Immunoprecipitation and chromatin fractionation

G. Aim of this study

II. MATERIALS AND METHOD

9. Immunoprecipitation and chromatin fractionation

Cells were harvested and lysed in NETN buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.5% NP-40, and 5 mM EDTA) with protease and phosphatase inhibitors. The lysate was sonicated and centrifuged at 13,000rpm for 15 min at 4°C. The supernatant was measured by Bradford assay and the equal amount of protein lysate was incubated with primary antibody for overnight at 4°C.

The immunoprecipitates were captured by Protein A sepharose Fast-Flow (GE Healthcare). The beads were washed four times with NETN buffer and boiled in 2X sample buffer.

For chromatin fractionation, cells were harvested and lysed in NETN buffer excluding 5mM EDTA with protease and phosphatase inhibitors on ice for 20min at 4℃. The lysate was centrifuged at 13,000 rpm for 15 min 4℃. The pellet was incubated with MNase (25U) and Benzonase (25U) for 20 min at 37℃ shaking incubator. The supernatant was collected by centrifugation at 4℃ and processed to western blotting.

10. Micrococcal Nuclease (MNase) assay

10⁷ HeLa cells were seeded on the day before treating with Neocarzionstain (NCS). Next

15 MNase (Thermo) was added in digestion buffer. The reaction was stopped by adding MNase stop buffer (20mM EDTA). Protease K and SDS were added and incubated in the samples overnight at 37℃. Genomic DNA was purified by using Nucleospin PCR clean up kit (Macherey-Nagel) and processed to running on agarose gel.

11. Homologous recombination (HR) and Non-homologous end joining (NHEJ) repair assay

U2OS-DR-GFP (HR) and U2OS-EJ5-GFP(NHEJ) reporter cells were seeded in 12 well washed with PBS three times and stained with propidium iodide (PI) and analyzed by Fluorescence-activated cell sorting (FACS).

13. Chromatin Immunoprecipitation (ChIP)

Cells were harvested after the indicated treatment (4-OHT or Shield1) and cross-linked with 1% formaldehyde for 20 min. Cells were lysed with SDS lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris, pH 8.1) supplemented with protease and phosphatase inhibitor cocktail (Thermo) for 10 min on ice. Cell lysates were sonicated by Bioruptor (Diagenode) and centrifuged at 13,000 rpm for 15 min. The supernatants were collected and diluted for overnight incubation with primary antibody. After overnight incubation, 20μl of Protein A agarose/salmon sperm DNA (Millipore) was added to each sample and incubated with rotation at 4℃. After 1h incubation, beads were washed with the following washing buffer: low salt immune complex wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 150 mM NaCl), high salt immune complex wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 500 mM NaCl), LiCl immune complex wash buffer (0.25 M LiCl, 1% IGEPAL-CA630, 1% deoxycholic acid (sodium salt), 1 mM EDTA, 10 mM Tris, pH 8.1), and TE buffer (10 Mm Tris-HCl, 1mM EDTA, pH 8.0).

After washing the beads, the immune complex was eluted in elution buffer (1% SDS, 0.1 M NaHCO3) for 30 min at RT. The eluates were incubated with 10μl of 5M NaCl to reverse histone-DNA crosslinks by heating at 65℃ for 4 h, followed by incubation with proteinase K at 45℃ for 1 h. DNA was purified with Nucleospin PCR clean up kit (Macherey-Nagel) and processed for

14. FokI assays

For the recruitment of RSF1-GFP, HDAC1-EGFP, H2AX-GFP, or Mre11-YFP, 2-6-5 cells were seeded on coverglass bottom dish (SPL) on the day before transfection and transfected with siRSF1 using Lipofectamine RNAiMax (Invitrogen). Next day, the indicated GFP-tagged construct was transfected with Lipofectamine 2000 (Invitrogen) and incubated for 48 h. Cells were harvested after 48 h transfection and fixed with 4% paraformaldehyde (Sigma), followed by imaging analysis.

