Depletion of RSF1 decreased histone H2A and H2B exchange at the sites of DSB

PART I. ATM-dependent chromatin remodeler RSF1 facilitates DNA damage checkpoints

6. Depletion of RSF1 decreased histone H2A and H2B exchange at the sites of DSB

Given that RSF1 is required for chromatin relaxation and nucleosome stability, I examined histone mobility upon DNA damage in RSF1-depleted cells. Previously, Dinant et al. found that SPT16 subunit of FACT is involved in H2A histone exchange at UV-induced DNA damage (Dinant et al., 2013). Interestingly, RSF complex is initially identified as the binding protein of FACT complex (LeRoy et al., 1998). Since chromatin is condensed in RSF1 depleted cells under DNA damage, the histone mobility upon DNA damage as a result of condensed chromatin in RSF1-depleted cells was examined. In order to visualize histone mobility at DSB site, I applied a fluorescence recovery after photobleaching (FRAP) to microirradiation in order to compare histone mobility at double strand breaks in living cells. First, the half of GFP signal in nucleus was photobleached by 488nm laser and local double strand break was introduced by 405nm laser.

Interestingly, GFP-H2A and GFP-H2B showed the enhanced fluorescence recovery at DSB sites, compared to fluorescence intensity in undamaged sites, which indicates that H2A and H2B dimer exchange is enhanced at the local DSB sites (Figure 7A). Next, the fluorescence intensity in RSF1-depleted cells after FRAP was measured. γH2AX was used the indicator of double strand break. In control cells, H2A and H2B dimer was rapidly exchanged at DSB sites, while H3 was unchanged at DSB sites. As expected, histone exchange of H2A and H2B dimer, but not H3 was significantly decreased in RSF1 depleted cells (Figure 7B, C, and D). Thus, these results showed that RSF1 as chromatin remodeler is required for chromatin dynamics for efficient repair upon DNA damage.

Figure 7. histone H2A and H2B exchange was decreased in RSF1 depleted cells at the site of DSB. (A) Recovery of fluorescence of GFP-H2A and GFP-H2B after photobleaching with 488nm laser was increased at local DSB sites. (B) Relative fluorescence intensity of H2A and GFP-H2B was normalized by the fluorescence intensity before local DSB with 405nm laser and quantified. (C and D) The recovery of fluorescence of GFP-H2A and GFP-H2B was decreased at the sites of DSB in RSF1-depleted cells. (E) The exchange of H3 was not changed in control and RSF1-depleted cells. ***P < 0.005 by Student’s t-test. N.S., not significant.

7. RSF1 facilitates homologous recombination repair by recruiting HR factors

Finally, to evaluate the biological implication of RSF1 in DNA damage repair, I performed repair assay after RSF1 siRNA transfection. Interestingly, RSF1 depletion impaired both NHEJ and HR repair (Fig. 8A and B). Reintroduction of WT RSF1 rescued the efficiency of HR and NHEJ, while that of 3SA RSF1 failed to rescue both repair efficiencies. Since it has been shown that chromatin remodelers are required for homologous recombination (HR) repair by recruiting of HR factors, replication protein A (RPA) and Rad51 are key molecules in searching for homologous template in HRR (Ciccia and Elledge, 2010). Therefore, I focused on whether RSF1 is required for HR repair. I tested again whether chromatin remodeler RSF1 is critical for recruiting HR factors RPA32 and Rad51 in HRR. In stably RSF1-depleted cells, the recruitments of both HR factors RPA32 and Rad51 were dramatically declined upon DNA damage, which is conversed to wild type cells (Fig. 8C and D). Again, I confirmed that these HR factors was less accumulated in chromatin in RSF1 depleted cells, even if the protein level of both factors was unchanged in stably RSF1 depleted cells (Fig. 8E). Taken together, these findings suggest that RSF1 facilitates HR through recruiting HR factors RPA32 and Rad51.

Figure 8. RSF1 facilitates homologous recombination repair. EJ-GFP (A) and DR-GFP (B) cells were transfected with control siRNA (siCtrl) or RSF1 siRNA (siRSF1). At 48 hours after siRNA transfection, I-SceI combined with RSF1-V5 (WT) or 3SA-V5 was transfected to induce DSB.

ATM inhibitor (KU55933) was treated before transfection of I-SceI. GFP-positive cells were counted using FACS at another 72 h. The relative percentage of GFP positive cells was calculated.

