Volume 30, Issue 2, Pages 177-191 (July 2014) Roles for Pbp1 and Caloric Restriction in Genome and Lifespan Maintenance via Suppression of RNA-DNA Hybrids  Jayesh S. Salvi, Janet N.Y. Chan, Kirk Szafranski, Tony T. Liu, Jane D. Wu, Jonathan B. Olsen, Nurussaba Khanam, Betty P.K. Poon, Andrew Emili, Karim Mekhail  Developmental Cell  Volume 30, Issue 2, Pages 177-191 (July 2014) DOI: 10.1016/j.devcel.2014.05.013 Copyright © 2014 Elsevier Inc. Terms and Conditions

Developmental Cell 2014 30, 177-191DOI: (10.1016/j.devcel.2014.05.013) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 1 Pbp1 Localizes to and Maintains the Stability of rDNA Repeats (A) rDNA repeats on Chr. XII. Each unit yields RNAPI-transcribed 35S precursor rRNA (processed into 25S, 18S, and 5.8S) and RNAPIII-transcribed 5S rRNA. CEN, centromere; TEL, telomere; IGS, intergenic spacer; rightward red fork, replication fork block; red rectangle, intergenic promoter E-pro; blue arrows, RNAPII-dependent ncRNA transcription; red circle, DNA replication origin; black arrow, RNAPI-dependent rRNA transcription. Primers amplifying IGS regions (P1–P5) are shown. (B) Relative rates of loss (±SD; ∗p < 0.01) of ADE2 marker gene from rDNA repeats. Representative images are shown. See Table S1 for full counts and statistical analyses. (C) Pbp1-TAP and Sir2-TAP localization analysis. Representative gel shows PCR products for rDNA sequences (as shown in 1A) or the internal repetitive locus control CUP1 amplified from input and TAP-immunoprecipitated (IP) chromatin. rRC, rRNA-coding region; arrowheads, nonspecific background bands. Graph shows fold enrichments relative to untagged control (mean ± SD; n = 3; ∗p ≤ 0.05). (D) ChIP analysis of TAP-tagged Pbp1, Pbp4, and Lsm12 localization to IGS1, IGS2, and rRC regions and assessment of dependence on Sir2. Fold enrichments for rDNA sequences (normalized to CUP1) are shown relative to untagged control. Sir2-TAP served as positive control. See also Figure S1 and Table S1. Developmental Cell 2014 30, 177-191DOI: (10.1016/j.devcel.2014.05.013) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 2 Pbp1 Interacts with rDNA IGS ncRNA and Paradoxically Sustains Its Accumulation in sir2Δ and trf4Δ Cells (A) PCR products of rDNA IGS1 sequences (P2 region as in Figure 1A) as well as CUP1 control sequences amplified from input or chromatin immunoprecipitated using anti-Sir2 or anti-diacetylated histone H3 (K9, K14) antibodies in ChIP. Relative fold enrichments for rDNA sequences, normalized to CUP1 and inputs, are shown below gels. (B and C) Semiquantitative reverse transcriptase (RT) PCR products for IGS1 ncRNA and ACT1 control amplified from input RNA or RNA immunoprecipitation (RIP) using indicated TAP-tagged proteins. Relative fold enrichments are shown below gels. (D) Relative RNA levels (Mean ± SE; n = 3; ∗p < 0.05) as revealed by reverse transcription coupled to quantitative PCR employing primer pairs amplifying various rDNA regions (as indicated in Figure 1A). Results are normalized to ACT1 control and values are presented relative to levels detected in wild-type cells. (E) Coimmunoprecipitation analysis examining interactions between Pbp1, Pbp1(1–297), and Pbp1(1–50) with Pab1. (F) RIP analysis examining the interaction of Pbp1, Pbp1(1–297), and Pbp1(1–50) with IGS1 ncRNA. Relative fold enrichments are shown below gels. (G) Effects of Pbp1 truncations on the rate of ADE2 marker loss (±SD; ∗p < 0.01; n.s., not statistically significant). See also Figure S2 and Table S1. Developmental Cell 2014 30, 177-191DOI: (10.1016/j.devcel.2014.05.013) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 3 Role for Pbp1 in the Suppression of RNA-DNA Hybrids within the Intergenic Spacers of rDNA Repeats (A) qPCR of rDNA sequences (shown in schematic) amplified from anti-RNA-DNA hybrid immunoprecipitates. Results are calculated as % input, normalized to CUP1, and presented relative to wild-type (±SD; n = 3). (B) ChIP-qPCR analysis using an anti-RNAPII (Rpb3 subunit) antibody. Values presented are relative to levels detected in wild-type cells (±SD; n = 3). (C) Effects of Pbp1 truncations on RNA-DNA hybrid levels as revealed by ChIP qPCR analysis employing the anti-RNA-DNA hybrid antibody (±SD; n = 3). (D) RNA-DNA hybrid levels were detected as in (A) except that all cells were cultured under stressful saturation conditions instead of standard logarithmic growth (±SD; n = 3). See also Figure S3. Developmental Cell 2014 30, 177-191DOI: (10.1016/j.devcel.2014.05.013) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 4 Artificial Overexpression of RNaseH1 Suppresses Intergenic RNA-DNA Hybrids and Aberrant Recombination within the rDNA Repeats of Pbp1-Deficient Cells (A–C) Cells with indicated genotypes and expressing RNaseH1 (+) or empty (−) plasmid control were used. (A) ChIP-qPCR analysis amplified rDNA sequences (as in Figure 1A) immunoprecipitated using anti-RNA-DNA hybrid S9.6 antibody. Results are calculated as % input, normalized to CUP1, and presented relative to wild-type cells with empty vector (±SD; n = 3). p values for observed effects