15. RNA isolation and RNA sequencing

Total RNA was isolated from U2OS cells, treated with control siRNA and RSF1 siRNA, using RNeasy Plus Mini kit (Qiagen). The quality of RNA was checked using Bioanalyzer RNA ChIP (Agilent Technologies). mRNA library was prepared using Truseq stranded mRNA kit (Illumina), and RNA sequencing running was performed using Nextseq 500 (Illumina).

16. Reverse transcription and quantitative RT-PCR

For quantitative RT-PCR, total RNA was isolated using RNeasy Plus Mini kit (Qiagen).

2μg of total RNA was used for cDNA synthesis by amfiRivert cDNA Synthesis Platinum Master Mix (GenDEPOT). Quantitative PCR was performed on Roter-GeneQ (Qiagen) using Maxima SYBR Green qPCR master mix (Thermo) with the following primers.

Table 3. qPCR primers

Cells were resuspended in fractionation buffer (50mM Hepes pH 7.5, 150mM NaCl, 1mM EDTA, 0.2% NP-40) and incubated on ice for 5 min. The lysate was centrifuged at 1,000 g for 5 min. Supernatant (Fraction I) was isolated, and pellet was further extracted in fractionation buffer containing 0.5% NP-40 and incubated on ice for 40 min. The extract was centrifuged at 16,000 g for

15 min. Supernatant (Fraction III) was separated, and pellet was resuspended in 1X sample buffer and boiled for 10 min. Equal aliquots of each fraction were separated on 4-12% SDS-PAGE.

18. Nucleosome stability assay

Cells were suspended in lysis buffer A and incubated for 5 min on ice. The lysate was centrifuged at 1,300 g for 4 min. Pellet was resuspended in lysis buffer B (20mM Tris pH 8.0, 15%

glycerol, 1.5% Triton X-100, 0.8M NaCl) and incubated for 30 min on ice. The extract was centrifuged at 13,000 rpm for 10 min. Insoluble and soluble fractions were boiled in sample buffer.

19. Statistical analysis

Statistics and graphs were performed using GraphPad Prism (version 5.0). Unpaired student’s t test was applied to compare two individual groups, while one-way ANOVA was applied to compare multiple groups. Asterisks indicates each p-values (* P < 0.05; ** P < 0.01, *** P <

0.005).

III. RESULTS

PART I. ATM-dependent chromatin remodeler RSF1 facilitates DNA damage checkpoints and homologous recombination repair

1. RSF1 is recruited at DNA double-strand break sites

To understand the role of RSF1 in DDR, I first examined whether RSF1 is recruited at DSBs upon DNA damage. RSF1 formed DNA damage foci with DNA damaging agent phleomycin (radio-mimetic drug making DSBs), which is co-localized with γH2AX and pATM, as the major hallmarks of DSBs (Fig. 1A and B). Furthermore, to demonstrate whether its localization is specific to DSBs, 2-dimentional (2D) and 3-dimentional (3D) images were obtained by workstation program (Nikon). As expected, RSF1 and γH2AX are co-locolizaed at DSBs (Fig. 1A and B). It has been shown that RSF1 is over-expressed in cancer cells rather than in normal cells.(Fang et al., 2011; Hu et al., 2012; Liu et al., 2012; Mao et al., 2006; Shih Ie et al., 2005) To confirm clearly what the recruitment of RSF1 at DSBs is not specific in cancer cells, the endogenous RSF1 was immunostained at 10 min after micro-irradiation and found that RSF1 was accumulated at the micro-irradiated sites in normal (human RPE1) and cancer cells (U2OS) (Fig. 1C). Again, in order to exclude the possibility that the recruitment of RSF1 at micro-irradiated sites is caused by non-specific damaging effects other than by DNA double-strand breaks, the recruitment of RSF1 was validated in stably integrated LacO-LacI nuclease system (Shanbhag et al., 2010). Briefly, the fused mCherry-LacI with FokI endonuclease (mCherry-LacI-FokI) generates a single double-strand break on chromosome 1 integrated LacO repeats. To detect a single focus at DSB in a single cell, RSF1-GFP was transfected with mCherry-LacI-FokI (WT or inactive nuclease (D450A)). As expected, RSF1-GFP was recruited at the site of FokI-induced single break, whereas the nuclease-deficient