N.S., not significant. *P < 0.05, ***P < 0.005 by one-way ANOVA with Tukey HSD. (C) U2OS RSF1-depleted cells were pre-sensitized with BrdU for 30 hours, followed by laser micro-irradiation. These cells were immunostained with RSF1 and RPA32 or RSF1 and anti-Rad51, and imaged using confocal microscope. (D) RPE1 cells were transfected with RSF1 siRNA or control siRNA for 72 hours and treated with phleomycin (10 μg/ml) for 1 hour. The chromatin fractions were isolated at each time points released after treatment with phleomycin. The level of chromatin-bounded proteins was analyzed by western blots. (E) The total level of RSF1, RPA32 and Rad51 in stably RSF1 depleted and control cells (U2OS).

PART II. RSF1 promotes DSB-induced transcriptional silencing by recruiting EZH2 and HDAC1 at DSB sites

1. RSF1 leads to DSB-induced transcriptional silencing at DNA lesions

In general, chromatin remodeling factors are key determinant for transcriptional regulation in biological processes. Recent studies indicate that gene transcription is influenced by DSB and DSB repair itself is also regulated by transcriptional status on chromatin. These studies also implicate the crosstalk between transcription and DNA damage. In part I, I identified RSF1 as chromatin remodeling factor modifying chromatin structure to propagate DNA damage checkpoint signaling pathway. Interestingly, RSF1 has been originally identified as transcriptional regulator in a complex with FACT (LeRoy et al., 1998). Thus, I assumed that RSF1 regulates transcriptional status at DSB sites, resulting in the efficient repair.

Previously, it has been reported that ATM regulates transcriptional silencing and RSF1 interacts with ATM (Pessina and Lowndes, 2014; Shanbhag et al., 2010), I tested whether RSF1 regulates ATM-mediated transcriptional silencing at DSB sites. Using FokI system, I examined the transcription status at DSB sites in RSF1 depleted cells. The nascent transcripts in the stably incorporated in IFII cell line are visualized by YFP-MS2. As I expected, ATM inhibition perturbed DSB-induced transcriptional silencing. Indeed, the results showed RSF1 depletion caused the aberrant transcription at DSB sties (Figure 9A, B, and C). In order to confirm the result, the nascent transcripts were immunostained by 5-Ethynyl Uridine (5EU) in order to visualize the nascent RNAs at the local DNA damage site (Figure 9D). As consistent to FokI system, the amount of transcripts at DSB sites was significantly reduced, compared to the amount in undamaged regions. In addition, RSF1 depletion reduced the loss of nascent RNAs at DSB sites. BrdU was used as the control to

show the defect of HR in RSF1 depleted cells, since the exposed BrdU, only followed by DNA resection, is immunostained by BrdU antibody (Figure 9D). To verify the function of RSF1 in DSB-induced transcriptional silencing, I added back the siRNA-resistant wild type RSF1 and quantified YFP-MS2 intensity. RSF1 addition in RSF1 depleted cells rescued the DSB-induced transcriptional silencing at DSB sites (Figure 9E). Furthermore, the loss of RNAPII pS2, which is transcriptional elongation marker, was also reported as the marker for DSB-induced transcriptional silencing (Shanbhag et al., 2010). Thus, I also examined the level of RNA PII pS2 at DSB sites in RSF1-depleted cells and found that RNAPII pS2 was not reduced after DNA damage in RSF1-RSF1-depleted cells (Figure 9F). Taken together, RSF1 depletion prevents DSB-induced transcriptional silencing and causes the aberrant transcription at DSB sites.

Figure 9. RSF1 leads to DSB-induced transcriptional silencing at DNA lesions. (A) IFII stable cell line was pre-treated with doxycycline to induce transcription and induced DSB by treating withShield-1 and 4-OHT. Cells were fixed at 4 h after DSB induction. ATM inhibitor (KU55933) was treated before DSB induction. (B) Quantification *P < 0.05, ***P < 0.005 by one-way ANOVA with Newman-Keuls. (C) The level of RSF1 in siRNA-treated and control cells (IFII). (D) U2OS cells were pre-sensitized with BrdU and immunostained with γH2AX, 5EU (RNA) and BrdU. (E) siRSF1-resistant RSF1 rescued transcription silencing at DSB. (F) IFII cells were transfected with siRSF1 and treated with doxycycline, followed by treatment with Shield1 and tamoxifen (4-OHT).

