Physical proximity of chromatin to nuclear pores prevents harmful R loop accumulation contributing to maintain genome stability

Abstract

During transcription, the mRNA may hybridize with DNA, forming an R loop, which can be physiological or pathological, constituting in this case a source of genomic instability. To understand the mechanism by which eukaryotic cells prevent harmful R loops, we used human activation-induced cytidine deaminase (AID) to identify genes preventing R loops. A screening of 400 Saccharomyces cerevisiae selected strains deleted in nuclear genes revealed that cells lacking the Mlp1/2 nuclear basket proteins show AID-dependent genomic instability and replication defects that were suppressed by RNase H1 overexpression. Importantly, DNA-RNA hybrids accumulated at transcribed genes in mlp1/2 mutants, indicating that Mlp1/2 prevents R loops. Consistent with the Mlp1/2 role in gene gating to nuclear pores, artificial tethering to the nuclear periphery of a transcribed locus suppressed R loops in mlp1∆ cells. The same occurred in THO-deficient hpr1∆ cells. We conclude that proximity of transcribed chromatin to the nuclear pore helps restrain pathological R loops.

Keywords: Mpl1/2; R loop; genome instability; nuclear pores; transcription.

Fig. S1.

Screening of the selected 400 deletion strains for mutants showing AID-induced hyperrecombination. (A) Cells were transformed with plasmid p313LZGAID, grown in galactose to induce AID expression, and plated on appropriate media to assess the amount of total and recombinant cells, respectively. Wild-type as well as hpr1∆ and mft1∆ THO mutants were used as controls. Representative results obtained with one of the 96-well plate are shown. Positions corresponding to WT, hpr1∆, mft1∆, and mlp1∆ are indicated. (B) For each candidate strain, four independent transformants were grown in either glucose (without AID expression; −AID) or galactose (with AID expression; +AID) and serial dilutions plated on selective media. Strains were assigned to the following categories: (i) strains showing much higher recombination level upon AID expression than without AID expression (Rec. +AID > Rec. −AID) and (ii) strains showing similar recombination levels with or without AID expression (Rec. +AID = Rec. −AID). Representative examples are shown for the different categories. Only strains assigned to the first category (Rec. +AID > Rec. −AID, Right) were selected for further analysis. Worthy of note, the sensitivity of our assay did not allow appreciating AID-dependent recombination increases in strains with high basal recombination level in the absence of AID expression (Left), these strains being consequently assigned to the second category and not retained for further analysis. (C) Recombination analysis of the 22 selected candidates using the L-lacZ direct repeats plasmid-borne system. Wild-type and mft1∆ cells were used as controls. Recombination frequencies were obtained as the median value of six independent colonies. The average and SEM of two independent fluctuation tests are shown for each genotype. Recombination frequency fold-increase obtained for each mutant relative to the wild type in AID expression condition is shown on the right.

Fig. 1.

Lack of Mlp1/2 leads to AID-dependent genome instability that is suppressed by RNase H1 overexpression. (A) Recombination analysis using the L-lacZ direct-repeat plasmid-borne system under the control of the LEU2 promoter in wild-type, mlp1∆, mlp2∆, and mlp1∆ mlp2∆ cells that do not express AID (−AID), express AID (+AID), or express both AID and RNase H1 (+AID +RNH1). A scheme of the system is shown (Upper). Average and SEM of independent fluctuation tests are shown (n ≥ 3). Fold increases vs. the wild type with AID are shown on the left. Statistical analyses using a two-tailed unpaired Student t test. *P < 0.05; **P < 0.01; ***P < 0.001. All values are provided in Table S2. (B) Recombination analysis using the L-lacZ system under the control of the GAL1 promoter in cells that do not express AID (−AID) or express AID (+AID) in conditions of low or high transcription. Fold increases with respect to levels under low transcription without AID are shown on the left. Expression levels of the recombination system under high transcription in the different strains were not significantly different (Fig. S2). Details as in A. (C) Recombination analysis using the chromosomal his3-based direct-repeat recombination system. ****P < 0.0001. Details as in A. (D) Percentage of S/G2 cells containing Rad52-YFP foci. Fold increases with respect to the wild type without AID expression are shown on the left. Average and SEM of independent experiments are shown (n ≥ 3). Details as in A. (E) Plasmid loss in cells that do not express RNase H1 (−RNH1) or do express it (+RNH1). Average and SEM of independent experiments are plotted (n ≥ 3). Details as in A.