mutant form of FokI (D450A) did not make a focus of RSF1-GFP at the site (Fig. 1D). The recruitment of RSF1 at DSBs was again confirmed using K230-ZFN, which is zinc finger nuclease generating unique DSB on chromosome 3 specifically (Xu et al., 2012). Accumulation of RSF1 at DSB together with γH2AX was validated again with Chromatin immunoprecipitation assay (Fig.

1E). Furthermore, SNF2h ATPase, given that SNF2h is recruited in PARP1 dependent manner (Smeenk et al., 2013), was also recruited at DSB on chromosome 3. Thus, these results strongly indicate that RSF1 is recruited at DSBs upon DNA damage.

Figure 1. RSF1 is recruited at DNA double-strand break site(s). (A and B) U2OS cells were immunostained with anti-RSF1 and anti-γH2AX at 2 h after treatment of phleomycin (+Phleo) (50 μg/ml) or untreatment of phleomycin (-Phleo). Scale bar, 10 μm. The damage foci were analyzed in 2-dimentional (2D) and 3-dimentional (3D) image with Workstation (Nikon). (C) U2OS and RPE1 cells were pre-sensitized with BrdU (10 μM) for 30 h, followed by laser micro-irradiation. Both cell lines were immunostained with anti-RSF1 and anti-γH2AX at 10 min after micro-irradiation. (D) Diagram of the recruitment of GFP-fused proteins and mCherry-LacI-FokI (wild type and D450A mutant) on chromosome 1p3.6, stably integrated with LacO repeats, and co-localization of GFP-fused proteins and FokI wild type (WT) or FokI mutant (D450A) in live cells. H2AX-GFP and Mre11-YFP used as controls. (E) Enrichment of RSF1, SNF2h and γH2AX on site-specific DSB on chromosome 3. DSB was induced by transfection with K230-ZFN in U2OS cells, and fold enrichment by ChIP was divided by input.

2. ATM-dependent accumulation of RSF1 at DSBs

Based on the large scale proteomic analysis of ATM and ATR substrates and mass spectrometry-based quantitative proteomics, RSF1 is one of the potential substrates for ATM/ATR and its phosphorylation is upregulated upon DNA damage (Beli et al., 2012; Matsuoka et al., 2007).

Since the recruitment of RSF1 is rapidly occurred at DSBs after micro-irradiation, the effect of ATM on the function of RSF1 upon DNA damage was investigated. The foci formation of RSF1 and pATM was examined after treatment with ATM inhibitor, KU55933, prior to adding to phleomycin. The average number of foci formation of RSF1 and pATM in pre-incubated cells with KU55933 was compared to the average number of foci in control cells without KU55933.

Consistently, the number of RSF1 and pATM foci was increased in the treatment with phleomycin., while the foci formation of RSF1 and pATM upon DNA damage was impaired after treatment with KU55933 (Fig. 2A and B). These results suggest that ATM inhibition diminished the foci formation of RSF1 upon DNA damage.

