Nuclear organisation and replication timing are coupled through RIF1-PP1 interaction

Three-dimensional genome organisation and replication timing are known to be correlated, however, it remains unknown whether nuclear architecture overall plays an instructive role in the replication-timing programme and, if so, how. Here we demonstrate that RIF1 is a molecular hub that co-regulates both processes. Both nuclear organisation and replication timing depend upon the interaction between RIF1 and PP1. However, whereas nuclear architecture requires the full complement of RIF1 and its interaction with PP1, replication timing is not sensitive to RIF1 dosage. The role of RIF1 in replication timing also extends beyond its interaction with PP1. Availing of this separation-of-function approach, we have therefore identified in RIF1 dual function the molecular bases of the co-dependency of the replication-timing programme and nuclear architecture

Importance of Polη for damage-induced cohesion reveals differential regulation of cohesion establishment at the break site and genome-wide.

Genome integrity depends on correct chromosome segregation, which in turn relies on cohesion between sister chromatids from S phase until anaphase. S phase cohesion, together with DNA double-strand break (DSB) recruitment of cohesin and formation of damage-induced (DI) cohesion, has previously been shown to be required also for efficient postreplicative DSB repair. The budding yeast acetyltransferase Eco1 (Ctf7) is a common essential factor for S phase and DI-cohesion. The fission yeast Eco1 ortholog, Eso1, is expressed as a fusion protein with the translesion synthesis (TLS) polymerase Polη. The involvement of Eso1 in S phase cohesion was attributed to the Eco1 homologous part of the protein and bypass of UV-induced DNA lesions to the Polη part. Here we describe an additional novel function for budding yeast Polη, i.e. formation of postreplicative DI genome-wide cohesion. This is a unique Polη function not shared with other TLS polymerases. However, Polη deficient cells are DSB repair competent, as Polη is not required for cohesion locally at the DSB. This reveals differential regulation of DSB-proximal cohesion and DI genome-wide cohesion, and challenges the importance of the latter for DSB repair. Intriguingly, we found that specific inactivation of DI genome-wide cohesion increases chromosomal mis-segregation at the entrance of the next cell cycle, suggesting that S phase cohesion is not sufficient for correct chromosome segregation in the presence of DNA damage

Polη is required for formation of cohesion in response to DSB during G2.

<p>(A) The experimental system used for detection of DI genome-wide cohesion activated by γ-IR. Cells harboring the temperature sensitive (ts) <i>smc1-259</i> allele together with <i>pGAL-SMC1</i> (<i>smc1^ts</i>/Smc1^WT system) were grown in YEP media supplemented with 2% raffinose (YEPR) at 21°C, arrested in G2/M by addition of benomyl and kept at G2/M throughout the experiment. Expression of <i>pGAL-SMC1</i> was induced in one half of the culture by addition of galactose (2%). DNA damage was induced by to γ-IR (350 Gy), and loading of cohesin and establishment of DI-cohesion allowed during 60 min at permissive temperature. The temperature was raised to 35°C whereby cohesion created during S phase, or after DNA damage by <i>smc1^ts</i> was degraded. γ-IR is denoted by a red arrow. (B) Detection of DI-cohesion after γ-IR using the s<i>mc1^ts</i>/Smc1^WT system, as in A, in WT or <i>rad30Δ</i> strains. Samples for analyzis of sister separation at the <i>URA3</i> locus on Chr. V by the TetR-GFP/Tet-O system were collected, and separation scored in ≥200 cells/time point. A red arrow denotes time point for γ-IR. Blue lines represent WT and green lines <i>rad30Δ</i> cells. Dashed lines indicate damage induction. (C) System for detection of DI genome-wide cohesion induced by a single DSB, in cells harboring the <i>smc1^ts</i> allele and the <i>pGAL-HO</i> that creates a DSB at the <i>MAT</i> locus on Chr. III. These cells are compared with cells in addition containing <i>pGAL-SMC1</i>. Cells were arrested in G2/M (as in A), when galactose was added to induce the expression, if present, of <i>pGAL-SMC1</i> and <i>pGAL-HO</i>. After 90 min, the temperature was raised to 35°C to destroy cohesion created during S phase or after DNA damage by <i>smc1^ts</i>. Induction of a DSB on Chr. III is indicated by magenta arrowheads. (D) DI-cohesion was determined after induction of a DSB induced at the <i>MAT</i> locus on Chr. III by expression of <i>pGAL-HO</i> as described in C, otherwise as in B. (E) Assay for detection of DI genome-wide cohesion formed by a non-cleavable version of Scc1 (Scc1^NC), expressed from the <i>GAL</i> promoter (<i>pGAL-scc1^NC</i>). Cells grown in YEPR at 25°C were arrested in G2/M as in A. Galactose addition to one half of the culture induced expression of <i>pGAL-HO</i> and <i>pGAL-scc1^NC</i>. Cohesin loading and cohesion formation was allowed during 90 min, when the cells were released into YEPD to allow repair and re-entry into the cell cycle. Provided the DSB was repaired, cells re-entered the cell cycle and went through mitosis. DI-cohesion formed via Scc1^NC prevented sister separation regardless. Eventually also these cells entered G1 but then with mis-segregated chromosomes. Induction of a DSB on Chr. III is indicated by magenta arrowheads. (F) Formation of DI-cohesion was examined using the Scc1^NC system as in 1E otherwise as in B and D.</p

Polη is not required for homologous recombination-mediated postreplicative DSB repair.