Cells were fixed at 5 h after DSB and performed ChIP analysis. N.S., not significant. **P < 0.01 by Student’s t-test

2. SNF2h is dispensable to DSB-induced transcriptional silencing at DSB lesions

Next, I tested if SNF2h ATPase, the binding partner of RSF1, is required for DSB-induced transcriptional silencing. First, the fluorescence intensity of YFP-MS2 in IFII cell line was quantified in SNF2h-depleted cells. The fluorescence intensity of YFP-MS2 in RSF1-depleted cells was increased as consistent to Figure 9, while the intensity in SNF2h-depleted cells was not changed, compared to the intensity in control cells (Figure 10A). The level of phosphorylation of RNAPII at S2 after the induction of DSB was checked in RSF1- and SNF2h- depleted cells. In RSF1-depleted cells, the level of RNAPII pS2 was highly accumulated at DSB sites. However, the level of phosphorylation of RNAPII at S2 in SNF2h-depleted cells showed no difference, which is consistent to figure 2A. The total RNAPII level was also immunostained as control (Figure 10B).

Thus, these results indicate that RSF1, but not SNF2h, is required for DSB-induced transcriptional silencing at DSB lesions.

Figure 10. SNF2h is not required for DSB-induced transcriptional silencing at sites of DSB. (A) IFII cell line transfected with siRSF1 and siSNF2h was fixed after DSB induction. Doxycycline was pre-treated before induction. The relative fluorescence intensity of YFP-MS2 in RSF1- and SNF2h- depleted cells was quantified. (B) DSB-induced cells were immunostained with RNA Polymerase II pS2 and total RNA polymerase II in RSF1- and SNF2h- depleted cells. Relative mean of fluorescence intensity (RMFI) of YFP-MS2, normalized by RMFI in undamaged region, was quantified in RSF1- and SNF2h- depleted cells. N.S., not significant. *P < 0.05, ***P < 0.005 by one-way ANOVA with Tukey HSD.

3. RSF1 promotes DSB-induced transcriptional silencing by regulating ATM activity.

Since ATM kinase is the major determinant to promote DSB-induced transcriptional silencing, I also examined the level of pATM at DSB sites. Furthermore, our previous data (Figure 6H) showed that the retention of ATM on chromatin was reduced in RSF1 depleted cells. Thus, transcriptionally silenced-DSB sites were immunostained with pATM. The level of pATM was significantly enriched at FokI-induced site in control cells, while the level of pATM was reduced or absent at DSB sites in RSF1 depleted cells (Figure 11A). Consistent to Figure 10, I checked the level of pATM in SNF2h depleted cells and found that the level was unchanged in SNF2h depleted cells, compared to control cells (Figure 11B). Thus, I concluded that RSF1 promotes DSB-induced transcriptional silencing by regulating the level of pATM at DSB sites.

Figure 11. RSF1 depletion reduced the level of pATM, resulting in the failure of DSB-induced transcriptional silencing. IFII cell line was transfected with siRSF1 (A) and siSNF2h (B) and induced transcription by doxycycline treatment, followed by DSB induction. Cells were fixed and immune-stained with pATM (S1981). RMFI of pATM at FokI-induced site in RSF1- (A) and SNF2h- (B) depleted cells was quantified. N.S., not significant. **P < 0.01 by Student’s t-test.

4. Screening RSF1-interacting proteins identified that RSF1 recruits transcriptional repressors at DSB sites

Since RSF1 independently promotes transcriptional silencing at DSB sites without SNF2h, I assumed that RSF1 promotes DSB-induced transcriptional silencing by recruiting other histone modifiers. Previously, the mass spectrometry analysis of RSF1 showed that RSF1 is tightly associated with many proteins that are involved in DDR (Figure 12A). Based on the analysis, 34 proteins of RSF1-interacting proteins were selected, including chromatin remodeling complexes, transcription, and DSB repair (Figure 12B). First, I screened the binding partners of RSF1 that are specifically recruited at DSB sites, selected by using micro-irradiation. Most of RSF1-binding proteins were recruited at DSB sites, except SWI/SNF Related, Matrix Associated, Actin Dependent Regulator Of Chromatin Subfamily C Member 1 (SMARCC1) and HDAC4. Next, I selected the proteins that are recruited by RSF1 at DSB sites. RSF1 depletion impaired the recruitment of 7 proteins at DSB sites, and, interestingly, most of them were the complexes involved in transcriptional repression (Figure 12C and D). These results showed that RSF1 promotes DSB-induced transcriptional silencing by recruiting the proteins that are involved in transcriptional repression.

