(A) The CAG-70 repeat locus analyzed. Double underlines indicate the qPCR amplicons used for qPCR: light blue is across CAG, dark blue is CAG adjacent; see S2B Fig for a comparison. (B) DRIP using the S9.6 antibody to RNA:DNA hybrids in wild-type, thp2Δ, trf4Δ, and rnh1Δrnh201Δ strains with and without RNase H treatment. The presence of the CAG locus was determined by qPCR using the across-CAG primers. Each bar represents the mean ± SEM of at least 3 biological replicates; each data point represents an individual biological replicate (Table G in S1 Tables). (C) qRT-PCR detecting rCUG and rCAG transcript levels. A consistent amount RNA per sample was reverse transcribed into cDNA by strand-specific RT-PCR and qPCR was used to quantify cDNA levels at the indicated locus. rCUG transcripts result from readthrough transcription from the URA3 gene [12], rCAG transcripts result from cryptic transcription from the G4T4 locus direction. Each bar represents the mean ± SEM of at least 3 biological replicates; each data point represents an individual biological replicate; *p < 0.05, compared to the same transcript in wild-type cells, by t test (Table H in S1 Tables). (D) Rate of FOAR × 10−6 in indicated mutants in RNase H1 overexpression conditions. The PMET25-RNH1 strain was grown in the presence of methionine (RNH1 repressed) or absence of methionine (RNH1 induced). Each data point represents an individual biological replicate; *p < 0.05, compared to no induction condition in the same mutant, by t test. Average of at least 3 experiments ± SEM is shown (Table A in S1 Tables). See S1B Fig and Table I in S1 Tables for RNase H1 expression levels in the various strains. (E) RNAPII ChIP at the CAG-70 repeat locus in the indicated strains either without (RNH1 endogenous promoter) or with RNase H1 overexpression (RNH1 expressed under the PMET25 promoter, induced in the absence of methionine). The IP/INPUT signal at the CAG repeat was determined by qPCR using the primer set adjacent to CAG-70. Each bar represents the mean ± SEM of at least 3 biological replicates; each data point represents an individual biological replicate; *p < 0.05 compared to wild type compared to no RNase H1 overexpression, by t test (Table J in S1 Tables). ChIP, chromatin immunoprecipitation; DRIP, DNA:RNA immunoprecipitation; FOA, fluoroorotic acid; RNAPII, RNA polymerase II.
https://doi.org/10.1371/journal.pbio.3001940.g003
