Genome-wide screen identifies pathways that govern GAA/TTC repeat fragility and expansions in dividing and nondividing yeast cells

Abstract

Triplex structure-forming GAA/TTC repeats pose a dual threat to the eukaryotic genome integrity. Their potential to expand can lead to gene inactivation, the cause of Friedreich's ataxia disease in humans. In model systems, long GAA/TTC tracts also act as chromosomal fragile sites that can trigger gross chromosomal rearrangements. The mechanisms that regulate the metabolism of GAA/TTC repeats are poorly understood. We have developed an experimental system in the yeast Saccharomyces cerevisiae that allows us to systematically identify genes crucial for maintaining the repeat stability. Two major groups of mutants defective in DNA replication or transcription initiation are found to be prone to fragility and large-scale expansions. We demonstrate that problems imposed by the repeats during DNA replication in actively dividing cells and during transcription initiation in nondividing cells can culminate in genome instability. We propose that similar mechanisms can mediate detrimental metabolism of GAA/TTC tracts in human cells.

Figure 1. Genome-wide Screen Methodology

(A) Query strain to uncover genotypes prone to GAA/TTC fragility. The 230 GAA/TTC repeats were positioned on the left arm of chromosome V with GAA as a template for lagging strand synthesis. The arrangement of LYS2, hphMX cassette, and CAN1 on chromosome V are shown. The location of HIS3 ORF under control of MATa-specific promoter and rpl28-Q38K allele on chromosomes IV and VII are depicted. (B) Schematics of the screen. MATα query strains carrying GAA/TTC tract were crossed with MATa tester strains from YKO, yTHC, and DAmP libraries. Diploids were selected by replica plating on media containing both G418 and Hygromycin B. After sporulation of diploids, MATa haploids were selected using histidine drop-out media containing cycloheximide. Haploids harboring both GAA/TTC tract and mutations of interests were further selected by G418 and Hygromycin B containing media. Cells were transferred to canavanine-containing media to score for arm-loss events. (C) Example plate for the screen. Columns are duplicates of query strains. Each row is one tester strain. The level of arm loss in TET-RFA2 (hyperfragile), Δmrc1 (hyperfragile), TET-SEC4 (no change in fragility), and wild-type are shown. (D) Experimental assay to verify results from the screen. ADE2 is placed between CAN1 and LYS2. Mutations in CAN1 will manifest as Can^RAde⁺ white colonies, whereas arm loss events will give rise to Can^RAde⁻ red papillae. See also Table S1.

Figure 2. Physical Detection of GAA/TTC-Induced DSBs

Location of GAA/TTC tracts on chromosome V is depicted. Intact chromosome V is ~585 kb, DSB at the tract results in a 43 kb broken fragment. Chromosomal DNA was separated by CHEF. Both intact and broken chromosomal V were detected by southern blot hybridization using a HPA3-specific probe. Lanes: 1, wild-type strain with (GAA)₂₀; 2, wild-type strain with (GAA)₂₃₀; 3, TET-TAF4 strain with (GAA)₂₃₀; 4, TET-RFA2 strain with (GAA)₂₃₀.

Figure 3. GAA/TTC Fragility Increases in Nondividing Cells

(A) Growth curves of wild-type and TET-TAF4 strains. Wild-type and TET-TAF4 strains enter stationary phase at ~43 and 49 hr after inoculation, correspondingly. Error bars indicate SEM. (B) GAA/TTC fragility frequencies of wild-type and TET-TAF4 strain in dividing and nondividing cells. Values are the median frequencies obtained from fluctuation tests of at least eight cultures carried out at the indicated time-points. Error bars represent 95% confidence intervals. (C) Detection of DSBs in wild-type and TET-TAF4 strains at different time-points during stationary phase. Zero hour corresponds to the time when strains stopped growing. Twenty hour and 70 hr indicate the time each strain spent in stationary phase. Lanes: 1, wild-type strain with (GAA)₂₀; 2, wild-type strain with (GAA)₂₃₀; 3, TET-TAF4 strain with (GAA)₂₃₀. Positions of DSBs and unbroken chromosome V are indicated by arrows. See also Table S4 and Figure S1.

Figure 4. Expansion Dynamics of (GAA)₁₀₀ Repeats in Actively Dividing and Nondividing Cells in Wild-type and TET-TAF4 Strains

(A) Growth curves for wild-type and TET-TAF4 strains. Error bars represent SEM. (B) Frequencies of expansion of (GAA)₁₀₀ in dividing and arrested cells. Values are the median frequencies obtained from fluctuation tests of at least eight cultures. Error bars represent 95% confidence intervals. See also Table S5.

Figure 5. Model for GAA/TTC Fragility and Expansion in Dividing and Nondividing Cells

(I) Replication-associated pathway for GAA/TTC (red line) instability. Hoogsteen base pairing in the triplex is indicated as blue dots. DNA replication helicase (green ring), DNA polymerase (solid gray oval), and attacking nuclease (solid orange pacman) are shown. (II) Transcription-associated pathway for GAA/TTC instability. Transcription initiation factors (solid brown ovals), the newly synthesized RNA (blue line) forming abnormal RNA-DNA hybrid (R-loop), and RNA polymerase II (solid purple ovals) are depicted. A detailed description is presented in the text. See also Figure S2.