BHPF-mediated downregulation of YTHDF2 induces apoptosis via activation of STING1-NFκB/UTX-TNFα axis. A and B, Effects of apoptosis inhibitor Q-VD-OPh on BHPF-induced CVP defects (A) and apoptosis (B) in 34 hpf zebrafish embryos with quantification (N denotes number of embryos for each experimental group). C, Effects of YTHDF2 depletion (ythdf2-MO) on apoptosis (images with quantification) and tnfα mRNA levels in zebrafish embryos. D, Schematic illustration of the unbiased DNA oligo affinity protein assay (DAPA)-mass spectrometry method. E, Venn diagram showing overlap of the DAPA-MS identified zebrafish tnfα promoter-bound proteins with the predicted zebrafish tnfα promoter transcription factors expressed in CVP region. The heat map depicting the abundance of proteins binding to tnfα promoter fragment with or without BHPF exposure (right panel). The data is log10 transformed. F, Western blot results showing the binding affinity of p65 to tnfα promoter fragment upon BHPF exposure (up panel) or depletion of YTHDF2 (down panel). G, ChIP-qPCR and RT-qPCR analysis showing association of p65, UTX, and H3K27me3 levels on tnfα promoter and tnfα mRNA expression in 34 hpf embryos under BHPF exposure with or without JSH-23 treatment. H, ChIP-qPCR analysis showing the levels of tnfα promoter-bound p65 and H3K27me3 in 34 hpf wild type zebrafish embryos and ythdf2 morphants. I, ChIP-qPCR and RT-qPCR analysis showing association of p65 on tnfα promoter and tnfα mRNA expression in 34 hpf embryos under BHPF exposure with or without H-151 treatment. J, Effects of m6A binding ability of YTHDF2 on YTHDF2 and sting1 mRNA interaction in embryos expressing wild type (ythdf2-flag WT) or m6A binding defective mutant (ythdf2-flag MUT); crebbp was used as a positive control. K, Effects of YTHDF2 on sting1 mRNA stability. Wild-type embryos, ythdf2 morphants with or without 8 hours of Actinomycin D (ActD) treatment were harvested for RT-qPCR analysis. L, Line chart showing the levels of microinjected sting1 mRNA (wild type and m6A modification sites mutant) in embryos at indicated time points. M, Effects of H-151, JSH-23, and GSK-J4 on BHPF-induced CVP defects with quantification (N denotes number of embryos for each experimental group). N, Schematic illustration of the YTHDF2-STING1-NFκB/UTX-TNFα apoptosis axis upon BHPF exposure. Suppression of any single step within this pathway (H-151 for STING, JSH-23 for NFκB, GSK-J4 for UTX, tnfα morpholino for TNFα, and Q-VD-OPh for apoptosis) rescued BHPF-induced CVP defects. Scale bar, 20 μm (A–C, M). Molecular biology experiment materials are zebrafish whole embryos (D–L). Zebrafish embryos were treated with BHPF from 4 hpf to the indicated time. Data are mean ± s.d. Student's t test, ns represents P > 0.05, * represents P < 0.05, ** represents P < 0.01, *** represents P < 0.001.
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