. We report here a strategy to identify HIF-1-target genes based on an integrative genomic approach combining computational strategies and experimental validation. To identify HIF-1-target genes microarrays data sets were used to rank genes based on their differential response to hypoxia. The proximal promoters of these genes were then analyzed for the presence of conserved HIF-1-binding sites. Genes were scored and ranked based on their response to hypoxia and their HIF-binding site score. Using this strategy we recovered 41% of the previously confirmed HIF-1-target genes that responded to hypoxia in the microarrays and provide a catalogue of predicted HIF-1 targets. We present experimental validation for ANKRD37 as a novel HIF-1-target gene. Together these analyses demonstrate the potential to recover novel HIF-1-target genes and the discovery of mammalian-regulatory elements operative in the context of microarray data sets.

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(A) Metabolic map of HIF-target genes. The KEGG pathway database was employed to map 81 HIF-target genes defined as the core response to hypoxia to pathways. Metabolism related processes are shown here (black objects) and those over-represented with a P < 0.05 are shown with a dotted white frame. Upregulated genes are shown as red nodes while downregulated genes as green nodes. Previously identified HIF-1-target genes are indicated by a diamond shape. (B) Mitochondrial map. Genes within the top 200 predicted HIF targets that localize to the mitochondria were mapped according to their functional annotation. Upregulated genes are shown as red nodes and downregulated as green nodes. Genes indicated in bold responded to hypoxia in at least three cell types. Dotted lines from iron–sulfur protein assembly and repair illustrate a few of the iron–sulfur proteins that are key to mitochondrial function, such as NADH dehydrogenase. (C) Graphical representation of protein-protein interaction network and Reactome pathways (D) enrichment for previously validated HIF-1 targets (red) and novel predicted targets (yellow). Only proteins/pathways that were significantly enriched (P < 0.05) are shown. See Supplementary Table S6 for analysis details.
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Figure 4: (A) Metabolic map of HIF-target genes. The KEGG pathway database was employed to map 81 HIF-target genes defined as the core response to hypoxia to pathways. Metabolism related processes are shown here (black objects) and those over-represented with a P < 0.05 are shown with a dotted white frame. Upregulated genes are shown as red nodes while downregulated genes as green nodes. Previously identified HIF-1-target genes are indicated by a diamond shape. (B) Mitochondrial map. Genes within the top 200 predicted HIF targets that localize to the mitochondria were mapped according to their functional annotation. Upregulated genes are shown as red nodes and downregulated as green nodes. Genes indicated in bold responded to hypoxia in at least three cell types. Dotted lines from iron–sulfur protein assembly and repair illustrate a few of the iron–sulfur proteins that are key to mitochondrial function, such as NADH dehydrogenase. (C) Graphical representation of protein-protein interaction network and Reactome pathways (D) enrichment for previously validated HIF-1 targets (red) and novel predicted targets (yellow). Only proteins/pathways that were significantly enriched (P < 0.05) are shown. See Supplementary Table S6 for analysis details.

Mentions: In the top 200 predicted HIF-target genes, 81 responded to hypoxia in at least three cell types (Figure 3). We hypothesize that these genes are likely to be part of the core response to hypoxia. In addition, these genes have a higher probability of being HIF-1 targets since their response to hypoxia was detected in multiple experiments. Of the 81 genes, 22 genes were previously reported to be HIF-target genes and 60 genes (74%) were upregulated in hypoxia. In addition, 80 (99%) of the genes contained a CpG island in their proximal promoter, significantly higher than the 51% CpG containing promoters in our entire promoter database. CpG islands are thought to be non-tissue specific (64), further supporting the selection of the 81 genes as a shared response to hypoxia. The Kyoto Encyclopedia of Genes and Genomes (KEGG) database was used to identify over-represented pathways and processes within the 81 genes. In Figure 4A a metabolic map of HIF-1-target genes was constructed from the resulting analysis (Supplementary Table S5). The metabolic map contained several known HIF-1-target genes, such as LDHA, ALDOC and GAPDH and also several novel targets such as GYS1 in starch and sucrose metabolism (65) and fumarate hydratase (FH) in the TCA cycle (66). One of the key cellular responses to HIF-1 in hypoxia is a metabolic shift from oxidative phosphorylation to glycolytic production of ATP. To highlight direct targets that may be involved in HIF-1 regulation of mitochondria function the predicted HIF targets that localize to the mitochondria (see ‘Materials and Methods’ section) were used to create a mitochondrial map (Figure 4B). Eight of eleven genes were downregulated in hypoxia and are involved in critical steps of mitochondrial function including two ribosomal subunits, cytochrome c, the iron transporter SLC25A28 and the iron–sulfur assembly and repair protein ISCU.Figure 3.


An integrative genomics approach identifies Hypoxia Inducible Factor-1 (HIF-1)-target genes that form the core response to hypoxia.