Figure 12. Screening RSF1-interacting proteins identified that RSF1 recruits transcriptional repressors at DSB sites. (A) Clusters of RSF1-interacting proteins, including chromatin remodeling factors, transcription factors, and DDR factors, by mass spectrometry analysis (B) RSF1-interacting proteins and proteins predicted in the network of RSF1 based on mass spectrometry was examined the recruitment at DSB sites by microirradiation. U2OS cells were transfected with GFP-tagged RSF1-interacting proteins and microirradiated, followed by fixation at 10 min after microirradiation. SPT16 was immunostained with anti-SPT16 because of its size of cDNA, which was unable to clone into GFP-tagged vector. (C) The selected proteins interacting with RSF1 was examined their recruitment at DSBs in RSF1-depleted cells. (D) RSF1-interacting proteins recruited at DSB sites in RSF1-dependent manner are involved in gene silencing or transcription of DNA.

5. Transcriptional repressors, recruited by RSF1, are involved in DSB-induced transcriptional silencing at DSB sites

Based on the screening results in Figure 12, EZH2 and HDAC1 were selected because these proteins are mainly known as the writers of histone modifications for transcriptional repression. Since RSF1 regulates transcriptional silencing at DSB sites, I examined whether the transcriptional regulators depending on RSF1 recruitment are also required for its silencing. Using FokI system, EZH2 and HDAC1 depletion showed the significant reduction in transcriptional silencing (Figure 13-1 A, B and C). Next, I confirmed that these regulators are regulated by the pre-existed transcription status using 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB), an inhibitor of RNA polymerase elongation. When I pre-treated DRB before micro-irradiation and inhibited the ongoing transcription, the transcriptional regulators that are associated with RSF1, except RSF1 itself, were not recruited at DSB sites (Figure 13-1 D and E). In order to confirm the result, other transcriptional inhibitors such as actinomycin D and amanitin were treated before micro-irradiation. As shown in Figure 13-2, the recruitment of RSF1 at DSB sites was solely dependent on the treatment of actinomycin D, which is also known as intercalating agent. This result explains that its recruitment at DSB sites may be regulated by chromatin structure, rather than by transcriptional status (Figure 13-2). These results suggest that transcriptional regulators, associated with RSF1, are recruited at transcriptionally active sites and involved in transcriptional silencing at DSB.

Figure 13-1. Transcriptional repressors, recruited by RSF1, are involved in DSB-induced transcription silencing at DSB sites. (A) IFII stable cells were transfected with siRNAs (EZH2, and HDAC1) and induced transcription, followed by DSB induction. ATM inhibitor was pre-treated before DSB induction. (B) Quantification. **P < 0.01, ***P < 0.005 by Student’s t-test. (C) The level of EZH2 and HDAC1 in siRNA treated and control cells (IFII). (D) The recruitment of EZH2 and HDAC1 was examined by microirradiation, and the recruitment at DSB sites was impaired after treatment of DRB (100μM), transcription inhibitor. (E) Quantification.

Figure 13-2. RSF1 recruitment at DSB sites was dispensable to pre-existed transcriptional status at DSB sites. (A) Live cell imaging of RSF-GFP at DSB sites after pre-treatment with Actinomycin D (ActD), DRB, and α-amanitin before micro-irradiation. (B) RMFI of RSF1-GFP at DSB sites normalized by the undamaged region was quantified and graphed. ns, not significant.

