Fig. 2: Characterizing HIF-1 binding sites.
From: The hypoxia response pathway promotes PEP carboxykinase and gluconeogenesis in C. elegans
a Diagram of the pck-1 locus and the different promoters used to drive Venus expression. The oval indicates HIF-1, and the black line beneath it indicates the HIF-1 binding site identified by ChIP-seq. The inverted triangle indicates the site of the HRE motif. Arrows indicate the TSS. Boxes indicate exons. The yellow arrow indicates sequences encoding the fluorescent Venus reporter. The black bar under the gene indicates sequences removed in the pck-1(ok2098) deletion. b–g Fluorescence images of animals expressing the transgenes indicated above the images and with the indicated genotype (wild-type, top row; egl-9 mutant, bottom row) under normoxic conditions. Venus fluorescence is indicated in yellow. Fluorescence from mCherry, used as an internal control in which fluorescence is not expected to change as it is not regulated by HIF-1, is magenta. Scale bar indicates 100 microns. Similar results were obtained in three independent trials. h Graph of Venus/mCherry fluorescence ratios for the indicated genotype under normoxic conditions. Dot color indicates a specific reporter as per panel (a) Error bars indicate mean ± SEM. ***P < 0.001, *P < 0.05 ANOVA/Bonferroni Multiple Comparison two-sided test for the indicated comparisons. N = 267 total animals (25–51/column). i Violin plot of histogram counting the number of HIF-1-binding sites (ChIP-seq peaks) against the number of other transcription factors (TFs) known to bind to each site’s region of the genome (within 400 bps). The top violin plot indicates all HIF-1 binding sites, whereas the middle (green) and bottom (red) violin plots indicate only the functional HIF-1 binding sites identified by BETA alone or the combined LOA and BETA approaches, respectively. The solid line indicates median, whereas dotted lines indicate quartiles. HOT sites (peaks near the binding site of 15 or more other transcription factors) are indicated by the blue dotted line. ****P < 0.0001, *P < 0.05 ANOVA/Kruskal–Wallis Multiple Comparison two-sided test for the indicated comparisons. j Graph of the fold change in enrichment (log2) of each indicated transcription factor bound within 400 bps of a HIF-1 binding site (ChIP-seq peak center). TFs with overrepresented or underrepresented binding near HIF-1 sites compared to chance (P < 0.0001 via a one-sided Bootstrap test adjusted for multiple comparisons via Benjamini–Hochberg using FDR values based on 1 × 105 bootstrapped datasets) are highlighted in yellow or cyan, respectively. TF data are from the modERN/ModENCODE consortium.
