a, ERK robustly phosphorylates the Mediator component MED24 on two serines in the C-terminal end of the protein directly adjacent to a hormone receptor-interaction domain; values taken from (Fig. 4a). Data are presented as the mean, n = 2 biologically independent samples. b, Expression values for Med24 at the indicated developmental stages44. c, A schematic outlining the strategy for making Med24-conditional ES cells and where single RNA guides are targeted in the endogenous locus. Med24 expression is maintained by a doxycycline-inducible promoter, whereby stable integration of the cassette can be selected for by either neomycin (top) or hygromycin (bottom). d, e, Western blot analysis of the expression levels of MED24 in wild-type ES cells expressing either construct depicted in c before deletion of endogenous MED24. Selection of exogenous MED24 with neomycin (d) gives slightly higher expression levels than selection with hygromycin (e). Data are representative of two independent experiments. All mutant phenotypes were reproduced in knockout cell lines rescued with either construct. ChIP–seq and transcriptome analysis was performed using cells expressing the neomycin rescue. f, Western blot analysis confirming disruption of endogenous MED24 from hygromycin-selected cells. C14, C15, C2 and C5 are homozygous-mutant cell lines. Data are representative of two independent experiments. g, Western blot analysis confirming disruption of endogenous MED24 from neomycin selected cells. WT1, WT3, WT5 and WT6 are homozygous-mutant cell lines. Data are representative of two independent experiments. h, GO analysis for MED24-bound regions showing that bound regions are associated with genes involved in peri-implantation development. MED24 peaks were located within traditional enhancers (from Extended Data Fig. 3c) and ontologically annotated with GREAT. n = 3 biologically independent samples. i, Motif analysis for MED24-binding regions within repressed and activated enhancers showing enrichment for ESRRB (repressed) and ETS–AP-1-factor (activated) motifs. n = 636 (activated) and 1,726 (repressed). j, Quantification of the levels of NANOG–eGFP expression upon the depletion of MED24. Data are mean + s.e.m., n = 6 biologically independent samples. k, ChIP-qPCR analysis of RNAPII binding to the Nanog enhancer in either wild-type or MED24-knockout cells; enrichment at an unbound region between the proximal enhancer and TSS serves as a negative control. Data are mean + s.e.m., n = 3 biologically independent samples. l, PCA of the nascent transcriptional response in wild-type and MED24-knockout samples following 2 h ERK stimulation. n = 3 biologically independent samples.