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. 2021 Feb 15;29(6):2134–2150. doi: 10.1016/j.ymthe.2021.02.017

Figure 4.

Figure 4

HIF-1α was the primary direct target of c-Myc and WDR5

(A) Re-analysis of the ChIP-seq datasets regarding Myc-WT and Myc-WBM in HEK293 cells (GEO: GSE60897). Pie chart and histogram show the percentage and number of different binding regions mapped for WT and WBM of c-Myc. (B) Venn diagram shows the overlap of genes binding with Myc-WT and Myc-WBM. (C) ChIP-seq datasets show the number and binding sites of genes interacting with WDR5 in HEK293 cells (GEO: GSE60897). (D) Overlapped genes interacting with the promoter-TSS region of both WDR5 and Myc-WT are shown in the Venn diagram. (E) Left panel: mutual regulated mRNAs in Ramos cells, a lymphoma cell line, with overexpression of Myc-WT and Myc-WBM (GEO: GSE126207). Right panel: WDR5 in QBC939 cells was silenced by shRNA, and mRNA sequencing was performed to screen the upregulated or downregulated genes (GEO: GSE149536). (F) Flowchart of screening downstream genes of c-Myc and WDR5 by the bioinformatics method. Upper panel: a total of 30 overlapped genes were screened out by the results in the ChIP-seq datasets and RNA-seq, which are shown in (A)–(E). Bottom panel: the overlapped 30 genes had only 2 common genes with EMT-involved genes in GO analysis, which were HIF1A and LEF1. (G) In QBC939 cells, mRNAs of HIF1A and LEF1 were detected with qPCR after silencing WDR5 (upper two panels) or overexpressing Myc-WT/WBM (bottom panel). (H) HIF-1α expression was detected with western blot after silencing WDR5 (upper two panels) or overexpressing Myc-WT/WBM (bottom panel) in QBC939 cells. (I) ChIP assay revealed that WDR5 knockdown (upper two panels) or Myc-WBM mutation (bottom panel) inhibited the recruitment of c-Myc on the HIF1A promoter. ∗p < 0.05, ∗∗p < 0.01, by one-way ANOVA. (J and K) mRNA correlations between HIF-1α and WDR5 (J) or c-Myc (K) were analyzed with a Pearson correlation test. (L and M) Protein correlations between HIF-1α and WDR5 (L) or c-Myc (M) were analyzed with a Pearson correlation test. (N) Protein levels of HIF-1α in CCA patients with double-positive, single-positive, and double-negative expression of c-Myc and WDR5. ∗∗∗p < 0.001, by one-way ANOVA.