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

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

A Comparison of the Rest Complex Binding Patterns in Embryonic Stem Cells and Epiblast Stem Cells

<div><p>We detected and characterized the binding sites of the representative Rest complex components Rest, Sin3A, and Lsd1. We compared their binding patterns in mouse embryonic stem (ES) cells and epiblast stem (EpiS) cells. We found few Rest sites unique to the EpiS cells. The ES-unique site features were distinct from those of the common sites, namely, the signal intensities were weaker, and the characteristic gene function categories differed. Our analyses showed that the Rest binding sites do not always overlap with the Sin3A and Lsd1 binding sites. The Sin3A binding pattern differed remarkably between the ES and EpiS cells and was accompanied by significant changes in acetylated-histone patterns in the surrounding regions. A series of transcriptome analyses in the same cell types unexpectedly showed that the putative target gene transcript levels were not dramatically different despite dynamic changes in the Rest complex binding patterns and chromatin statuses, which suggests that Rest is not the sole determinant of repression at its targets. Nevertheless, we identified putative Rest targets with explicitly enhanced transcription upon Rest knock-down in 143 and 60 common and ES-unique Rest target genes, respectively. Among such sites, several genes are involved in ES cell proliferation. In addition, we also found that long, intergenic non-coding RNAs were apparent Rest targets and shared similar features with the protein-coding target genes. Interestingly, such non-coding target genes showed less conservation through evolution than protein-coding targets. As a result of differences in the components and targets of the Rest complex, its functional roles may differ in ES and EpiS cells.</p></div

Overlap of the Rest binding sites between the ES and EpiS cells.

<p>Overlap of the Rest binding sites, associated with cording genes or non-coding genes, between the ES and EpiS cells, except for the R+/S−/L− sites. The numbers of genes associated with such sites are shown in parentheses.</p

Rest binding site characterization.

<p>(A) The Rest binding sites detected through ChIP Seq overlap between the ES and EpiS cells. We used ES cell data as the standard for this comparison. The parentheses show the numbers of protein-coding genes associated with such sites. (B) GO terms enriched for the ES-unique targets. The number of genes in the indicated GO category and statistical significance for such enrichment are shown in the third and fourth columns, respectively. We used GO terms for which the number of genes was 100–500 and the number of Rest target genes in the indicated category was not less than 20. (C) The ES-unique, EpiS-unique, and common detected binding sites intensities using the ChIP tag counts normalized to the input tag counts for the detected binding sites. Boxplots were drawn for the indicated site categories based on the ChIP Seq tag counts in ES (left) and EpiS cells (right). The statistical significance for the differences is also shown in the top margin. (D) The consensus sequences detected around the ES-unique or common Rest binding sites. The sequence logo and statistical significance are shown in the third and fourth columns, respectively.</p

Co-binding patterns of Rest complex components.

<p>Number of Rest binding sites, associated with coding genes or non-coding genes, categorized by Sin3A and Lsd1 co-binding to the indicated category in ES or EpiS cells. The numbers of genes associated with such sites are shown in parentheses.</p

Rest complex ChIP Seq.

<p>Examples of the Rest complex binding sites detected and associated with protein-coding genes in ES and EpiS cells. ChIP Seq tags for Rest, Sin3A, and Lsd1 in the indicated cells are shown. (A), (B), and (C) show examples of the ES-unique, common, and EpiS-unique sites in the vicinity of “Snap23”, “C2cd5 and Etnk1”, and “Cdh23”, respectively.</p

Rest binding sites surrounding the lncRNA.

<p>Examples of the Rest binding sites associated with lncRNA in the indicated cell types. Rest ChIP Seq tags in the indicated cells are shown. (A), (B), and (C) show examples of the ES-unique, common, and EpiS-unique site in the vicinity of “1500002O10Rik”, “4930524C18Rik”, and “1700025J12Rik and 4933432K03Rik”, respectively.</p

Putative Rest lncRNA targets.

<p>(A) Rest binding sites associated with lncRNAs that overlap between ES cells and EpiS cells. We used ES cells data as the standard for this comparison. The numbers of non-coding-genes associated with such sites are shown in parentheses. (B) Consensus sequences surrounding the ES-unique or common Rest binding sites associated with lncRNA. The sequence logo and statistical significance are shown in the third and fourth columns, respectively. (C) Intensities of the detected binding sites measured using the tag counts for the detected binding sites. We show boxplots for the indicated binding site categories based on the ChIP Seq tag counts in the ES (left panel) and EpiS cells (right panel). The statistical significances for the differences are also shown in the top margin. (D) ChIP Seq intensity changes in the active chromatin and enhancer modifications between the ES and EpiS cells. The statistical significance for the differences was evaluated using Wilcoxon’s signed rank test, which is shown in the top margin. (E) Rest binding site evolutionary conservation. We show the average phastCons score distribution from the −50 base to the +50 base in the Rest binding sites <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095374#pone.0095374-Siepel1" target="_blank">[55]</a>. Boxplots are shown for the indicated categories. The statistical significance for the differences was evaluated using Wilcoxon’s signed rank test, which is shown in the top margin. (F) The Rest binding site associated with lncRNA. Rest ChIP Seq tags in the indicated cells are shown in upper two lanes. A heat map of the phastCons score is shown in the third lane, wherein the more conserved sites are bluer.</p

Transcription effects from Rest complex binding.

<p>(A and C) Boxplots of the transcript levels measured using TSS tag counts in ES (left lanes) or EpiS cells (right lanes) for the indicated category are shown. (B and D) The TSS tag count fold changes between the ES and EpiS cells for the indicated category are shown. The statistical significance for the differences was evaluated using Wilcoxon’s signed rank test, which is shown in the top margin.</p