Genome-wide identification and annotation of HIF-1α binding sites in two cell lines using massively parallel sequencing

Abstract

We identified 531 and 616 putative HIF-1α target sites by ChIP-Seq in the cancerous cell line DLD-1 and the non-cancerous cell line TIG-3, respectively. We also examined the positions and expression levels of transcriptional start sites (TSSs) in these cell lines using our TSS-Seq method. We observed that 121 and 48 genes in DLD-1 and TIG-3 cells, respectively, had HIF-1α binding sites in proximal regions of the previously reported TSSs that were up-regulated at the transcriptional level. In addition, 193 and 123 of the HIF-1α target sites, respectively, were located in proximal regions of previously uncharacterized TSSs, namely, TSSs of putative alternative promoters of protein-coding genes or promoters of putative non-protein-coding transcripts. The hypoxic response of DLD-1 cells was more significant than that of TIG-3 cells with respect to both the number of target sites and the degree of induced changes in transcript expression. The Nucleosome-Seq and ChIP-Seq analyses of histone modifications revealed that the chromatin formed an open structure in regions surrounding the HIF-1α binding sites, but this event occurred prior to the actual binding of HIF-1α. Different cellular histories may be encoded by chromatin structures and determine the activation of specific genes in response to hypoxic shock.

Electronic supplementary material: The online version of this article (doi:10.1007/s11568-011-9150-9) contains supplementary material, which is available to authorized users.

Keywords: ChIP-Seq; HIF-1 alpha; Hypoxia; Transcriptome.

Fig. 1

Identification of HIF-1α binding sites. a Representative sites identified as “hypoxia-responsive”, “hypoxia-enriched” and “non-hypoxia-responsive” HIF-1α binding sites. The definitions for each group are described in the text. The former two categories were considered for identifying HIF-1α target genes. Blue and red horizontal bars indicate 36-bp sequence tags that were mapped to the forward and reverse strands, respectively. The genomic coordinates and RefSeq IDs of the indicated transcript models are shown in the margin. b Validation of the identified binding sites by ChIP-Seq analysis using real-time PCR. The fold enrichment of the signals determined for immunoprecipitated samples compared with the background noise is shown on a log scale for 29 selected cases. Genomic regions as specified in Supplementary Table 1 were used for the validation analysis. For details, see “Materials and methods” and Supplementary Fig. 2. Error bars represent the standard deviations of triplicate experiments. (Color figure online)

Fig. 2

Characterization of RefSeq transcripts. a Genomic locations of the identified HIF-1α binding sites in DLD-1 and TIG-3 cells compared to the RefSeq transcript models. Positions relative to the RefSeq genes are shown. When a RefSeq gene contained multiple transcript models, all of the transcript models were considered. b Distributions of the distances from the identified binding sites to the 5′-ends of the RefSeq transcript models. Blue and red bars represent DLD-1 and TIG-3 cells, respectively. When a RefSeq gene contained multiple transcript models, all of the transcript models were considered and counted redundantly. (Color figure online)

Fig. 3

Correlations of HIF-1α target genes and their expression levels. a Venn diagram of the number of identified HIF-1α target genes in DLD-1 and TIG-3 cells. The upper panel shows the total number of genes for which HIF-1α binding sites were identified in the flanking regions. The lower panel shows the identified target genes with expression levels that were induced more than twofold under hypoxia, as determined on the basis of digital TSS tag counts. b Fold changes in the expression levels of the identified target genes in HIF-1α knock-down cells. The fold changes were calculated as the TSS tag concentration in knock-down cells/TSS tag concentration in normal cells. The bars below the axis represent cases in which gene expression was repressed in HIF-1α knockdown cells in comparison to normal cells (83% of all cases). See Supplementary Fig. 4 for details of the validation analysis. c Distributions of the fold changes in expression levels of the genes bound by HIF-1α in DLD-1 and TIG-3 cells, as determined by the digital TSS tag counts. The statistical significance of the difference in distributions was evaluated using the Wilcoxon rank sum test (P = 1e–13)

Fig. 4

Fold change in the amount of RNA present in polysome fractions in response to hypoxic stimuli. Distributions of the fold changes of transcripts in the polysome fractions in response to hypoxia, as determined by digital RNA-Seq tag counts. Fold changes in HIF-1α-bound target genes for which transcriptional induction was observed (left), HIF-1α-bound genes for which no transcriptional induction was observed (middle) and all RefSeq genes (right) are shown. Statistical significance in the differences in the distributions were evaluated using the Wilcoxon rank sum test and are shown in the margin

Fig. 5

Nucleosome structures in regions surrounding HIF-1α binding sites. Nucleosome occupancy in regions surrounding the identified HIF-1α binding sites. The calculated nucleosome occupancy scores (y-axis) are plotted against every genomic coordinate (x-axis). The computational procedure used to calculate the nucleosome occupancy score is described in “Materials and methods”. The putative binding site of HIF-1α, represented by HRE, was defined as position 0 (x-axis). Blue and red lines indicate the nucleosome occupancy scores under hypoxia and normoxia, respectively. Only the HIF-1α binding sites for which the associated TSCs were induced by more than twofold were used in the plot for each cell type. For details regarding the Nucleosome-Seq datasets, see Supplementary Fig. 9. (Color figure online)

Fig. 6

Profiles of RNA polymerase II binding and histone modification in the regions surrounding HIF-1α binding sites. Average tag concentrations (y-axis) obtained in the ChIP-Seq analyses of pol II (a, e), H3K4me3 (b, f), H3Ac (c, g) and H3K27me3 (d, h) are plotted for each genomic coordinate (x-axis). The observed profiles are shown for DLD-1 (a–d) and TIG-3 cells (e–h). In each panel, red and blue lines represent the tag counts under hypoxia and normoxia for the IP samples, respectively. Green and sky blue lines represent the results obtained for the background control (whole cell extract samples). The putative binding site of HIF-1α, represented by HRE, was defined as position 0 (x-axis). For each panel, the tag counts for 290 and 221 binding sites, which have HRE among the HIF-1α target TSCs (either geneic or intergenic TSCs), were averaged in DLD-1 and TIG-3 cells, respectively. (Color figure online)