Fig. S2.

Expression of the L-lacZ direct-repeat plasmid-borne system used in Fig. 1B in wild-type, mlp1∆, mlp2∆, and mlp1∆ mlp2∆ cells. (A) Scheme of the recombination system. (B) Total RNA was isolated from transformed cells grown in galactose-containing media to midlog phase. Northern blot analysis was performed using a radioactively labeled fragment of the LacZ gene as probe. A probe against the constitutively expressed SCR1 gene was used as loading control. Representative results are shown for each genotype. (C) Quantification of L-lacZ expression as shown in B. LacZ signal was normalized to the SCR1 levels of each sample. Average and SEM of three independent experiments are plotted.

Fig. 2.

R loops accumulate at transcribed genes in mlp1/2 mutants. (A) Schematic view of the analyzed genes and amplicons. (B) DRIP using the S9.6 antibody in wild-type, mlp1∆, mlp2∆, and mlp1∆ mlp2∆ asynchronously growing cells. Where indicated, samples were treated with RNase H (RNH). S9.6 signal were normalized to the wild-type value in each experiment. Average and SEM of independent experiments are shown (n ≥ 3). Statistical analyses as in Fig. 1A. *P < 0.05; **P < 0.01; ***P < 0.001. (C) DRIP in the his3-based direct-repeat recombination system. The S9.6 signal at the GNC4 and GAL1 genes were used as controls. Details as in B.

Fig. 3.

Replication progression is impaired in mlp1∆ cells. (A) BrdU incorporation upon release of G1-arrested cells was analyzed at early replication origin ARS508 by immunoprecipitation and real-time-qPCR in wild-type and mlp1∆ cells. A schematic drawing of the genomic region and amplicons are depicted (Top). Experiments were done in cells treated with 20 mM hydroxyurea (HU) not overexpressing RNase H1 (−RNH1) or overexpressing RNase H1 (+RNH1). Quantification of BrdU incorporation relative to a late replication locus is plotted for each region. Average and SEM of independent experiments are shown (n = 3). The P values calculated by the Wilcoxon signed-rank test are shown for each condition. Plotted values are normalized to the average signal obtained for the wild type at the 25-min time-point in the +510 region for each experiment. The graphs obtained without normalization to the wild-type value are shown in Fig. S4. (B) BrdU incorporation upon release of G1-arrested cells at ARS1021. Signal in the +625 region was used for normalization. Details as in A.

Fig. S3.

Cell-cycle progression analyses of mlp1∆ cells. (A) Flow cytometry analyses (FACS) of mlp1∆ and wild-type cells during release from α-factor–mediated G1 arrest in normal condition (Top), in the presence of 20 mM hydroxyurea (+HU; Middle), and in cells expressing human AID (+AID; Bottom). FACS analyses were performed with a FACSCalibur (BD Bioscience) using CellQuest software. (B) Chromosome XII species revealed by hybridization with ADE5 in pulse-field electrophoresis (PFGE) of DNA from mlp1∆ and wild-type cells released from α-factor–mediated G1 arrest in normal condition (Upper) and in the presence of 20 mM hydroxyurea (+HU; Lower). Nonlinear chromosomes (NLCs), which include replication intermediates, correspond to the signal coming from the gel well. The NLC signal was quantified with respect to the total signal of each lane. FLC, full-length linear chromosomes. Representative results and quantification from three independent experiments are shown. Average and SEM are plotted. PFGE was performed using standard protocol.

Fig. S4.

Representation of the BrdU incorporation analyses shown in Fig. 3 and at ARS1211 without normalization with wild-type values. (A) BrdU incorporation upon release of G1-arrested cells was analyzed at early replication origin ARS508 by immunoprecipitation and real-time-qPCR in wild-type and mlp1∆ cells. A schematic drawing of the genomic region and localization of the amplified regions are depicted (Top). The analysis was performed in cells treated with 20 mM hydroxyurea (HU) that did not overexpress RNase H1 (−RNH1) or did overexpress RNase H1 (+RNH1). Quantification of BrdU incorporation relative to a late replication locus is plotted for each region. Average from three independent experiments and corresponding SEM are shown. The P values calculated by the Wilcoxon signed-rank test are shown for each condition. (B) BrdU incorporation upon release of G1-arrested cells at the early replication origin ARS1211. Other details as in A. (C) BrdU incorporation upon release of G1-arrested cells at the early replication origin ARS1021. Other details as in A.

