Figure 5.

MYC mediates mRNA cap methylation of Wnt/β-catenin signaling pathway transcripts by recruiting CDK7 to gene promoters. (A) m⁷G-mRNA levels of Wnt pathway transcripts in IMECs following CDK7 or CDK9 siRNA depletion normalized to total mRNA input (n=3, biological). Depletion was confirmed using Western blot in right panel. Error bars indicate SEM and asterisks indicate significance (*p < 0.05, **p < 0.01). (B) m⁷G-mRNA levels of Wnt pathway transcripts in P493-6 cells at the 0 h (MYC off) or 24 h time point (MYC on) following treatment with either 100nM SNS-032 or 50nm/250nM THZ1. Normalized to total mRNA input (n=3, biological). Inhibition was confirmed using Western blot in right panel. Error bars indicate SEM and asterisks indicate significance (§p < 0.05, ¶p < 0.01 relative to 0 h DMSO and *p < 0.05, #p < 0.01 relative to 24 h DMSO). (C) Chromatin immunoprecipitations (ChIPs) for MYC, CDK7, and phospho-S5 RNA Pol II were performed in P493-6 cells at three time points of MYC activation (0, 1, and 24 h). qPCR performed to detect levels of each factor at GSK3B and APC promoters (normalized to input, ACTB control) (n=3, biological). (D) Western blot detecting global phospho-S5 levels in P493-6 cells. Phospho-S5 levels quantified in right panel (n=3, biological). Error bars indicate SEM and asterisks indicate significance (*p < 0.05, **p < 0.01, ***p < 0.001)