Benita Y, Kikuchi H, Smith AD, Zhang MQ, Chung DC, Xavier RJ - Nucleic Acids Res. (2009)

(A) Metabolic map of HIF-target genes. The KEGG pathway database was employed to map 81 HIF-target genes defined as the core response to hypoxia to pathways. Metabolism related processes are shown here (black objects) and those over-represented with a P < 0.05 are shown with a dotted white frame. Upregulated genes are shown as red nodes while downregulated genes as green nodes. Previously identified HIF-1-target genes are indicated by a diamond shape. (B) Mitochondrial map. Genes within the top 200 predicted HIF targets that localize to the mitochondria were mapped according to their functional annotation. Upregulated genes are shown as red nodes and downregulated as green nodes. Genes indicated in bold responded to hypoxia in at least three cell types. Dotted lines from iron–sulfur protein assembly and repair illustrate a few of the iron–sulfur proteins that are key to mitochondrial function, such as NADH dehydrogenase. (C) Graphical representation of protein-protein interaction network and Reactome pathways (D) enrichment for previously validated HIF-1 targets (red) and novel predicted targets (yellow). Only proteins/pathways that were significantly enriched (P < 0.05) are shown. See Supplementary Table S6 for analysis details.
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Related In: Results  -  Collection

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Figure 4: (A) Metabolic map of HIF-target genes. The KEGG pathway database was employed to map 81 HIF-target genes defined as the core response to hypoxia to pathways. Metabolism related processes are shown here (black objects) and those over-represented with a P < 0.05 are shown with a dotted white frame. Upregulated genes are shown as red nodes while downregulated genes as green nodes. Previously identified HIF-1-target genes are indicated by a diamond shape. (B) Mitochondrial map. Genes within the top 200 predicted HIF targets that localize to the mitochondria were mapped according to their functional annotation. Upregulated genes are shown as red nodes and downregulated as green nodes. Genes indicated in bold responded to hypoxia in at least three cell types. Dotted lines from iron–sulfur protein assembly and repair illustrate a few of the iron–sulfur proteins that are key to mitochondrial function, such as NADH dehydrogenase. (C) Graphical representation of protein-protein interaction network and Reactome pathways (D) enrichment for previously validated HIF-1 targets (red) and novel predicted targets (yellow). Only proteins/pathways that were significantly enriched (P < 0.05) are shown. See Supplementary Table S6 for analysis details.
Mentions: In the top 200 predicted HIF-target genes, 81 responded to hypoxia in at least three cell types (Figure 3). We hypothesize that these genes are likely to be part of the core response to hypoxia. In addition, these genes have a higher probability of being HIF-1 targets since their response to hypoxia was detected in multiple experiments. Of the 81 genes, 22 genes were previously reported to be HIF-target genes and 60 genes (74%) were upregulated in hypoxia. In addition, 80 (99%) of the genes contained a CpG island in their proximal promoter, significantly higher than the 51% CpG containing promoters in our entire promoter database. CpG islands are thought to be non-tissue specific (64), further supporting the selection of the 81 genes as a shared response to hypoxia. The Kyoto Encyclopedia of Genes and Genomes (KEGG) database was used to identify over-represented pathways and processes within the 81 genes. In Figure 4A a metabolic map of HIF-1-target genes was constructed from the resulting analysis (Supplementary Table S5). The metabolic map contained several known HIF-1-target genes, such as LDHA, ALDOC and GAPDH and also several novel targets such as GYS1 in starch and sucrose metabolism (65) and fumarate hydratase (FH) in the TCA cycle (66). One of the key cellular responses to HIF-1 in hypoxia is a metabolic shift from oxidative phosphorylation to glycolytic production of ATP. To highlight direct targets that may be involved in HIF-1 regulation of mitochondria function the predicted HIF targets that localize to the mitochondria (see ‘Materials and Methods’ section) were used to create a mitochondrial map (Figure 4B). Eight of eleven genes were downregulated in hypoxia and are involved in critical steps of mitochondrial function including two ribosomal subunits, cytochrome c, the iron transporter SLC25A28 and the iron–sulfur assembly and repair protein ISCU.Figure 3.

Bottom Line: The proximal promoters of these genes were then analyzed for the presence of conserved HIF-1-binding sites.We present experimental validation for ANKRD37 as a novel HIF-1-target gene.Together these analyses demonstrate the potential to recover novel HIF-1-target genes and the discovery of mammalian-regulatory elements operative in the context of microarray data sets.

View Article: PubMed Central - PubMed

Affiliation: Center for Computational and Integrative Biology, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA.

ABSTRACT
The transcription factor Hypoxia-inducible factor 1 (HIF-1) plays a central role in the transcriptional response to oxygen flux. To gain insight into the molecular pathways regulated by HIF-1, it is essential to identify the downstream-target genes. We report here a strategy to identify HIF-1-target genes based on an integrative genomic approach combining computational strategies and experimental validation. To identify HIF-1-target genes microarrays data sets were used to rank genes based on their differential response to hypoxia. The proximal promoters of these genes were then analyzed for the presence of conserved HIF-1-binding sites. Genes were scored and ranked based on their response to hypoxia and their HIF-binding site score. Using this strategy we recovered 41% of the previously confirmed HIF-1-target genes that responded to hypoxia in the microarrays and provide a catalogue of predicted HIF-1 targets. We present experimental validation for ANKRD37 as a novel HIF-1-target gene. Together these analyses demonstrate the potential to recover novel HIF-1-target genes and the discovery of mammalian-regulatory elements operative in the context of microarray data sets.

Show MeSH
Related in: MedlinePlus