Yra1 connection to irreparable DSBs and the Nuclear Periphery

Yra1 localizes a random locus to the Nuclear Periphery

Studies from the Gasser laboratory have shown that yeast irreparable DSBs relocate to the nuclear periphery and interact with the STUbL Slx5-Slx8 associated with nuclear pores (Cook et al., 2009; Horigome et al., 2016; Nagai et al., 2008).

Because Yra1 is Ubiquitinated by Slx5-Slx8 (Project background), we investigated whether Yra1 could have a role in DDR by facilitating DSB relocation to nuclear pores. We first tested by zoning assays whether Yra1 is able to relocate a DNA locus to the nuclear periphery if artificially tethered to it (in collaboration with the Gasser laboratory). In this gain of function assay performed in the presence of endogenous Yra1, LexA-Yra1 is expressed from a plasmid in a strain containing LexA-binding sites on Chromosome VI next to LacO repeats bound by LacI-GFP protein (description in Figure 4.1 A). The movement of the DNA locus is followed with respect to the nuclear periphery visualized by Nup49-CFP or Nup49-GFP dividing the nucleus into 3 zones. LexA-Yra1 significantly relocates a DNA locus to the nuclear periphery (enrichment in Zone 1) in comparison to the LexA-empty control that shows random distribution of the locus within the 3 zones (Figure 4.1 B (Horigome et al., 2015)). The LexA-Yra1 mutants are also able to relocate the DNA locus to the periphery (Figure 4.1 B). Interestingly, when distinguishing cell cycle stages, the LexA-yra1(1-210) mutant does not relocate the DNA locus in G1 indicating the importance of the C-terminal region in Yra1 localization at the nuclear periphery in the G1 phase of the cell cycle (Figure 4.1 C). Not enough cells expressing yra1Δintron and LexA-yra1allKR could be quantified due to the dominant negative effect of these constructs on growth, as shown by spot test analysis of transformants in the Nup49-GFP background (Figure 4.1 D). We generated an additional strain for the zoning assay that contains the Nup49-mCherry tag. However, the Nup49-mCherry is sicker than the Nup49-GFP strain, making the dominant negative effect of LexA-yra1allKR construct even stronger (Figure 4.1 D). The LexA-yra1Δintron construct heavily affects the growth of the GFP strain and prevents growth when introduced into the Nup49-mCherry tagged background (no transformants were obtained). For these as well as microscope equipment reasons, we used a Nup49-CFP tagged strain when the

quantifications were performed at the Susan Gasser laboratory, and the Nup49-GFP tagged strain when the quantifications were performed at our Bioimaging facility.

Figure 4.1: Yra1 relocates a randomly distributed locus to the Nuclear Periphery in G1 and S phase.

A) Description of the zoning assay. The nucleus is divided into 25 Z-stacks. The brighter green dot corresponds to the LacI-GFP with respect to the nuclear periphery labelled with Nup49-GFP. Zone 3:

the dot is inside the nucleus, Zone 1: the dot is at the nuclear periphery, Zone 2: the dot is in between Zone 3 and Zone 1. Cells were transformed with LexA-Yra1 constructs that bind LexA binding sites near the LacO array recognized by LacI-GFP. Image modified from (Horigome et al., 2015).

B) Zoning assay with LexA-yra1 mutants considering all cell cycle phases. Nup49-GFP or Nup49-CFP strains were transformed with the plasmids HA-LexA-empty, HA-LexA-Yra1 WT, HA-LexA-yra1(1-210), HA-LexA-yra1Δintron and HA-LexA-yra1allKR. The graph shows the percentage of foci in each Zone and the dotted line indicates the random distribution. At least 50 images were analysed for each condition; the analysis was restricted to round nuclei with the LacI-GFP in the same plane as Nup49-GFP. The Table shows how many cells were quantified for each condition.

C) Zoning assay with LexA-yra1(1-210) in G1 and S phase. The Nup49-CFP strain was transformed with HA-LexA-empty, HA-LexA-yra1 WT and HA-LexA-yra1(1-210). The percentage of foci in each Zone in G1 versus S phase cells is shown; the dotted line indicates the random distribution. The Table shows how many cells were quantified for each condition. This experiment was performed by Chihiro Horigome.

