Absolute gene occupancies by RNA polymerase III, TFIIIB, and TFIIIC in Saccharomyces cerevisiae

Abstract

A major limitation of chromatin immunoprecipitation lies in the challenge of measuring the immunoprecipitation effectiveness of different proteins and antibodies and the resultant inability to compare the occupancies of different DNA-binding proteins. Here we present the implementation of a quantitative chromatin immunoprecipitation assay in the RNA polymerase III (pol III) system that allowed us to measure the absolute in vivo occupancy of pol III and its two transcription factors, TFIIIC and TFIIIB, on a subset of pol III genes. The crucial point of our analysis was devising a method that allows the accurate determination of the immunoprecipitation efficiency for each protein. We achieved this by spiking every immunoprecipitation reaction with the formaldehyde cross-linked in vitro counterparts of TFIIIB-, TFIIIC-, and pol III-DNA complexes, measuring the in vitro occupancies of the corresponding factors on a DNA probe and determining probe recovery by quantitative PCR. Analysis of nine pol III-transcribed genes with diverse sequence characteristics showed a very high occupancy by TFIIIB and pol III (pol III occupancy being generally approximately 70% of TFIIIB occupancy) and a TFIIIC occupancy that ranged between approximately 5 and 25%. Current data suggest that TFIIIC is released during transcription in vitro, and it has been proposed that TFIIIB suffices for pol III recruitment in vivo. Our findings point to the transient nature of the TFIIIC-DNA interaction in vivo, with no significant counter-correlation between pol III and TFIIIC occupancy and instead to a dependence of TFIIIB-DNA and TFIIIC-DNA complex maintenance in vivo on pol III function.

FIGURE 1.

The formation of Myc-tagged promoter complexes on a SUP4 gene: EMSA analysis. A, the ³²P-labeled modified SUP4 probe was incubated with C128-Myc BRα fraction (lanes 2–5) and with purified recombinant Brf1 and Bdp1 (lanes 2, 4, and 5), in the presence of unlabeled wild type SUP4 tDNA (lane 2) or SUP4 tRNA gene carrying a G56 substitution in its boxB that eliminates TFIIIC binding (lanes 3–5). Heparin was added to strip TFIIIC for lane 5. The locations of free probe (DNA), TFIIIB-DNA, TFIIIC-DNA, and TFIIIB-TFIIIC-DNA complexes are indicated at the left. B, to form a TFIIIB-TFIIIC-DNA complex, the same probe as in A was incubated with a Tfc4-Myc DEAE Sephadex fraction and with recombinant TFIIIB (lanes 2 and 3). For lane 3, the complex was treated with formaldehyde prior to the addition of heparin. The location of each complex is indicated at the left. The bracket indicates heparin-resistant complexes that were quantified together to determine the fraction of probe bound by TFIIIC. C, the same probe as in A was incubated with a Bdp1-Myc BRα fraction and with recombinant Brf1 and TBP. For lane 2, the complex was treated with formaldehyde prior to the addition of heparin. The brackets at the right indicate the complexes that were quantified together to determine the fraction of probe bound by TFIIIB. D, the same probe was incubated with a C128-Myc DEAE Sephadex fraction and with recombinant TFIIIB to form a pol III-TFIIIB-TFIIIC-DNA complex. The addition of ATP, CTP and UTP (lanes 2–5) allowed the formation of a stalled heparin-resistant elongating complex, which was stripped with heparin to remove pre-elongating pol III and TFIIIC (lanes 2–5) and subsequently cross-linked with formaldehyde (lanes 3–5). The locations of TFIIIB-DNA and pol III-TFIIIB-DNA complexes are indicated at the left. The bracket indicates the complex that was quantified to determine the fraction of probe bound by pol III. Lanes 1 and 2 are from a separate, preliminary experiment.

FIGURE 2.

Gene occupancy by TFIIIC measured in terms of its τ_A (subunit Tfc4; τ₁₃₁) and τ_B (Tfc6; τ₉₁) subcomplexes. The occupancy of the Myc-tagged TFIIIC subunits Tfc4 and Tfc6 was compared on tRNA genes tI(AAT)BL and tP(AAG)CR, SNR6 and ZOD1. The values are expressed as “percent occupancy,” which was calculated as percent recovery of the corresponding genes after immunoprecipitation with anti-Myc antibody, corrected for the efficiency of immunoprecipitation as measured by the recovery of a ³²P-labeled SUP4 probe used to assemble the Myc-tagged TFIIIC-containing complexes spiking the immunoprecipitation reaction. (The DNA probe contains unique flanking sequences that allow recovery to be measured by qPCR.) The mean values from at least two independent chromatin immunoprecipitations with S.E. are shown.

FIGURE 3.

The elimination of purines within the first 12 bp of a modified SUP4 transcription unit induces the loss of both TFIIIC and TFIIIB. A, sequence of the modified SUP4 genes TA30 and TA30pyr, showing the position of the TATA box, the transcriptional start site (for TA30) and boxA. The nontranscribed strand of TA30pyr contains only pyrimidine nucleotides up to bp +17. B, primer extension performed on total RNA extracted from yeast cells transformed with plasmids expressing TA30 and TA30pyr. The transcriptional start site of TA30 transcript and the first base of the 5′ end-processed transcript (+13) are mapped in lane 1, whereas lane 2 shows a considerably lower level of signal for TA30pyr (at bp +1/+2 and bp +18). C, occupancy by TFIIIC, TFIIIB, and pol III was measured on TA30 and TA30pyr in two different backgrounds: in pRS316 (left panel) and in the same plasmid containing two additional tRNA genes positioned ∼ 1600 bp from the modified SUP4 genes (right panel). The values are expressed as “percent occupancy,” as described in the legend to Fig. 2.

FIGURE 4.

Occupancy of the SCR1 gene by TFIIIC, TFIIIB, and pol III. A, schematic representation of SCR1, indicating the position of the transcriptional start site, the TATA box, boxA, boxB, and the terminator. The position of the overlapping amplicons used to measure occupancy is shown below the gene. B, occupancy by TFIIIC, TFIIIB, and pol III of the overlapping SCR1 segments indicated in A. The values are expressed as “percent occupancy” (described in the legend to Fig. 2).