are indicated on graph. (B) Relative rates of loss (±SD) of the ADE2 marker gene from rDNA repeats. p values are shown and see Table S2 for full counts and statistical analyses. (C) ChIP-qPCR analysis amplified rDNA sequences immunoprecipitated with anti-RNAPII (Rpb3 subunit) antibody. Results are calculated as % input normalized to CUP1 control and presented values are relative to levels detected in wild-type cells with empty vector (±SD; n = 3). (D) Effects of deleting endogenous RNaseH enzymes on rDNA stability in wild-type and pbp1Δ cells. Relative rates of loss (±SD; ∗p < 0.01) of the ADE2 marker gene from rDNA repeats are shown. See Table S1 for full counts and statistical analyses. See also Tables S1 and S2. Developmental Cell 2014 30, 177-191DOI: (10.1016/j.devcel.2014.05.013) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 5 Caloric Restriction Decreases RNA-DNA Hybrid Levels at the rDNA Repeats of Pbp1-Deficient Cells via Endogenous RNaseH and Additional Factors (A and B) Cells were cultured under standard (2% glucose; −ve) or caloric restriction (CR) (0.05% glucose; +ve) conditions and analyzed via ChIP-qPCR to amplify indicated rDNA sequences immunoprecipitated with the anti-RNA-DNA hybrid antibody. Results are calculated as % input, normalized to CUP1, and presented relative to wild-type non-CR conditions (±SD; n = 3). CR-dependent percent changes in hybrid levels are shown in (B). (C) Effects of CR on the levels of rDNA IGS1 ncRNA. Shown are relative ncRNA levels (±SD; n = 3) as revealed by RT-qPCR employing primer pairs amplifying the P1 and P4 IGS1 regions of rDNA. Results are normalized to ACT1 control and values are presented relative to levels detected in wild-type non-CR cells. (D) Effects of CR on the rate of loss (±SD; ∗p < 0.01; n.s., not significant) of ADE2 marker gene from the rDNA repeats of cells lacking Pbp1 and/or Sir2. See also Table S3. Developmental Cell 2014 30, 177-191DOI: (10.1016/j.devcel.2014.05.013) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 6 Deletion of the Pbp1-Interacting and G4DNA-Stabilizing Factor Stm1 Abolishes RNA-DNA Hybrid Accumulation and Aberrant Recombination at the rDNA Repeats of pbp1Δ Cells (A) Anti-RNA-DNA hybrid ChIP-qPCR examining the impact of Fob1 loss on CR-dependent hybrid suppression. Results are calculated as % input, normalized to CUP1, and presented relative to wild-type cells (±SD; n = 3). (B and C) Representative protein detection in silver-stained gel is shown with the 1, 4, 12, and s respectively marking Pbp1, Pbp4, Lsm12, and Stm1 positions (B). LC-MS/MS analysis results indicated with the number of unique peptides followed by percent coverage of the protein sequence (C). Details of proteomic analyses are in Table S4. (D) Western blots for coimmunoprecipitation analysis assessing physical interactions between native Pbp1-TAP and Stm1-Myc13. (E) ChIP examining the localization of Stm1-TAP to the rDNA IGS1 in wild-type and various mutant backgrounds. Relative fold enrichments are shown below the gel. (F) Schematic: IGS1 and IGS2 are depicted with their three G4-DNA sites (green arrows) as well as the location of primer pairs used in ChIP analysis. Graph: anti-RNA-DNA hybrid ChIP-qPCR analysis assessing genetic interactions between Pbp1 and Stm1. Results are calculated as % input, normalized to CUP1, and presented relative to wild-type (±SD; n = 3). (G) Relative rates of loss (±SD; ∗p < 0.01) of the ADE2 marker gene from rDNA repeats are shown. Full counts and statistical analyses are in Table S1. (H) Analysis of rDNA replication intermediates. Gels/schematics revealing X-shaped as well as blocked large Y and small y-shaped intermediates are presented. Graph shows relative levels (normalized to Fob1 RFB; ± SD; n = 3) of Y/y intermediates at or corresponding to the block sites of pbp1Δ cells. See also Figure S4 and Tables S1, S4, and S5. Developmental Cell 2014 30, 177-191DOI: (10.1016/j.devcel.2014.05.013) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 7 Intersection of CR, Stm1/Pif1/G4DNA, and RNaseH in rDNA Repeat Regulation and Lifespan Maintenance (A and B) Effects of CR on Stm1 localization to different rDNA regions in several genetic backgrounds. Shown are ChIP analyses amplifying rDNA sequences or CUP1 control from input and TAP-immunoprecipitated (IP) chromatin. Relative fold enrichments for P1 and P4 are shown below gels. (C and D) Effect of CR on RNA-DNA hybrid accumulation in cells with indicated genotypes. Shown are ChIP-qPCR analyses amplifying indicated rDNA sequences. Results are calculated as % input, normalized to CUP1, and presented relative to wild-type non-CR-treated cells (±SD; n = 3). Key CR-dependent percent changes are shown on graph. (E–H) Replicative lifespan plots. Mean lifespans are indicated in parentheses and full statistical analysis is in Table S6 (∗p < 0.01). (I) Model for Pbp1/CR-dependent RNA-DNA hybrid suppression at G4DNA-containing loci. See text for details. See also Table S6. Developmental Cell 2014 30, 177-191DOI: (10.1016/j.devcel.2014.05.013) Copyright © 2014 Elsevier Inc. Terms and Conditions