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.
Highlights
(encoding the spliceosomal U6 snRNA), RPR1, SCR1, SNR52, and RNA170
Transcription of the tRNA genes accounts for 10 –15% of nucleoside triphosphates consumed by nuclear transcription (2); reflecting this high energetic cost, polymerase III (pol III) transcription is regulated in response to environmental stress and nutrient availability through multiple signaling pathways that converge on Maf1, the central repressor of pol III transcription (3)
The TFIIIB-DNA complex suffices for pol III recruitment during rapidly reiterating rounds of transcription in vitro (16); the low levels of enrichment of TFIIIC subunits relative to TFIIIB subunits in chromatin immunoprecipitation (ChIP) analysis (17–19) may indicate that, once bound to DNA, TFIIIB suffices for pol III recruitment in vivo (17)
Summary
Yeast Strains and Plasmids—Thirteen copies of the c-Myc epitope were inserted at the C termini of RET1, BDP1, TFC4, TFC1, TFC6, TFC7, and TFC8 in yeast strain BY4727 (ATCC) by homologous recombination of a 13Myc-KanMX PCR product (29) generated with primers specified in supplemental Table S2. QPCR was performed by titrating total input DNA and ChIP DNA in the presence of [␣-32P]dCTP, using the primers listed in supplemental Table S3 (M13 forward and reverse primers were used for amplification of TA30 and TA30pyr in pRS316) and quantified by PAGE and phosphorimaging plate analysis. For protein-DNA complex formation with Myc-tagged TFIIIC subunits, reactions were supplemented with 100 fmol each of recombinant Brf, TBP, and Bdp (rTFIIIB) (35) and incubated for 40 min at room temperature. For in vitro assembled Myc-tagged TFIIIB-containing complexes, the BR␣ fraction from a BDP1-Myc cell extract was incubated with probe DNA, 100 fmol of recombinant TBP, and 100 fmol of recombinant Brf. For in vitro assembled Myc-tagged pol III-containing complexes, the DEAE Sephadex fraction from a RET1-Myc cell extract was incubated with the DNA probe and 100 fmol of rTFIIIB and highly purified TFIIIC (16). The samples were loaded onto an 8% polyacrylamide, 8 M urea gel and quantified by phosphorimaging plate analysis
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