Fig. 5: MED15 T603 phosphorylation regulates senescence by modulating FOXA1-mediated repression of downstream genes.

a Transcription factor motif analysis of T603A-downregulated genes and T603D-upregulated genes identified via RNA-seq. b Workflow of HA IP-MS in HA-MED15 knock-in and WT cell nuclear extracts. c Scatter plot of three HA IP-MS replicates in terms of log₂ (fold change) correlation and log₁₀ (P value) for three sets of experiments: HA IP of HA-MED15 293T cells vs 293T WT control cells. IP experiments were performed with nuclear extracts isolated from 293T cells (n = 3). d Co-IP assay using FLAG beads to pull down FLAG-FOXA1 in FLAG-FOXA1-overexpressing and control 293T cells. e Endogenous co-IP assay using a FOXA1 antibody to pull down MED15 and pMED15 in WT and T603A mutant HaCaT cells treated without or with TGFβ for 3 h. f Pie chart showing Pol II and FOXA1 ChIP-seq binding sites, promoter regions defined as −1000 bp to +200 bp relative to the transcription start site (TSS). n = 2 biologically independent ChIP-seq replicates in WT and T603A mutant HaCaT cells. g Heatmap of the expression of MED15 pT603 downstream genes in MED15 T603A mutant HaCaT cells and MED15 T603D mutant MCF7 cells. h Heatmap of Pol II and FOXA1 binding peaks on MED15 pT603 downstream genes identified via ChIP-seq. i Average enrichment profiles of FOXA1 ChIP-seq signals at MED15 pT603 downstream genes in the reference genome hg19. ChIP-seq signals are normalized to CPM (counts per million mapped reads). j Violin plot of the FOXA1 binding intensity to MED15 pT603 downstream genes. ****P < 0.0001, t-test. k Average enrichment profiles of Pol II ChIP-seq signals at MED15 pT603 downstream genes in the reference genome hg19. ChIP-seq signals are normalized to CPM. l Violin plot of the Pol II binding intensity to MED15 pT603 downstream genes. ****P < 0.0001, Kolmogorov–Smirnov (K–S) test. m ChIP-seq track plot showing FOXA1 and Pol II genomic binding associated with the IGF1R, OAS1 and CD47 genes in WT and T603A HaCaT cells. The RNA-seq track plot shows corresponding changes in gene expression in cells expressing different MED15 T603 mutants. ChIP-seq and RNA-seq signals are normalized to CPM.