In order to confirm again whether the foci formation of RSF1 depends on ATM upon DNA damage, human A-T cells (ATM knockout fibroblast) was immunostained with RSF1 and γH2AX after treatment with phleomycin. As expected, the percentage of foci formation of RSF1 (containing over 10 foci) was reduced in ATM-deficient cells after treatment with phleomycin rather than in ATM wild type cells (Fig. 2C and D). Since it had been reported that IR can induce the foci formation of γH2AX by all three PIKKs (ATM, ATR, and DNA-PK) (Wang et al., 2005), γH2AX positive cells were counted in ATM wild type and ATM deficient cells. As expected, γH2AX-positive cells were not changed dramatically in ATM wild type cells versus ATM deficient cells upon DNA damage, whereas the foci formation of RSF1 is significantly reduced in ATM knock-out cells (Fig. 2C and D). Next, the accumulation of RSF1 was examined by micro-irradiation in ATM- or ATR-depleted cells using short hairpin RNA (shRNA) transiently expressed. The accumulation of

RSF1 was impaired in ATM-depleted cells, while ATR depletion did not affect the accumulation of RSF1 (Fig. 2E and F). These data therefore prove that ATM, rather than ATR, is required for accumulation of RSF1 at DSB sites in DDR.

Figure 2. The accumulation of RSF1 at DSBs is dependent on ATM activity. (A) U2OS cells were untreated (Ctrl) or treated with ATM inhibitor (KU55933) (20 μm) prior to phleomycin treatment (+Phleo) or untreatment (-Phleo) and immunostained with pATM (pS1981) and anti-RSF1. (B) Quantitative analysis of result in (A). ‘Before’ means untreatment of phleomycin; ‘After’

means treatment of phleomycin. The average number (Mean ± S.E.M.) of foci per cell was counted from over 100 cells. ***P < 0.005 by Student’s t-test. (C) Human fibroblast wild type (WT) or ATM knock-out (KO) cell was untreated (-Phleo) or treated with phleomycin (+Phleo) for 2 h and immunostained with anti-γH2AX and anti-RSF1. Scale bar, 10μm. (D) Quantitative analysis of result in (C). The percentage of foci positive cells (>10 foci per cell) was counted. **P < 0.01, ***P

< 0.005 by Student’s t-test. (E) U2OS cells were transfected with EGFP, shATM, or shATR and pre-sensitized with BrdU for 30 h, followed by laser micro-irradiation. Cells were immunostained with anti-RSF1 and anti-pATM at 1 hour after micro-irradiation. Scale bar, 10 μm. (F) The level of pATM (pS1981) and pATR (pS428) was analyzed by western blot.

3. The putative motifs pSQ of RSF1 by ATM is required for its accumulation at DSBs

As mentioned previously, RSF1 has the putative motifs (pS524, pS1226, pS1325) for ATM.

I thus focused on whether these motifs are important for accumulation of RSF1 at DSBs. To evaluate whether the motifs of RSF1 by ATM is important in DDR, the level of RSF1 phosphorylation was examined by phspho-specific antibody (α-pSQ/TQ). The phosphorylation level of RSF1 is increased in DNA-damaging condition (Fig. 3A). Next, the recruitment of RSF1 at DSB sites was examined after treatment with ATM inhibitor KU55933 prior to micro-irradiation.

Consistently, accumulation of RSF1 was reduced in ATM inhibition, compared to its recruitment in normal condition (Fig. 3B and C). Thus, these data suggested that the recruitment of RSF1 at micro-irradiated sites is dependent on ATM activity. Next, the effect of putative motifs of RSF1 on its recruitment at DSB sites was examined. After substituting three serine residues (S524, S1226, and S1325) to alanine, I tested the accumulation of RSF1 wild type and 3SA mutant after micro-irradiation. Interestingly, over-expression of the 3SA mutant showed the reduced accumulation at the DSBs, whereas RSF1 WT was accumulated gradually at DSBs. Again, after 3SA mutant was transfected into shRSF1 stable cell line, the accumulation of this mutant is declined at DNA lesions compared to wild type (Fig. 3B and C). These results suggest that RSF1 is phosphorylated by ATM upon DNA damage and these motifs by ATM are important for its accumulation at DSBs.

Figure 3. The putative pSQ motifs of RSF1 by ATM are required for its accumulation at DSBs.

