Differential dependencies of human RNA polymerase II promoters on TBP, TAF1, TFIIB, and XPB.

General transcription factors (GTFs) are required for RNA polymerase II (Pol II) to initiate transcription at promoters. In this study, we determined the effects of acute depletion of TBP, TAF1, TAF4, TFIIB, and XPB in HAP1 cells. We performed precision nuclear run-on sequencing (PRO-Seq) and quantified nascent transcripts arising from more than 70,000 promoters. The average dependencies for each factor across all promoters varied widely even though levels of depletions were similar. Many of the effects could be attributed to the presence or absence of core promoter elements. Depletion of TBP had a large effect on only a small fraction of Pol II and Pol III promoters. TFIIB depletion also led to readthrough transcription downstream of the 3' ends of genes. We conclude that promoter activity is influenced by recruitment of TFIID, sequence-specific transcription factors, and interaction of the preinitiation complex (PIC) with the +1 nucleosome. Overall design: Five HAP1 cell lines were developed in which the endogenous locus for TBP, TAF1, TAF4, TFIIB or XPB were tagged with FKBP12 as described in the methods. To assay the effects of depleting each of the GTFs on nascent transcription we performed PRO-Seq as described in the methods. Briefly, each of the cell lines were treated with either DMSO (control) or 400 nM dTAGV-1 for 2 h. Nuclei was then isolated and PRO-Seq libraries were prepared in duplicates (one experiment was performed for TAF4).