Genomic location of the human RNA polymerase II general machinery: evidence for a role of TFIIF and Rpb7 at both early and late stages of transcription

Abstract

The functions ascribed to the mammalian GTFs (general transcription factors) during the various stages of the RNAPII (RNA polymerase II) transcription reaction are based largely on in vitro studies. To gain insight as to the functions of the GTFs in living cells, we have analysed the genomic location of several human GTF and RNAPII subunits carrying a TAP (tandem-affinity purification) tag. ChIP (chromatin immunoprecipitation) experiments using anti-tag beads (TAP-ChIP) allowed the systematic localization of the tagged factors. Enrichment of regions located close to the TIS (transcriptional initiation site) versus further downstream TRs (transcribed regions) of nine human genes, selected for the minimal divergence of their alternative TIS, were analysed by QPCR (quantitative PCR). We show that, in contrast with reports using the yeast system, human TFIIF (transcription factor IIF) associates both with regions proximal to the TIS and with further downstream TRs, indicating an in vivo function in elongation for this GTF. Unexpectedly, we found that the Rpb7 subunit of RNAPII, known to be required only for the initiation phase of transcription, remains associated with the polymerase during early elongation. Moreover, ChIP experiments conducted under stress conditions suggest that Rpb7 is involved in the stabilization of transcribing polymerase molecules, from initiation to late elongation stages. Together, our results provide for the first time a general picture of GTF function during the RNAPII transcription reaction in live mammalian cells and show that TFIIF and Rpb7 are involved in both early and late transcriptional stages.

Figure 1. TAP of the RNAPII general transcription machinery

(A) SYPRO-stained SDS gels showing the CDK7- and XPB/ERCC3-TAP eluates. Although the identity of most visible bands is known, only interaction partners that were validated through examination of the literature are shown. Gene symbols and common names are provided. Tagged polypeptides are indicated by an asterisk. Western blots validating the presence of the TFIIH subunits as well as XPG and BCR in both CDK7- and XPB/ERCC3-TAP eluates are shown. (B) TAP-tagged TFIIS/TCEA1 co-purifies with Rpb1 carrying a hypophosphorylated CTD. The N-20 antibody, which detects both the hypophosphorylated (IIa) and hyperphosphorylated (IIo) forms of RNAPII in a whole cell extract (WCE) by being directed to the N-terminal part of Rpb1, revealed the presence of hypophosphorylated RNAPII in both the Rpb11- and TFIIS/TCEA1-TAP eluates. (C) Amino acid sequence alignment of the TFIIS/TCEA1 and TFIIS.1/TCEA2 proteins. The peptides sequences obtained by MS/MS that identify the TFIIS.1/TCEA2 protein in the Rpb11–TAP eluate are boxed. (D) In vitro transcription reactions were reconstituted using calf thymus RNAPII in the presence of the classically purified GTFs TBP, TFIIB, TFIIF and TFIIH. The linearized DNA template carries the AdML promoter and directs the synthesis of a 391-nt transcript. The TAP-tagged GTFs (TFIIAαβ–TAP, TFIIE56–TAP and XPB–TAP) and RNAPII (Rpb11–TAP) were used to replace their cognate highly purified counterparts after their omission from the reconstituted system (All). A control reaction performed with an eluate obtained from non-induced cells is included.

Figure 2. Occupancy of regions proximal to the TIS and further downstream TRs of selected human genes by the GTF and RNAPII subunits obtained by ChIP

Fold enrichment of regions located close to the TIS (left-hand matrix) and further downstream TRs (right-hand matrix) over control regions are represented for each TAP-tagged factor, including subunits of RNAPII (Rpb11) and the GTFs TFIIA (TFIIAαβ), TFIIB, TFIIE (TFIIE56), TFIIH (XPB), TFIID/STAGA (TAF10 and TAF13), the Mediator (SRB7) and TFIIS/TCEA1. Colour code: black:> 5-fold enrichment; grey: significantly enriched with less than 5-fold enrichment values; white: not significantly enriched; X: not determined. The number of regions situated close to the TIS and further downstream TRs occupied by each factor is indicated as a ratio (observed/total tested). The results shown for TFIIS/TCEA1 were obtained by the analysis of cells cross-linked after an irradiation with a total dose of 12.5 J/m² of UVC (TFIISexp). Student’s t tests were performed to determine the significance of the enrichment values obtained in four experiments, including at least two independent ChIP assays.

Figure 3. Occupancy of regions proximal to the TIS and further downstream TRs of selected human genes by the TFIIF subunit RAP30 and the RNAPII subunit Rpb11 as determined by ChIP

Fold enrichment of regions situated close to the TIS (A) and further downstream TRs (B) over control regions are represented for nine selected genes. Student’s t tests were performed to determine the significance of the enrichment values obtained in four experiments, including at least two independent ChIP assays (^*P < 0.05, ^**P ≤ 0.001).

Figure 4. Occupancy of regions proximal to the TIS and further downstream TRs of selected human genes by Rpb7 as determined by ChIP

(A, B) Fold enrichment of regions situated close to the TIS (A) and further downstream TRs (B) over control regions are represented for Rpb7 and Rpb11 when cells are cultured under normal conditions. (C, D) Fold enrichment of regions situated close to the TIS (C) and further downstream TRs (D) after a heat shock (1 h at 42 °C) (HS) are represented for Rpb7 and Rpb11. (E, F) The distribution along the genes ENO1 (E) and HSPA8 (F) is represented for Rpb7 and Rpb11 cultured under normal conditions or after a heat shock (HS). Colour code: black: > 10-fold enrichment; dark grey: between 5- and 10-fold enrichment; light grey: significantly enriched with less than 5-fold enrichment values; white: not significantly enriched. Student’s t tests were performed to determine the significance of the enrichment values obtained in four experiments, including at least two independent ChIP assays (^*P < 0.05, ^**P ≤ 0.001).