Fig. S5.

Replication progression is impaired in mlp1∆ cells. (A) BrdU incorporation upon release of G1-arrested cells was analyzed at early replication origin ARS508 by immunoprecipitation and real-time-qPCR in wild-type and mlp1∆ cells. A schematic drawing of the genomic region and localization of the amplified regions are depicted (Top). The analysis was performed in untreated cultures (−HU) or in cultures treated with 20 mM hydroxyurea (+HU). Quantification of BrdU incorporation relative to a late replication locus is plotted for each region. Average from three independent experiments and corresponding SEM are shown. The P values calculated by the Wilcoxon signed-rank test are shown for each region. (B) BrdU incorporation upon release of G1-arrested cells at the early replication origin ARS1211. Other details as in A.

Fig. 4.

R loop-dependent genome instability in mlp1 mutants mimicking constitutive intra-S checkpoint activation or blind to checkpoint activation. (A) Recombination analysis using the L-lacZ system under the control of the LEU2 promoter in mlp1S1710D (mlp1D) and mlp1S1710A (mlp1A) cells that do not express AID (−AID), express AID (+AID), or express both AID and RNase H1 (+AID +RNH1). *P < 0.05; **P < 0.01. Details as in Fig. 1. (B) Percentage of S/G2 cells containing Rad52-YFP foci. Details as in A. (C) Plasmid loss in cells that do not express RNase H1 (−RNH1) or do express RNase H1 (+RNH1). ***P < 0.001. Details as in A. (D) DRIP using the S9.6 antibody. Where indicated, samples were pretreated with RNase H (RNH). *P < 0.05; **P < 0.01; ***P < 0.001. Details as in Fig. 2. Experiments of B–D were performed concomitantly with those shown in Figs. 1 and 2, so values for wild-type cells are the same.

Fig. 5.

Artificial tethering to the nuclear pore is sufficient to suppress cotranscriptional R loops in mlp1∆ and hpr1∆ cells. (A) Scheme of the artificial tethering system. Binding of a LexA–Nup60 fusion protein to LexA binding sites (BS) inserted downstream of the GAL1 gene anchors the GAL1 to the nuclear pore. (B) Time-course analysis of GAL1 mRNA upon transcription induction in wild-type and mlp1∆ cells expressing either LexA or LexA–Nup60. Representative results are shown. (C) Quantification of GAL1 mRNA induction as shown in B. GAL1 signal was normalized to the SCR1 levels of each sample. Average and SEM of independent experiments are plotted (n ≥ 3). The P values were calculated by the Wilcoxon signed-rank test. (D) Time-course ChIP of the RNAP II large subunit Rpb1. In each experiment, ChIP values were normalized to the 0 time-point of the wild type. Location of the amplicons relative to GAL1 gene is indicated (Upper). Details as in C. (E) Time-course DRIP analysis with the S9.6 antibody. DRIP values were normalized to the 0-min time-point in each strain. Details as in D. (F) DRIP using the S9.6 antibody in wild-type, mlp1∆, and hpr1∆ cells grown to midlog phase in galactose medium and expressing either LexA or LexA–Nup60. *P < 0.05; **P < 0.01. Details as in Fig. 2. DRIP data of the rDNA locus is shown in Fig. S6. (G) Model for R loop formation in the nucleoplasm. Gating of a transcribed locus to the NPC prevents formation of R loops. This likely occurs thanks to the prompt export of the nascent mRNP that minimizes the probability of back-hybridization to the template DNA. In gene gating defective mutants such as mlp1∆, transcribed genes remain in the nucleoplasm and R loops accumulate, leading to moderate genome stability.

Fig. S6.

DNA–RNA immunoprecipitation using the S9.6 antibody in wild-type, mlp1∆, and hpr1∆ cells grown in galactose and expressing either LexA or LexA–NUP60 fusion protein at the rDNA locus. Where indicated, samples were treated with RNase H (RNH) before immunoprecipitation. Average of signal values of R loop detection obtained from at least three independent experiments and the corresponding SEM are shown. Statistical analyses were performed with a two-tailed unpaired Student t test. *P < 0.05; **P < 0.01.