**P < 0.01, ***P < 0.005 by Two-way ANOVA with Bonferroni posttests.

6. RSF1 depletion impaired EZH2 recruitment at DSB sites.

Previous reports revealed that the recruitment of EZH2 as the downstream of ATM is important for ATM-mediated transcriptional silencing at DSB sites (Shanbhag et al., 2010). Based on the results in figure 11 showing that RSF1 may regulate DSB-induced transcriptional silencing by regulating ATM activity, I predicted that RSF1 regulates transcriptional silencing by recruiting transcriptional repressors such as EZH2 at DSB sites. In order to examine the prediction, the live imaging of emGFP-EZH2 was monitored by micro-irradiation. The result showed that RSF1 depletion significantly reduced EZH2 enrichment at DSB sites (Figure 14A and B). Next, the interaction of RSF1 with EZH2 was tested in the absence and presence of DNA damage. In the presence of DNA damage, RSF1 binds more tightly to EZH2, and DRB pre-treatment followed by DNA damage perturbed its interaction (Figure 14C). In order to determine the epistatic regulation of RSF1 and EZH2 on DSB-induced transcriptional silencing, RSF1 and EZH2 were double-knocked down, and the fluorescence intensity of YFP-MS2 at FokI localized sites was quantified. Since the double knock down didn’t show the addictive effect on transcriptional silencing, I could conclude that these two proteins are existed in the same pathway (Figure 14D). Thus, these data suggest that RSF1 recruits EZH2 at active transcription site upon DNA damage.

Figure 14. RSF1 depletion impaired EZH2 (polycomb transcriptional repressor) recruitment at DSB sites. (A and B) U2OS cells were transfected with siRSF1 and emGFP-EZH2 and pre-sensitized with BrdU, followed by laser irradiation. DRB was treated for 2 h before micro-irradiation. RMFI of emGFP-EZH2 was quantified and graphed in (B). ***P < 0.005 by two-way ANOVA with Bonferroni. (C) U2OS cells were pretreated with DRB, followed by DNA damage.

RSF1 was immunoprecipitated and immunoblotted with GFP-EZH2 and ɣH2AX. DRB-treatment impaired the interaction of RSF1 with EZH2. (D) Failure of DSB-induced transcriptional silencing in RSF1 and EZH2 double knockdown. YFP-MS2 at DSB sites was quantified and normalized by YFP-MS2 at undamaged sites in each siRNA-treated cell. **P < 0.01, ***P < 0.005 by one-way ANOVA with Tukey HSD.

7. SANT domain of EZH2, interacting with RSF1, is important to its recruitment at DSB sites.

Next, in order to search for the domain interacting with RSF1, EZH2 deletion mutants were generated and purified in vitro. The interaction of EZH2 deletion mutants with RSF1 was examined by far western. Interestingly, SANT domain is required for EZH2 to interact with RSF1 (Figure 15A). Furthermore, EZH2 deletion mutants were examined by microirradiation whether SANT domain interacting with RSF1 is important for the recruitment of EZH2 at DSB sites. The live image of EZH2 deletion mutants using micro-irradiation was monitored, and its subcellular localization of EZH2 deletion mutants showed that SANT domain is important for EZH2 to be localized in nucleus. The micro-irradiation analysis suggests that SANT-CXC domain is required for the recruitment of EZH2, while SET domain is required to retain EZH2 at the sites (Figure 15B).

These data suggest that SANT domain of EZH2 is important for its recruitment at DSB sites by interacting with RSF1.

Figure 15. RSF1 directly interacts with SANT domain of EZH2, and its interaction is required for the recruitment at DSB sites. (A) GST-EZH2 deletion mutants were purified in insect cells and blotted in NC memberane. The membrane was incubated with GST-RSF1 purified from insect cells for overnight and detected with anti-RSF1. (B) U2OS cells were transfected with emGFP-EZH2 deletion mutants and micro-irradiated. Relative fluorescence intensity of each EZH2-deletion mutants at micro-irradiated sites was normalized by background intensity of cells and quantified.

8. RSF1 depletion induces DSB-induced transcriptional silencing by the reduction in H2A ubiquitination at transcriptionally active region upon DNA damage

During development, polycomb group proteins are critical for regulating Hox gene expression, originally discovered in Drosophila. EZH2 is known as the component of PRC2, and it is reported that PRC2 complex recruits PRC1 at gene promoters and promotes the ubiquitination of H2AK119 on the promoters Furthermore, this ubiquitination recruits PRC2 complex again to promote trimethylation on H3K27 for transcriptional silencing (Simon and Kingston, 2013). Since EZH2 recruitment was reduced in RSF1 depleted cells, I examined whether the recruitment of PRC1 is also decreased as the result of the defect in EZH2 recruitment at DSB sites. The cells were fixed at 10 minutes after micro-irradiation, and the frequency of the recruitment of RING1B and its

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