(A) U2OS cells were irradiated at 10Gy and harvested at 1 hour after irradiation. Whole cell extracts were lysed and immunoprecipitated with RSF1 antibody. The immunoprecipitated was detected with pSQ/TQ antibody. γH2AX antibody used as a DNA damage control. (B) RSF1-GFP (WT) or RSF1 mutant (3SA) was transfected in U2OS cells and pre-sensitized with BrdU for 30 h, followed by laser micro-irradiation. ATM inhibitor KU55933 was pre-treated for 12 hours prior to micro-irradiation. Scale bar, 10 μm. (C) Quantitative analysis of fluorescence intensity at DSBs.

The accumulated fluorescence intensity at micro-irradiated sites was normalized the background around the sites. NFU stands for normalized fluorescence unit. The values (Mean ± S.E.M.) were calculated and plotted against time from at least 10 individual cells.

4. Depletion of RSF1 attenuates DNA damage checkpoint signals

In general, DNA damage induces ATM/ATR activation, followed by the phosphorylation of γH2AX and recruitment of the mediators, including MDC1, BRCA1, 53BP1 and others (Ciccia and Elledge, 2010). Thus, the accumulation of RSF1 in ATM-dependent manner upon DNA damage led us to examine whether RSF1 affects in DNA damage checkpoint signaling. Stably RSF1-depleted HeLa cell line was used to check the DNA damage checkpoint signaling. Surprisingly, the depletion of RSF1 attenuated DNA damage checkpoints (pNBS1, pChk1/2), especially in the propagation of γH2AX (pS139) (Fig. 4A). This result was confirmed again with U2OS cell line that was transiently knock-downed RSF1 by siRNA (Fig. 4B). Next, the retention of γH2AX and pATM after micro-irradiation was monitored. The γH2AX retention was dramatically abrogated in RSF1-depleted cells compared to control cells, whereas signals of pATM seemed to be reduced at little in RSF1 depleted cells (Fig. 4C). Next, the cell cycle progression was analyzed to check whether the depletion of RSF1 affects the cell cycle progression upon DNA damage. FACS analysis demonstrates that cell cycle progression in RSF1 depleted cells with treatment of phleomycin for 12 hours showed the

Figure 4. Depletion of RSF1 attenuated DNA damage checkpoints. (A) Stably RSF1-depleted (shRSF1) or control (shCtrl) cells (HeLa) were treated with phleomycin (50 μg/ml) and harvested at each indicated time points. Whole cell extracts were directly lyzed with sample buffer. The activation of DNA damage checkpoints was examined by western blot with the indicated antibodies.

U, untreated. (B) U2OS cells were transfected with siRNA for Rsf-1 (siRsf-1) or for control (siCtrl) and harvested at the indicated time points after treatment with phleomycin (Phleo) (50 μg/ml).

Activation of DNA damage checkpoints were estimated by western blot with the indicated antibodies. (C) Stably RSF1-knock-downed or control U2OS cells were immunostained with anti-RSF1 and anti-γH2AX, or anti-anti-RSF1 and anti-pATM at 1 hour after laser micro-irradiation. Scale bar, 10 μm. (D) FACS profiles of stably RSF1-depleted (shRSF1) or control (shCtrl) cells during continuous DNA damage with phleomycin. (E) Stably RSF1-depleted cells (U2OS) were treated with phleomycin for 2 h and immunostained with anti-γH2AX, anti-MDC1 and anti-53BP1. The average number (Mean ± S.E.M.) of foci per cell was counted from over 100 cells. **P < 0.01,

***P < 0.005 by Student’s t-test.

Figure 5. Depletion of RSF1 attenuated DNA damage checkpoints. Stably RSF1-knock-downed U2OS cells were transfected with EGFP, RSF1 WT and RSF1 3SA mutant and immunostained with anti-γH2AX (A), anti-MDC1 (B), and anti-53BP1 (C) after treatment with phleomycin (50 μg/ml).

