(A) MudPIT of Med26-associated proteins. See also Table S1. (B) Schematic of wild type and mutant Med26 proteins. (C) Western blot of FLAG-IPed complexes from HeLa cells expressing wild type and mutant Med26 (lanes 2-5, 7-10) and of rat liver Pol II (lane 1) (Serizawa et al., 1992). Lanes 1-3 and 4-5, respectively, are from different blots. (D) Proteins copurifying with FLAG-Med26 NTD, TFIIS NTD, or Elongin A NTD, analyzed by western blotting. WT, wild type. Here and in subsequent figures, IP and input (3% of total) were analyzed on different blots; hence it is not possible to make quantitative comparisons between IP and input samples.

(A) MudPIT of proteins bound to SEC components, NARG2, and KIAA0947. (B) Western blotting of proteins associated with FLAG-tagged NARG2, KIAA0947 and AF9. (C) Binding of NARG2 and KIAA0947 to ELL/EAF1. FLAG-IPed complexes from baculovirus-infected insect cells expressing the indicated proteins were analyzed by western blotting. Asterisk, IgH. (D) Binding of EAF1 to the Med26 NTD. Western blotting of FLAG-IPed complexes or lysates (Input) from baculovirus-infected insect cells, using anti-FLAG (M2) antibodies and Alexa Fluor 680-labeled anti-mouse IgG (light chain-specific) (red) or rabbit anti-cMyc antibodies and IR Dye™ 800-labeled goat anti rabbit IgG (green).

(A) Schematic of immobilized template assay. (B) Med26 NTD is dispensable for Mediator-dependent recruitment of TFIID to a promoter. Assays contained GAL4×5-MLT template and GAL4-VP16, TFIID, and Mediator (Med) with Med26 or Med26ΔNTD as indicated. (C) Mediator-dependent recruitment of ELL/EAF1 to GAL4- (lanes 1-3) or HNF4α-responsive promoters. Med(F-Med26 wt), Med26-Mediator. (D) Med26ΔNTD-Mediator [Med(F-Med26 ΔNTD)] does not recruit EAF1 to a promoter. (E) Free Med26 is not sufficient for EAF1 recruitment. Lanes 1-4, assays contained either free full length Med26 or Med26-Mediator. Lanes 5-8 show amounts of free Med26 and Med26-Mediator in binding assays. (F) Free Med26 NTD blocks Mediator-dependent EAF1 recruitment. Assays were performed with Med26-Mediator, with or without free Med26 NTD (His-Med26-NTD).

(A) Western blots of FLAG IPs or lysates (Input) from cells stably expressing the indicated FLAG-Med26 NTD proteins. Data is from the same experiment in Figure 1D. Negative and positive controls in the first two lanes of each panel are repeated from Figure 1D. (B) Defective binding of Med26(R61A,K62A)-Mediator to elongation factors. FLAG IPs from cells stably expressing the indicated FLAG-Med26 proteins analyzed by silver staining (Ag stain) or western blotting (WB). (C) Med26(R61A,K62A)- Mediator fails to recruit EAF1 to a promoter. Assays contained template, HNF4α, EAF1, and wild type or Med26(R61A,K62A)-Mediator. (D) TFIID blocks Mediator-dependent recruitment of EAF1 to a promoter. Assays contained template, HNF4α, EAF1, TFIID and Mediator.

(A) Dox-inducible shRNA-mediated silencing of endogenous Med26 expression in 293T cells stably expressing different shRNAs. Cells were incubated with or without Dox for 48 hours, and cell lysates were analyzed by western blotting. (B)(C) Med26 depletion decreases cell proliferation in 293T cells stably expressing Dox-inducible shRNAs. Cell number is expressed relative to cell number at day 5 in cultures without Dox. (D) Western blots for endogenous Med26 or tubulin 48 hrs after transfection of non-targeting (control) or Med26 siRNAs. (E) Effect of siRNA-mediated Med26 depletion on gene expression. (F) Effect of Med26 depletion on Hsp70 induction by heat shock. Data points are the average of three independent experiments; error bars show standard deviation. See also Figure S2 and Table S2.

(A) Occupancy of Med26 on the promoter of c-MYC or HSP70 gene under non-heat or heat shocked condition (7.5 min). (B)(C) Effect of Med26 depletion on AFF4, CDK9 and Pol II occupancy on the c-MYC or HSP70 gene under steady state or heat shocked conditions in HEK293T cells. Ig, IgG (TBP, Pol II, AFF4, CDK9 and Ser5p ChIPs) or IgM (Ser2p ChIP) control; Spec ab, specific antibody. (A)(B)(C) ChIP/input is average from two biological replicates, error bars show data range. (D) Med26, Pol II and SEC components in HEK293T cells treated with non-targeting siRNA or Med26 siRNA #4.

(A) Med26-wt (wild type), but not Med26-mut (R61A,K62A), rescues proliferation defect of Med26 depleted 293T cells expressing Dox-inducible Med26 shRNA #3 and exogenous wild type or mutant Med26, grown with or without Dox. (B) Wild type, but not Med26(R61A,K62A) rescues gene expression defect of Med26 depleted cells. (C) Effect of overexpressing Med26 or Med26(R61A,K62A) on Pol II occupancy at c-MYC in HeLa cells stably expressing wild type or mutant Med26. Ig, IgG (Pol II and Ser5p ChIPs) or IgM (Ser2p ChIP) control; Spec ab, specific antibody. (D) Model for Mediator and Med26 NTD function during the transition to productive elongation. In the initiation and/or early elongation complexes, the EAF binding site on the Med26 NTD is occluded by interaction with TFIID. As Pol II moves further from the initiation site and/or upon receipt of a suitable signal, the Med26 NTD is released from TFIID, allowing it to recruit ELL/EAF complexes. Green and red oval, Med26; N, Med26 NTD; black bars, binding sites for Med26 NTD on TFIID and EAF family members.