Fig. 4: MIR31HG modulates GLI2 expression via WDR5/MLL3/P300 complex-mediated H3K4me1 and H3K27ace modification in NSCLC cells.

A Expression level of H3K4me1/2/3 and H3K27Ace in NSCLC cells upon MIR31HG overexpression or knockdown detected by immunoblot. H3 was used as an internal control for nuclear protein. B CUT&Tag sequencing of H1299 genome precipitated by H3K4me1 or H3K27ace antibody zoomed in at the GLI2 enhancer area in H1299 cells. C CUT&Tag assay followed by RT-qPCR of the H3K4me1 + /H3K27ace+ region of GLI2 enhancer by H3K4me1 or H3K27ace antibody, or IgG as a control. The primers P1-P10 were designed for the GLI2 enhancer region, and N1-N3 locate in front of P1-P10 region where no peaks detected as negative control. D Amplification of MIR31HG transcripts upon WDR5 antibody precipitation, as detected by the RIP assay. IgG antibody-precipitated specimens were used as negative control. Whole cell lysate was labeled as the input and used as the positive control. E Schematic diagram (upper panel) showing Flag-tagged full-length WDR5 protein or its truncates and interaction between biotin-sense labeled MIR31HG and peptides detected by RNA pulldown assay (lower panel). F Interaction between WDR5 and H3K4me1 modification detected by co-IP immunoblotting. G, H Protein levels of H3K4me1, H3K27ace, and GLI2 in NSCLC cells followed by WDR5 (G) or P300 (H) inhibitor administration. α-Tubulin and H3 were used as internal controls for whole cell and nuclear protein, separately. I RT-PCR (up) and RT-qPCR (down) of MIR31HG following RIP assay precipitated by MLL3, P300, and IgG as the negative control. DMSO: solvent of WDR5 inhibitor; WDR5i WDR5 inhibitor applied at 20 µM. J Interaction between WDR5 and MLL3 detected by co-IP immunoblot in H520 and H1299 cells. K Interaction between MLL3 and P300 detected by co-IP immunoblot in NSCLC cells. Data are presented as mean ± SEM; significance abundance: * p < 0.05, ** p < 0.01, *** p < 0.001.