D) Spot test analysis of Nup49-GFP and Nup49-mCherry strains transformed with the plasmids HA-LexA-Yra1 WT, HA-LexA-yra1(1-210), HA-LexA-yra1Δintron, yra1allKR or HA-LexA-empty. Confluent cultures of transformants were serially diluted and spotted on Leu- plates at 25°C, 30°C and 34°C. E) Western blot analysis of the different HA-LexA-Yra1 fusions with an αHA antibody is shown below. wondered whether its relocation could indirectly depend on transcription. To address this possibility, we treated the cells with Phenanthroline (200µg/ml) to block transcription for 30 min before fixing the cells with paraformaldehyde. Phenanthroline treatment did not impair the ability of Yra1 to target the DNA locus to the nuclear envelope, indicating that the relocation of the locus is transcription independent (Figure 4.2 A).

Yra1 localizes a random locus to the nuclear periphery under DNA damage induction

Next, to test whether LexA-Yra1 increases the relocation of the DNA locus at the nuclear periphery in presence of DNA damage, we treated the cells with the DSB

agent Zeocin (250 µg/ml) for 1h30min. Yra1 did not increase the relocation of the locus to the Nuclear Envelope under DNA damage induction (Figure 4.2 A).

Yra1 localizes a random locus to the nuclear periphery in Δslx8, Δslx5 and Δsiz2

Since Yra1 is post-translationally modified by E3 Ubiquitin and SUMO ligases, we examined whether the relocation of the DNA locus to the nuclear periphery by HA-LexA-Yra1 is compromised in Δslx8, Δslx5 or Δsiz2. The HA-HA-LexA-Yra1 was able to relocate the locus to the nuclear periphery in Δslx8, Δslx5 and Δsiz2 (Figure 4.2 B).

The relocation of the DNA locus at the nuclear periphery by HA-LexA-Yra1 in the double mutants Δslx8Δtom1 and Δslx5Δtom1 that abrogate Yra1 ubiquitination (Project Background), and Δsiz1/Δsiz2 that abrogates Yra1 Sumoylation (Project Background), was not examined because the single mutants analyzed were already sick and affected the round nuclear shape important for the analysis.

Figure 4.2: Yra1 relocates a randomly distributed locus to the Nuclear Envelope in the presence of Phenantroline or Zeocin and independently of Slx5/Slx8/Siz2.

A) Zoning assay of LexA-Yra1WT under Phenantroline (200µg/ml) treatment for 30’ or Zeocin (250µg/ml) treatment for 1h30’ in the Nup49-GFP background. The percentage of foci in each Zone considering all cell cycle phases (G1/S/G2) is shown; the dotted line indicates the random distribution.

The Table on the right shows how many cells were quantified for each condition.

B) Zoning assay of LexA-Yra1WT in Δslx8, Δslx5 or Δsiz2 Nup49-GFP background. The percentage of foci in each Zone considering all cell cycle phases (G1/S/G2) is shown; the dotted line indicates the random distribution. The Table shows how many cells were quantified for each condition.

Yra1 is recruited to an irreparable DSB (HO cut)  

To obtain more direct evidence for a possible role of Yra1 in DDR, we induced an irreparable DSB using a galactose inducible HO endonuclease, which cuts the DNA at the MAT locus as described in (Nagai et al., 2008). Yra1 recruitment at the HO cut was examined by ChIP (Figure 4.3 A). Yra1 recruitment at DSBs is significant after 2h of HO GAL induction at regions close to the DSB (Figure 4.3 B and C).

Considering that the efficiency of the cut is nearly 100% after 30’ of HO induction, the recruitment after 2h suggests it may depend on extensive resection.

Figure 4.3: Yra1 is recruited to an irreparable DSB HO cut site.

A) Control for HO cut induction in the presence of Galactose after 30’, 60’, 120’, 240’ and the no cut control after 120’ in Glucose using a PCR assay specific for the HO cut site. The strain system (Nagai et al., 2008) is diagrammed on the right. The HO endonuclease, expressed from a GAL promoter, induces the HO cut at the MAT locus that cannot be repaired because of the deletion of the homologous HML and HMR regions (Δhml and Δhmr).

B) Yra1 recruitment at the HO cut site after 30’, 60’, 120’ and 240’ of HO endonuclease induction with Galactose using the above described strain (Nagai et al., 2008). The 2h Glucose time point was used as no cut control. ChIP values using αYra1 antibody are shown as fold change over no cut (Glucose 2h) at PMA1 (gene bound by Yra1), a Telomeric region, and at 0.6 Kb, 1.6Kb, 4.5Kb, 9.6Kb and 23Kb from the HO cut. The average of 4 experiments is shown with corresponding standard error of the mean and P value < 0.05 (*) or P value < 0.01 (**).

