Figure 7.

WDR5 suppressed PHD2 expression through HDAC2-induced H3K4 deacetylation

(A) Half-life detection of PHD2 mRNA was performed using 10 μM actinomycin D (Act D) for the indicated times. (B) Chromatin accessibility of PHD2 promoters was evaluated by quantitative real-time PCR with DNase I-pretreated nucleus of control, scramble, and WDR5-silenced QBC939 cells. Fold change was analyzed using 2^−ΔΔCt. (C) ChIP assay showed that WDR5-silenced QBC939 cells had elevated H3K4ac modification at the PHD2 promoter regions. H3K4ac antibody was used for immunoprecipitation (IP) after WDR5 knockdown, and quantitative real-time PCR of the PHD2 promoter in output was performed. (D) A CUT&Tag assay was performed to confirm that WDR5 knockdown promoted H3K4ac modification at PHD2 promoter regions. WDR5 was knocked down by shWDR5 in QBC939 cells, and H3K4Ac antibody was used to interact with the H3K4-acetylized protein. Log₂(RPM + 1) was used for quantification. (E) Overlay of H3K4ac at EGLN1 (PHD2) loci in scramble (purple) and WDR5 knockdown (green) QBC939 cells. Significantly increased regions are underscored (red box). (F) List of 10 DEGs with most significant change in CUT&Tag data. EGLN1 (PHD2) had the highest fold change after WDR5 knockdown. (G) CoIP of WDR5 with HDAC1–HDAC3 in QBC939 cells. (H) Effect of HDAC1–HDAC3 knockdown on the expression of PHD2 protein in QBC939 cells. (I) ChIP assays show that H3K4ac on PHD2 promoters in HDAC2 silenced QBC939 cells. H3K4ac antibody was used for IP after HDAC2 knockdown, and qPCR of the PHD2 promoter in output was performed. (J) ChIP assay revealed that WDR5 directly interacts with the promoter region of PHD2 gene. WDR5 antibody was used for IP, and qPCR of the PHD2 promoter in output was performed. (K) ChIP assay revealed that WDR5 knockdown attenuated HDAC2 binding to the PHD2 promoter. HDAC2 antibody was used for IP after WDR5 knockdown. (L) ChIP assay revealed that WDR5 overexpression disrupted H3K4ac at PHD2 promoter regions and knockdown or inhibition of HDAC2 reversed it. H3K4ac antibody was used for IP, and quantitative real-time PCR of PHD2 promoter in output was performed. (M) Expression of PHD2 in WDR5-overexpressed QBC939 cells that were treated with 50 μM SCA for 48 h or transfected with siHDAC2. Data were analyzed with two-way ANOVA in (A), or with one-way ANOVA in (B), (C), and (I)–(L). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. n.s., not significant.