Fig. 3. p63 directly binds to MED12 and the Mediator Kinase Module to activate the basal lineage program.

a, FLAG immunoprecipitation of transiently transfected p63-C-3xFLAG in HEK293T nuclear lysates. Ponceau staining and western blot of FLAG and endogenous MKM (MED12) and core Mediator (MED23) subunits are shown for input and IP samples. The two bands in the Ponceau under p63 correspond to denatured IgG fragments. b, FLAG immunoprecipitation of transiently transfected N-3xFLAG-p63 or p63-C-3xFLAG in HEK293T nuclear lysates in the presence or absence of ethidium bromide (EtBr, final concentration of 50μg/mL). Ponceau and western blot of endogenous MKM (MED12, CDK8) and core Mediator (MED1) subunits are shown for input and IP samples. c, MBP-pulldown of purified MBP or MBP-p63 incubated with nuclear lysates of endogenously tagged 3NHA-CDK8 HeLa cells. Nuclear lysate was equally split between MBP and MBP-p63 for pulldown. Ponceau (left) shows the immobilized MBP fusion proteins, and HA stains the 3xHA-tagged CDK8. Only pulldown results are shown, as the signal was undetectable in the 1% input. d, Western blot of MBP-pulldown of purified MBP-p63 incubated with Sf9 cell lysates expressing different individual human Mediator subunits. 1% input is shown in the left lane. e, MBP pulldown of MBP or MBP-p63 incubated with human MED12-expressing Sf9 lysates. f, Glycerol gradient sedimentation of purified MBP or MBP-p63 incubated with human 4-subunit MKM. Glycerol percentages are indicated above the blots; larger complexes will migrate farther down the gradient. Black dashed boxes show glycerol gradient eluted fractions of individual p63, MBP, or MKM, as well as MBP-MKM incubation samples which did not display shifted migration pattern. Red dashed boxes highlight p63-MKM glycerol gradients that eluted in size-shifted fractions. g, Scatterplot depicting gene expression changes upon MED12 or MED13/MED13L knockout. DESeq2-derived log₂FC for all expressed genes are plotted. Representative plot of 2 different sgRNA per gene. Select basal genes are highlighted. h, GSEA plots of MED13/MED13L double knockout using gene signatures derived from human basal PDAC tumors (Chan-Seng-Yue et al. 2020, left) and direct p63 gene targets in PDAC (Somerville et al. 2018, right). Complete GSEA analysis for all the sgRNA tested can be found in Supplementary Table 2.