The number of foci formation per cell was calculated and graphed (D). ***P < 0.005 by Student’s t-test. N.S., not significant.

5. Depletion of RSF1 caused the failure in chromatin relaxation upon DNA damage

To understand the mechanism of how RSF1 regulates ATM-dependent checkpoint signaling pathway, I examined the function of RSF1 as chromatin remodeling factor in response to DNA damage. In the context of chromatin, remodeling chromatin is the critical step to propagate DDR signaling pathway and recruit repair factors (Price and D'Andrea, 2013; Soria et al., 2012). Since γH2AX is not propagated in RSF1-depleted cells, I hypothesized that the reduction of γH2AX propagation was caused by chromatin condensation in RSF1 depleted cells. TSA treatment is well known drug that induces global chromatin relaxation by inhibiting class I and II HDACs and inducing hyper-acetylation at chromatin. Thus, I treated TSA in RSF1 depleted cells and examined whether the treatment with TSA rescued the failure of chromatin decompaction caused by RSF1 depletion. Interestingly, TSA treatment significantly rescued the defect in propagation of γH2AX upon DNA damage in RSF1 depleted cells, which indicates that the reduction of γH2AX propagation in RSF1-depleted cells was caused by defect in chromatin relaxation (Figure 6A). In order to visualize the chromatin status upon DNA damage, I performed Micrococcal Nuclease (MNase) assay. MNase assay is the conventional assay to measure the accessibility of chromatin.

Chromatin relaxation was usually measured by MNase assay and the relaxation was induced by neocarzinostatin (NCS) treatment, which mimics ionizing radiation. This assay showed the level of di-nucleosome was increased in RSF1-depleted cells, and the level of mono-nucleosome was reduced in RSF1-depleted cells under DNA damage (Figure 6B). In addition to MNase assay, H3 eviction at the site of DSB is another method to examine the chromatin status under DNA damage.

Histone eviction is commonly found at the sites of DSB in order to open and relax the chromatin structure (Goldstein et al., 2013). To examine the H3 eviction under DNA damage AsiSI cell line, which has the defined regions that are transcriptionally active and inactive, was used (Aymard et al., 2014). H3 eviction was analyzed by ChIP assay at DSB sites in RSF1 depleted cells in

transcriptionally active and inactive sites. In general, transcription regulation is determined by chromatin status. Thus, I assumed that H3 eviction will be mild in transcriptionally inactive site, compared to H3 eviction in transcriptionally active site. As I expected, the broad range of H3 in transcriptionally active sites was evicted in control cells upon DNA damage, while the narrow range of H3 in transcriptionally inactive sites was evicted upon DNA damage. Interestingly, RSF1 depletion prevented H3 eviction at DSB sites in both transcriptionally active and inactive sites (Figure 6C, D, and E). This result suggests that RSF1 is required to promote chromatin decondensation upon DNA damage.

Previous reports also showed that the loss of nucleosome stability at DSB sites is mediated by chromatin remodeling factor, p400 (Xu et al., 2010). Thus, I also tested if RSF1 depletion regulates nucleosome stability in response to DNA damage. In stably RSF1 knocked down cells, nucleosome was resistant to evict histones upon DNA damage (Figure 6F). In addition this resistance was rescued by the treatment of sodium butyrate (NaB), which induces chromatin relaxation (Figure 6G). Thus, these results indicate that the nucleosome is more stable and compacted in response to DNA damage in RSF1 depleted cells, compared to control cells.

In addition to chromatin remodeling factors, ATM kinase is also known as the major determinant of chromatin status in response to DNA damage. Previous studies showed that ATM kinase induces global chromatin relaxation upon DNA damage (Ziv et al., 2006). Since the above results showed that chromatin relaxation is mediated by RSF1, I tested if ATM is regulated by RSF1.

Because the results in figure 4 showed the mild reduction of pATM in RSF1-depleted cells, the retention of ATM on chromatin in RSF1-depleted cells was examined. ATM retention on chromatin

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