Fig 2: Effects of GTF depletion on initiation and elongation. (A) Phases of Pol II initiation. TFIID is recruited by core promoter elements and/or a positioned +1 nucleosome and through interactions with other TFs. TBP-dependent promoters have the most stable recruitment due to TBP interaction with its binding motif which is likely quickly stabilized by TFIIA. TAF1-dependency relies in part on TAF1 interaction with a G-rich downstream element and a positioned +1 nucleosome. XBP- and TFIIB-dependence occurs on the least stable interactions that require rapid initiation before loss of TFIID. After TFIID recruitment, the other GTFs associate to complete the PIC which then initiates. (B) RNA polymerase II elongation control with a depiction of the effect of compensatory P-TEFb release from the 7SK snRNP. Lifetimes and elongation rates for the four phases of the transcription cycle are indicated. P-TEFb dependent phosphorylation of DSIF normally occurs during the transition into productive elongation and is reversed downstream of the CPS. Termination occurs during promoter-proximal pausing and downstream of the CPS. Reduction of productive elongation leads to compensatory release of P-TEFb from the snRNP which we hypothesize leads to enhanced phosphorylation of DSIF to both increase gene body transcription and to delay termination downstream of the CPS. Green dashed outline and arrows indicate activity of released P-TEFb.

Fig 3: RUNX3 defends against endogenous oncogenic K-Ras. a H460-ERT2-RUNX3 and H460-ERT2-RUNX3-K94/171R cells were synchronized by serum deprivation and stimulated with 10% serum and 1 µM 4-OHT for the indicated durations (0, 4, and 8 h). Time-dependent subcellular localization of the expressed proteins was analyzed by double immunofluorescence staining (green = RUNX3; red = F-actin). b, c H460-ERT2-RUNX3 and H460-ERT2-RUNX3-K94/171R cells were serum-starved for 24 h, stimulated with 10% serum or 10% serum + 1 µM 4-OHT. Cells were harvested at the indicated time points, and the time-dependent interactions of RUNX3 with BRD2, p300, H4K12-ac, TFIID complex (TAF1, TAF7, and TBP), SWI/SNF complex (BRG-1 and BAF155), and MLL1/5 were measured by IP and IB. Expression of p14ARF, p53, and p21 was measured by IB. The binding of the proteins and H4K12-ac, H3K27-me3, and H3K4-me3 to the ARF promoter was measured by ChIP at the indicated time points. One-thirtieth of the lysates were PCR-amplified as input samples. d H460-vec, H460-ERT2-RUNX3, and H460-ERT2-RUNX3-K94/171R cells were treated with indicated si-RNA, serum-starved for 24 h, and then stimulated with 10% serum or 10% serum + 1 µM 4-OHT for the indicated durations. Apoptotic cells were detected by flow cytometry after Annexin V–FITC/PI staining. The levels of p53 were measured by IB

Fig 4: GTFs targeted for rapid depletion. (A) Cryo-EM structures of TFIID bound to promoter DNA (6MZM) (6) and assembled PIC containing TFIID and TFIIH (7ENC) (3). Targeted proteins have been colored in the structures as follows: TBP (blue), TAF1 (red), TFIIB (green), and XPB (purple). (B) Depletion of TBP, TAF1 and TFIIB at different concentrations of dTAGV-1. Also shown is XPB depletion over time. Protein levels were determined by western blots of whole cell lysates utilizing GTF-specific antibodies, except the anti-FKBP12 antibody was used for XPB. Actin or vinculin was used as a loading control. (C) Genome browser view of tracks depicting sense and divergent PRO-Seq transcript pileups for wildtype (WT) HAP1 cells or the indicated FKBP12 tagged HAP1 cell lines at the KISS1R locus. Cells were treated for 2 h with DMSO as a control (light colors) or 400 nM dTAGV-1 to deplete the tagged factor (dark color) before PRO-Seq was performed.