Volume 30, Issue 1, Pages 86-97 (April 2008) FACT-Mediated Exchange of Histone Variant H2AX Regulated by Phosphorylation of H2AX and ADP-Ribosylation of Spt16  Kyu Heo, Hyunjung Kim, Si Ho Choi, Jongkyu Choi, Kyunghwan Kim, Jiafeng Gu, Michael R. Lieber, Allen S. Yang, Woojin An  Molecular Cell  Volume 30, Issue 1, Pages 86-97 (April 2008) DOI: 10.1016/j.molcel.2008.02.029 Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 1 Isolation of H2AX-Associated Factors (A) Schematic diagram of purification of H2AX-associated factors. Nuclear extracts from HeLa-derived H2AX-expressing cells were fractionated over P11 ion-exchange column with BC buffer as indicated. A pool of 0.8 and 1.2 M KCl elutes containing ectopic H2AX was subjected to M2 agarose affinity purification. Lane 1, proteins purified from control HeLa cells; lane 2, proteins purified from H2AX-expressing cells. NE, nuclear extract; FT, flowthrough. (B) Nuclear localization of ectopic H2AX. (Top) Nuclear and cytoplasmic fractions were prepared from H2AX-expressing cells and analyzed by western blotting with FLAG antibody. α-tubulin and lamin-A/C were used as markers for cytoplasmic (C) and nuclear (N) fractions, respectively. (Bottom) Ectopic H2AX present in cells was stained with FLAG antibody and imaged by fluorescence microscopy. DAPI was used for nucleus staining. Left panels (1), HeLa-derived FH: H2AX cells; right panels (2), regular HeLa cells. (C) Mass spectrometry analysis of H2AX-bound polypeptides. After H2AX-associated factors were resolved by 4%–20% gradient SDS-PAGE, bands were excised and subjected to mass spectrometric analysis. Protein size markers (in kDa) are indicated on the left. Lane 1, proteins purified from control HeLa cells; lane 2, proteins purified from H2AX-expressing cells. (D) Immunoblots of H2AX-associated factors. Purified H2AX-associated factors were separated by 4%–20% gradient SDS-PAGE, and selected components were analyzed by western blotting with the indicated antibodies. Lane 1, input; lane 2, mock-purified control; lane 3, H2AX-associated factors. Molecular Cell 2008 30, 86-97DOI: (10.1016/j.molcel.2008.02.029) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 2 Preparation of Recombinant Proteins and Nucleosomes (A) Coomassie staining of recombinant histones. Reconstituted histone dimers (H2AX, H2A, f:H2A, or h:H2AZ with H2B) and octamers containing H2AX or H2A were electrophoresed on a 15% polyacrylamide gel and stained with Coomassie brilliant blue. (B) Diagram of 5′ biotinylated 207 bp 601 nucleosome positioning sequence. The gray oval and arrows indicate nucleosome position and restriction enzyme cleavage sites, respectively. (C) Purification of recombinant nucleosomes. Nucleosomes were reconstituted from recombinant histone octamers and 207 bp 601 nucleosome positioning sequence by salt gradient dialysis and purified through 5%–30% glycerol gradient centrifugation. Total 25 fractions (each 200 μl) were collected from the top (#1) to the bottom (#25). Nucleosome-containing fractions (14–18) were identified by 1.5% agarose gel electrophoresis and combined for histone exchange assays. (D) Purification of recombinant factors. FLAG-Spt16, His-SSRP1, FLAG-PARP1, and His-Ku70/Ku86 were expressed in Sf9 cells and purified as described (Li et al., 2004; Orphanides et al., 1999). His-NAP1 and FLAG-Tip60 were expressed in bacteria and purified. DNA-PK was purified from HeLa cells. Lane 1, protein molecular weight markers. (E) In vitro interaction of H2AX with its associated factors. Recombinant Spt16, SSRP1, PARP1, DNA-PK, Ku70/86, and Tip60 were mixed with H2AX and immunoprecipitated with anti-H2AX antibody. Bound proteins were detected by western blot analysis with the indicated antibodies (right panel). Lane 1, 10% of input proteins. Molecular Cell 2008 30, 86-97DOI: (10.1016/j.molcel.2008.02.029) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 3 H2AX-H2B Dimer Exchange by FACT and H2AX-Associated Factors (A) H2AX-H2B dimer integration by FACT in vitro. H2A nucleosomes (120 ng) were immobilized on