C) Yra1 recruitment at 0.6Kb from the HO cut site after 2h of HO endonuclease induction with Galactose using the same strain as above. The 2h Glucose time point was used as no cut control. ChIP values (using αYra1 antibody and Preimmune) are shown as fold change over an intergenic region. The average of 7 experiments is shown with corresponding error of the mean and P value < 0.05 (*) or P value < 0.01 (**).

Yra1 binds an irreparable DSB (HO cut) in the absence of transcription  

Since Yra1 is an mRNA export adaptor, we wondered whether the recruitment at DSBs could depend on transcription and performed the same Yra1 ChIP experiments in the presence of the transcription inhibitor Phenanthroline. We observed no significant difference in Yra1 recruitment at the DSB following a 15' treatment with Phenantroline (Figure 4.4 A). ChIP of Rad51 was also performed as control for induction of the cut (Figure 4.4 B), and qPCR analyses of PHO84 and YRA1 transcripts were used as control for transcription inhibition (Figures 4.4 C and D).

Furthermore, qPCR analysis does not reveal any transcript at the site of the HO cut (Figure 4.4 D). This observation is consistent with a study showing that resection at DSBs is paralleled by transcription inhibition of surrounding loci (Manfrini et al., 2015). These data suggest that Yra1 binding to resected DNA ends is not linked to transcription.

Figure 4.4: Yra1 binding at an irreparable DSB does not depend on transcription

A) Yra1 recruitment at 0.6 Kb from the HO cut site after 2h of HO endonuclease induction with Galactose and 2h Glucose as no HO cut control in the absence (-Phe ethanol treatment) or presence of Phenanthroline (200µg/ml) added for 15’ at the end of the 2h Galactose induction. ChIP values using αYra1 antibody or Preimmune at HO cut site (0.6 Kb) are shown as fold change over an intergenic region. The average of 5 experiments is shown with corresponding standard error of the mean and P value < 0.05 (*) or P value < 0.01 (**). The P value above the condition Glu 2h αYra1 (-) Phe refers to the condition Glu 2h Preimmune (-) Phe. The P value above the condition Gal 2h αYra1 (-) Phe refers to the condition Gal 2h Preimmune (-) Phe as well as to the condition Glu 2h αYra1 (-) Phe.

B) Rad51 recruitment around HO cut site (0.6, 1.6, 4.5 and 9.6 Kb from cut) after 2h in Galactose to induce HO endonuclease or 2h in Glucose as no cut control in the absence (-Phe, ethanol treatment) or presence of Phenantroline (+ Phe 200µg/ml) added for the last 15’. ChIP values (using αRad51 antibody) are shown as percentage of input. Average of 4 independent experiments is shown with relative standard error of the mean and P value < 0.05 (*).

C) RT-qPCR analysis of PHO84 transcripts in the absence (-Phe ethanol treatment) or presence of Phenantroline (200µg/ml) added for 15’ at the end of the 2h HO endonuclease induction with Galactose and 2h Glucose as no HO endonuclease induction (no cut control). Average and standard deviation of 4 independent experiments is shown.

D) RT-qPCR analysis of YRA1 and HO cut transcripts in the absence (-Phe ethanol treatment) or presence of Phenantroline (200µg/ml) added for 15’ at the end of the 2h of HO endonuclease induction with Galactose and 2h Glucose as no HO endonuclease induction (no cut control).

Yra1 post-translational modification by Slx5-Slx8 led us to define whether Yra1 ubiquitination changes during induction of the irreparable HO cut. To address this possibility, we performed a ubiquitination assay under irrepairable HO cut induction. Yra1 is ubiquitinated in the presence or not of HO cut induction, indicating that the overall level of Yra1 ubiquitination does not change (Figure 4.5). This does not exclude that Yra1 bound at the HO cut could be or not Ubiquitinated.

Figure 4.5: Yra1 ubiquitination level does not change under irreparable HO cut induction.

A) Yra1 ubiquitination assay in the HO irreparable strain (Nagai et al., 2008) transformed with the 2µ plasmid expressing cupper inducible His-Ubiquitin or Empty vector. His-Ubiquitin was induced with cupper overnight. His-Ubiquitinated proteins were affinity purified on Nickel beads and ubiquitinated forms of Yra1 were detected by Western blotting with an anti-Yra1 antibody. Western blotting of input samples with anti-Pgk1 was used as loading control and with anti-Yra1 to define Yra1 levels.

