Limits...
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.

Show MeSH

Related in: MedlinePlus

ANKRD37 response to hypoxia. (A) ANKRD37 mRNA levels were monitored by qPCR across seven cell lines and normalized to 18S rRNA. (B, C) ANKRD37 hypoxic induction over time during hypoxia. (B) Endogenous HIF1A protein levels determined after 0, 4, 8 and 12 h of hypoxic incubation by western blot. (C) mRNA levels of ANKRD37 and VEGFA measured by qPCR after 0, 4, 8 and 12 h of hypoxic incubation.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2724271&req=5

Figure 5: ANKRD37 response to hypoxia. (A) ANKRD37 mRNA levels were monitored by qPCR across seven cell lines and normalized to 18S rRNA. (B, C) ANKRD37 hypoxic induction over time during hypoxia. (B) Endogenous HIF1A protein levels determined after 0, 4, 8 and 12 h of hypoxic incubation by western blot. (C) mRNA levels of ANKRD37 and VEGFA measured by qPCR after 0, 4, 8 and 12 h of hypoxic incubation.

Mentions: To demonstrate the utility of our method in identifying novel HIF-1-target genes, we selected five genes that were previously unknown to be HIF-1 targets, ANKRD37 (ranked 8), KLF10 (ranked 24) WSB1 (ranked 32), GYS1 (ranked 34) and ELL2 (ranked 465). Using quantitative PCR, ANKRD37 was induced up to 35-fold in hypoxia compared to normoxia, KLF10 up to 2.5-fold, WSB1 up to 4.9-fold, GYS1 up to 7.8-fold, ELL2 was not induced in hypoxia and VEGFA that was used as a positive control was induced up to 8.3-fold (Figure 5 and Supplementary Figure S4A). The gene ANKRD37 was studied further due to its extreme induction in hypoxia and its high ranking within the predicted targets. ANKRD37 is a short (158 amino acids, 17 kDa) ankyrin repeat protein with unknown function that is conserved in mammals and zebrafish. ANKRD37 responded to hypoxia in four of six cell types and four HIF-1-binding sites were predicted within the proximal promoter. The response to hypoxia was validated under hypoxic conditions across seven cell lines. We observed a strong induction of ANKRD37 in all cell lines but most significantly in the colon cancer cell lines DLD-1, HCT116 and SW480 (Figure 5A). ANKRD37 hypoxic induction was measured after 4, 8 and 12 h of incubation in hypoxic conditions in HCT116 cells and compared to VEGFA. HIF-1 protein levels were determined by western blot and showed a significant induction after 4 and 8 h compared to normoxia and a slight decrease after 12 h (Figure 5B). ANKRD37 and VEGFA mRNA levels showed a similar profile that matched the HIF-1 induction; however, ANKRD37 was induced 3-fold over the level of VEGFA induction (Figure 5C).Figure 5.


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)

ANKRD37 response to hypoxia. (A) ANKRD37 mRNA levels were monitored by qPCR across seven cell lines and normalized to 18S rRNA. (B, C) ANKRD37 hypoxic induction over time during hypoxia. (B) Endogenous HIF1A protein levels determined after 0, 4, 8 and 12 h of hypoxic incubation by western blot. (C) mRNA levels of ANKRD37 and VEGFA measured by qPCR after 0, 4, 8 and 12 h of hypoxic incubation.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2724271&req=5

Figure 5: ANKRD37 response to hypoxia. (A) ANKRD37 mRNA levels were monitored by qPCR across seven cell lines and normalized to 18S rRNA. (B, C) ANKRD37 hypoxic induction over time during hypoxia. (B) Endogenous HIF1A protein levels determined after 0, 4, 8 and 12 h of hypoxic incubation by western blot. (C) mRNA levels of ANKRD37 and VEGFA measured by qPCR after 0, 4, 8 and 12 h of hypoxic incubation.
Mentions: To demonstrate the utility of our method in identifying novel HIF-1-target genes, we selected five genes that were previously unknown to be HIF-1 targets, ANKRD37 (ranked 8), KLF10 (ranked 24) WSB1 (ranked 32), GYS1 (ranked 34) and ELL2 (ranked 465). Using quantitative PCR, ANKRD37 was induced up to 35-fold in hypoxia compared to normoxia, KLF10 up to 2.5-fold, WSB1 up to 4.9-fold, GYS1 up to 7.8-fold, ELL2 was not induced in hypoxia and VEGFA that was used as a positive control was induced up to 8.3-fold (Figure 5 and Supplementary Figure S4A). The gene ANKRD37 was studied further due to its extreme induction in hypoxia and its high ranking within the predicted targets. ANKRD37 is a short (158 amino acids, 17 kDa) ankyrin repeat protein with unknown function that is conserved in mammals and zebrafish. ANKRD37 responded to hypoxia in four of six cell types and four HIF-1-binding sites were predicted within the proximal promoter. The response to hypoxia was validated under hypoxic conditions across seven cell lines. We observed a strong induction of ANKRD37 in all cell lines but most significantly in the colon cancer cell lines DLD-1, HCT116 and SW480 (Figure 5A). ANKRD37 hypoxic induction was measured after 4, 8 and 12 h of incubation in hypoxic conditions in HCT116 cells and compared to VEGFA. HIF-1 protein levels were determined by western blot and showed a significant induction after 4 and 8 h compared to normoxia and a slight decrease after 12 h (Figure 5B). ANKRD37 and VEGFA mRNA levels showed a similar profile that matched the HIF-1 induction; however, ANKRD37 was induced 3-fold over the level of VEGFA induction (Figure 5C).Figure 5.

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