magnetic beads and incubated with FACT (1 μg) or H2AX-associated factors in the presence of H2AX-H2B dimer. H2AX-H2B dimer integration was detected by western analysis of the immobilized nucleosomes with H2AX antibody after 30 and 60 min reactions. Histone H3 was used as loading control. (B) H2AX-H2B dimer dissociation by FACT in vitro. The assays were identical to Figure 3A, except that H2AX nucleosomes were used and free H2AX released from nucleosomes was analyzed. (C) Comparison of FACT and NAP1 activities. H2A nucleosomes were incubated with FACT or NAP1 in the presence of H2AX-H2B dimer (first panel), f:H2A-H2B dimer (second panel), or h:H2AZ-H2B dimer (third panel). The dimer integrations were analyzed by western blot of the immobilized nucleosomes with anti-H2AX, anti-FLAG (for H2A), and anti-H2AZ antibodies after 60 min exchange reactions. (D) Inhibition of H2AX-H2B and H2A-H2B dimer integration by Spt16 knockdown. 293T cells were transfected with Spt16 shRNA or nonspecific control shRNA by two consecutive transfections. Concentration of Spt16 present in cell lysates derived from these cells was checked by western blot analysis with anti-Spt16 antibody (first panel). Changes in nucleosomal integration of FLAG-H2AX, FLAG-H2A, and FLAG-H2AZ were monitored by western blot analysis of purified chromatin with anti-FLAG antibody (second panel). Histone H3 was used as loading control (third panel). Molecular Cell 2008 30, 86-97DOI: (10.1016/j.molcel.2008.02.029) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 4 Effect of H2AX Phosphorylation on H2AX-H2B Dimer Dissociation (A) Phosphorylation of recombinant H2AX. His-H2AX (200 ng), H2AX octamer (800 ng), and H2AX nucleosome (800 ng) were phosphorylated by purified DNA-PK or H2AX-associated factors in the presence of ATP (10 mM). Phosphorylation was detected by western blotting with γH2AX antibody. H2AX was detected by H2AX antibody as loading control. (B) Effect of H2AX phosphorylation on H2AX dissociation in vitro. The exchange reactions were identical to Figure 3B, except that H2AX nucleosomes were phosphorylated by DNA-PK or H2AX-associated factors. The reactions were analyzed by western blot analysis with anti-H2AX antibody, and relative intensity of bands was quantified by Phosphorimager (Bio-Rad). Lane 1, 50% (60 ng) of input H2AX nucleosomes. (C) In vitro interaction of H2AX with Spt16 and SSRP1. The interaction assays were as described in Figure 2E, but using both unmodified and prephosphorylated H2AX proteins as described in the Experimental Procedures. Lane 1, 5% of input Spt16/SSRP1. (D) Isolation of wild-type or mutant H2AX mononucleosomes. Mononucleosomes were prepared from 293T cells transfected with wild-type and mutant FLAG-H2AX expression vectors (lanes 4–7) or empty vector control (lanes 2 and 3) by MNase digestion of chromatin. Nucleosomal DNA was extracted and analyzed by 2% agarose gel electrophoresis (lanes 2–7). FLAG-H2AX nucleosomes were immunoprecipitated from total nucleosomes using M2 beads, and the amount of purified nucleosomes was normalized by 15% SDS-gel electrophoresis of inner histone octamers (lanes 8–13). To trigger the DNA damage response, cells were treated with 100 μM of etoposide for 2 hr. Lane 1, 100 bp DNA ladder (M). (E) In vivo interaction of Spt16 with nucleosomal H2AX. H2AX mononucleosomes were purified from H2AX-expressing cells (lanes 3–6) or control cells (lanes 1 and 2) as described in Figure 4D. Nucleosome-bound Spt16 and H2AX phosphorylation were detected by western analysis using Spt16 and γH2AX antibodies as indicated. Concentration of FLAG-H2AX was also measured to confirm that equal amount of nucleosome was used in all analyses. Molecular Cell 2008 30, 86-97DOI: (10.1016/j.molcel.2008.02.029) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 5 Structural Alteration of the Nucleosome by H2AX Phosphorylation (A) Phosphorylation effect on restriction enzyme digestion. H2AX nucleosomes (120 ng each) were reconstituted on biotinylated 207 bp 601 nucleosome positioning sequence and immobilized on