Experiment performed by Ivona Bagdiul.

B) Yra1 ChIP analysis of the samples treated as described in A. Yra1 recruitment was examined at 0.6Kb, 1.6Kb and 4.5Kb from the HO site in cells transformed with empty vector or with the 2µ His-Ubiquitin expressing plasmid and grown in glucose (no cut control) or galactose for 2h as indicated.

ChIP values using αYra1 antibody are shown as percentage of input. Black bars are the corresponding ChIP values using the Preimmune as non-specific binding control. Experiment performed by Ivona Bagdiul.

C) Control of the HO cut induction after 2h in Galactose and the no cut control after 2h in Glucose using a PCR assay specific for the HO cut site.

Yra1 mutants differently bind an irreparable HO cut  

It is possible that the sensitivity to DNA damage agents of Yra1 mutants could be due to impaired recruitment to DSB loci. To define whether the HA-Yra1 mutants are differentially recruited to an irreparable DSB, we integrated the YRA1WT, HA-yra1(1-210), HA-yra1Δintron, and HA-yra1allKR sequences into the irreparable HO DSB strain and examined the recruitment of these different HA-Yra1 proteins at the HO cut by ChIP using αHA antibody (Figure 4.6 A). Figure 4.6 D presents a spot test analysis of yra1 mutants integrated in the DSB background and confirms that persistent irreparable HO cut induction causes lethality. Chromatin immunoprecipitation under HO cut induction shows that the HA-yra1(1-210) is not recruited to the HO cut site, while the yra1Δintron is more recruited than the HA-Yra1 WT both in Glucose (no cut) and in Galactose (HO cut) (Figure 4.6 A). The increased binding of HA-yra1Δintron reflects increased protein levels compared to the HA-Yra1WT (Figure 4.6 B). The HA-yra1allKR mutant binds the HO cut to the same extent as HA-Yra1WT despite increased protein expression (Figure 4.6 A-B). In contrast to the HA-yra1Δintron, the other HA-yra1 mutants show no correlation between HO cut binding and protein expression levels, suggesting that the Yra1 C-terminal region could be important for Yra1 recruitment to the DSB. Since we observed Yra1 recruitment at the HO cut 2h after Gal induction, once there has been extensive resection, we wondered how RPA binding to the HO cut may vary in the different yra1 mutants (Figure 4.6 C). RPA binding slightly increases in the HA-yra1(1-210) and HA-yra1allKR and increases even more in the HA-yra1Δintron (Figure 4.6 C). These changes do not correlate with the differential recruitment of the HA-yra1 mutants to the HO cut site. Therefore, is not clear whether the RPA binding could be partially dependent on Yra1 recruitment.

Figure 4.6: Yra1 mutants are differentially recruited to the HO cut site  

A) ChIP using αHA antibody of HA-Yra1 WT, HA-yra1(1-210), HA-yra1allKR, HA-yra1Δintron, Yra1WT (no tag) at 0.6 Kb from the HO cut site after 2h of HO endonuclease induction with Galactose.

The 2h Glucose time point was taken as no cut control. ChIP values are shown as percentage of input.

The average of 3 independent experiments is shown with corresponding standard error.

B) Protein levels of HA-Yra1 WT, HA-yra1(1-210), HA-yra1allKR and HA-yra1Δintron after 2h in Glucose or Galactose to induce the HO cut. Yra1 proteins were detected with an αHA antibody and values normalized with Pgk1 protein levels. The average of 3 independent experiments is shown with corresponding standard error.

C) ChIP using αRPA antibody of Yra1 WT (WT), yra1(1-210), yra1allKR, HA-yra1Δintron, at 0.6 Kb from the HO cut site after 2h of HO endonuclease induction with Galactose. The 2h Glucose time point was taken as no cut control. ChIP values are shown as percentage of input. The average of 3 independent experiments is shown with corresponding standard error of the mean and P value < 0.05 (*) referred to the condition HA-YRA1 WT Gal 2h.

D) Spot test analysis of HA-YRA1 WT, HA-yra1(1-210), HA-yra1allKR, HA-yra1Δintron in the inducible HO cut background at 25°C, 30°C, 34°C in YEPD 2% Glu. Spot test in YEPD 2% Galactose (25°C) shows lethality of the strains when a persistent irreparable HO cut is induced. At least 4 independent clones were tested for each HA-yra1 mutant.