<p>(A) Representative examples of pulsed field gels run for analysis of DSB repair efficiency. G2/M arrested cells with the indicated genes deleted were isolated before and at indicated time points following 150 Gy γ-IR. Cells were lysed in agarose plugs and genomic DNA was resolved by pulsed field gel electrophoresis. Control cells containing two fragments of Chr. XVI (0–685 and 685–948 kb from left telomere) were added as loading control <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003158#pgen.1003158-Hamer1" target="_blank">[61]</a>. After Southern blotting, membranes were hybridized with a Chr. XVI probe, containing Chr. XVI left arm specific sequences. Approximate region of association with Chr. XVI is shown as a black bar above indication for Chr. XVI. The fraction of intact Chr. XVI in relation to control Chr. XVI was quantified using a phosphoimager. (B) Averages from quantification of pulse field gels as in (A) from ≥3 exp for each strain. The results are presented as the ratio of the intact Chr. XVI remaining at the specified time compared with unirradiated Chr. XVI measured before γ-IR, related to the fragmented Chr. XVI in each lane. Error bars represent standard deviation (SD) based on a minimum of three individual experiments and significant differences are indicated with <i>p</i>-values (students <i>t-test</i>). (C) G2/M arrested cells with indicated genotypes were exposed to 150 Gy γ-IR after which 300 cells were plated on YEPD plates. After 2 days colonies were counted for estimation of survival. The results are shown as the fractions of colonies surviving after γ-IR compared with non-irradiated controls of the same genotype. One representative experiment (from two performed) is shown.</p

Implications for the action of Polη in Eco1-dependent acetylation of Scc1.

<p>(A) Formation of DI- cohesion was examined using the experimental system described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003158#pgen-1003158-g001" target="_blank">Figure 1C</a>, except that instead of <i>smc1^ts</i> a ts allele of the Scc1 component of cohesin was utilized (<i>scc1-73</i>). From introduced plasmids either the <i>pGAL-SCC1</i> or an acetyl mimic Scc1 version, <i>pGAL-scc1-K84Q, K210Q</i> were expressed. A DSB was induced at the <i>MAT</i> locus on Chr. III by HO in indicated sample series. Sister separation was analyzed in samples collected at the indicated time points, by the TetR-GFP/Tet-O-system, and separation scored in ≥200 cells per time point. (B) DI-cohesion was determined as in (A) in corresponding strains with <i>rad30Δ</i>. (A, B) Blue lines represent WT and green lines <i>rad30Δ</i> cells. Dashed lines indicate damage induction.</p

DI-cohesion deficiency caused by lack of Polη is rescued by overexpression of <i>ECO1</i>, but Polη is not required for stabilization of Eco1.

<p>(A) Formation of DI-cohesion was examined using the experimental system described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003158#pgen-1003158-g001" target="_blank">Figure 1C</a>. Smc1^WT was expressed in all the strains, a plasmid containing <i>pGAL-ECO1</i> was introduced into half of the strains and a DSB was induced at the <i>MAT</i> locus on Chr. III, in indicated sample series. Samples for detection of sister separation by the TetR-GFP/Tet-O were collected at indicated time points, and separation scored in ≥200 cells per time point. (B) DI-cohesion was determined as in (A) in corresponding strains with <i>rad30Δ</i>. (A, B) Blue lines represent WT and green lines <i>rad30Δ</i> cells. Dashed lines indicate damage induction. (C) A schematic outline of the experimental set up used for C-E is shown. Eco1-myc₁₃ levels were determined by Western blotting on protein extracts prepared at indicated time points from WT and <i>rad30Δ</i> cells, in the absence or presence of γ-IR (150 Gy). Cdc11 was used as loading control. FACS profiles from selected time points are presented to show the cell cycle distribution throughout the experiment. One representative experiment (from two performed) is shown. (D) Quantification of the relative Eco1 level, normalized to Cdc11. The time point 0 h nocodazole (0N) for each strain is set to 1. (E) Quantification of the relative Eco1 level, normalized to Cdc11 with the time point 0 h +/− γ-IR set to 1 in each strain.</p

Polη is required for DI genome-wide cohesion but not for cohesin loading or establishment of DSB–proximal cohesion.