magnetic beads. After phosphorylation by DNA-PK, nucleosomes were digested with BanI (lanes 2–6) and BsiWI (lanes 8–12) for 2 hr (lanes 3, 4, 9, and 10) and 4 hr (lanes 5, 6, 11, and 12). After washing and Proteinase K digestion, DNA fragments released from beads were ethanol precipitated and analyzed by 2.5% agarose gel electrophoresis. Lanes 1 and 7, uncut DNA; lanes 2 and 8, free DNA digested with BanI and BsiWI. (B) M.SssI accessibility analysis of H2AX nucleosome. H2AX nucleosomes were reconstituted and phosphorylated as in Figure 5A and treated with M.SssI to methylate accessible CpG sites present in nucleosomal DNA (left panel). Naked DNA and unmodified H2AX nucleosomes were included as controls. Methylated DNA was treated with sodium bisulfite and sequenced to determine DNA methylation at CpG sites. Empty circles represent unmethylated (inaccessible) CpG dinucleotides, whereas filled circles represent methylated (accessible) CpG dinucleotides. Percentage of methylated CpG dinucleotides was calculated in right panel. Molecular Cell 2008 30, 86-97DOI: (10.1016/j.molcel.2008.02.029) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 6 Regulatory Effects of PARP1-Mediated ADP-Ribosylation of Spt16 in H2AX Exchange (A) In vitro ribosylation of Spt16 by PARP1. The reactions contained either recombinant Spt16 (0.5 μg) and PARP1 (0.5 μg) (lanes 1 and 2) or H2AX-associated factors (lanes 3 and 4) in the absence (lanes 1 and 3) or presence (lanes 2 and 4) of NAD+. The ADP-ribosylation of Spt16 was detected by immunoblot analysis using poly-ribose antibody. (B) Repression of FACT-dependent H2AX integration by ADP-ribosylation of Spt16. The exchange assays were identical to Figure 3A, except that recombinant Spt16 and Spt16 present in purified H2AX-associated factors were premodified by recombinant PARP1 or PARP1 in the purified factors, respectively. (C) Repression of FACT-dependent H2AX dissociation by ADP-ribosylation of Spt16. The exchange assays were the same as in Figure 6B, but with H2AX nucleosome and free H2A-H2B dimer. (D) Purification of ectopic H2AX nucleosome. Mononucleosomes containing FLAG-H2AX were purified from 293T cells with or without DNA damage as described in Figure 4D, but after cells were transfected with the shRNA vectors that express either the PARP1 shRNA (lanes 4, 5, 8, and 9) or the negative control shRNA (lanes 2, 3, 6, and 7). Purified mononucleosomes were analyzed by 2% agarose gel (lanes 2–5) and 15% SDS-gel electrophoresis (lanes 6–9). Lane 1, 100 bp DNA ladder (M). (E) Repressive effects of PARP1-mediated poly-ADP-ribosylation of Spt16. H2AX nucleosomes were purified as described in Figure 6D, and nucleosomal binding of Spt16 was examined by western blot analysis using anti-Spt16 antibody (α-Spt16). Immunoblots documenting the reduction in PARP-1 levels after transfection with the PARP1 shRNA vector are also shown (α-PARP1). β-actin expression levels were monitored as an internal control (α-actin), and equivalent gel loading of H2AX nucleosomes was confirmed by western blotting with anti-FLAG antibody (α-FLAG). Western blotting also detected H2AX phosphorylation in response to DNA damage (α-γH2AX). Molecular Cell 2008 30, 86-97DOI: (10.1016/j.molcel.2008.02.029) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 7 Summary of Regulatory Mechanisms for H2AX Exchange FACT facilitates both H2AX incorporation into and H2AX dissociation from the nucleosome. DNA-PK-mediated phosphorylation of nucleosomal H2AX facilitates FACT-induced dissociation of H2AX via structural destabilization of the nucleosome. In contrast, PARP1-mediated ADP-ribosylation of Spt16 induces dissociation of FACT from the nucleosome, resulting in stabilization of nucleosomal H2AX. Therefore, it is possible that ADP-ribosylation of Spt16 and phosphorylation of nucleosomal H2AX function as competing regulatory factors for proper regulation of FACT-induced H2AX exchange during DNA repair process. Molecular Cell 2008 30, 86-97DOI: (10.1016/j.molcel.2008.02.029) Copyright © 2008 Elsevier Inc. Terms and Conditions