<p>(A) Schematic illustration of Chr. V with the Tet-O array and the inserted HO cut-site (not to scale). (B) Formation of Chr. V DSB-proximal cohesion was examined in WT and <i>rad30Δ</i> cells using the <i>smc1^ts</i>/Smc1^WT system (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003158#pgen-1003158-g001" target="_blank">Figure 1C</a>). Smc1^WT was expressed in all groups, and a DSB induced 4 kb from the Tet-O array on Chr. V, in half of the cultures, by expression of <i>pGAL-HO</i>. Blue lines represent WT and green lines <i>rad30Δ</i> cells. Dashed lines indicate damage induction. (C) The Chr. V DSB was induced in all groups and Smc1^WT expressed in indicated sample series, otherwise as in B. (D) Scc1-FLAG binding determined by ChIP on chip after DSB induction for 90 min on Chr. VI (206 kb from the left telomere, indicated by red arrow heads) in G2/M arrested cells of indicated genotype. Orange peaks display significant chromosomal binding sites where the x-axis show chromosomal positions and the y-axis show log₂ of signal strength. (E) Chromosomal association of G2-expressed Scc1-HA analyzed by ChIP-sequencing in WT and <i>rad30Δ</i> cells in the presence and absence of DSB induction at the <i>MAT</i> locus on Chr. III. Shown are, to the left Chr. V 500–570 kbp and to the right Chr. VIII 100–130 kb, from the left telomeres respectively. Orange peaks display significant chromosomal binding sites, the x-axis show chromosomal positions and the y-axis show linear fold enrichment.</p

The DI-cohesion function of Polη is not mediated via its polymerase activity or through PCNA-interaction.

<p>(A) Polη with relative positions of the five conserved polymerase motifs (I–V), the polymerase associated domain (PAD), the ubiquitin-binding motif (UBZ), the nuclear localization signal (NLS) and the PCNA-binding box (PIP). Mutated residues analyzed in this study are indicated with arrows (not to scale). (B) UV-sensitivity was analyzed in <i>rad30Δ</i> cells carrying the <i>RAD30 or rad30</i>-<i>D30A</i>, -<i>E39A</i> or -<i>D155A</i> genes on a low copy-number <i>CEN LEU2</i> plasmid. Exponentially growing cells were plated on YEPD plates in 10-fold dilutions, left untreated or UV irradiated (50 J/m²) as indicated, and then incubated 3 days at 25°C. (C) DI-cohesion measured with the Scc1^NC system (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003158#pgen-1003158-g001" target="_blank">Figure 1E</a>) in response to one DSB on Chr. III in WT and <i>rad30Δ</i> cells in comparison with <i>rad30Δ</i> cells carrying the <i>RAD30</i>, <i>rad30</i>-<i>D30A</i>, -<i>E39A</i> or -<i>D155A</i> plasmids. Sister chromatid separation was scored with the TetR-GFP/Tet-O-system. (D) <i>rad30Δ</i> strains harboring polymerase mutant versions of <i>rad30</i> exit the G2/M arrest simultaneously. Percentages of Pds1-myc₁₃ positive cells were determined by <i>in situ</i> staining with an anti-myc antibody. The percentage of positive cells at each time point is related to the percentage at complete G2 arrest for each strain, which is set to 100%. (E) Polη-myc₁₃ levels were analyzed by Western blotting. Whole cell extracts were isolated from WT cells (<i>RAD30-no tag</i>), cells harboring <i>RAD30-myc₁₃</i> or cells with differently mutated <i>rad30-myc₁₃</i> tagged alleles as indicated. Cdc11 was used as loading control. One representative experiment (from four performed) is shown. (F) The requirement for Polη interaction with PCNA in DI-cohesion was tested in <i>pol30</i>-<i>K164R</i>, <i>rad30</i>-<i>D570A</i> and <i>rad30</i>-<i>F627A</i>, <i>F628A</i> mutants using the Scc1^NC system (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003158#pgen-1003158-g001" target="_blank">Figure 1E</a>).</p

A regulatory role for the cohesin loader NIPBL in nonhomologous end joining during immunoglobulin class switch recombination.

DNA double strand breaks (DSBs) are mainly repaired via homologous recombination (HR) or nonhomologous end joining (NHEJ). These breaks pose severe threats to genome integrity but can also be necessary intermediates of normal cellular processes such as immunoglobulin class switch recombination (CSR). During CSR, DSBs are produced in the G1 phase of the cell cycle and are repaired by the classical NHEJ machinery. By studying B lymphocytes derived from patients with Cornelia de Lange Syndrome, we observed a strong correlation between heterozygous loss-of-function mutations in the gene encoding the cohesin loading protein NIPBL and a shift toward the use of an alternative, microhomology-based end joining during CSR. Furthermore, the early recruitment of 53BP1 to DSBs was reduced in the NIPBL-deficient patient cells. Association of NIPBL deficiency and impaired NHEJ was also observed in a plasmid-based end-joining assay and a yeast model system. Our results suggest that NIPBL plays an important and evolutionarily conserved role in NHEJ, in addition to its canonical function in sister chromatid cohesion and its recently suggested function in HR