Modulation of nucleosome-binding activity of FACT by poly(ADP-ribosyl)ation - PubMed (original) (raw)

Modulation of nucleosome-binding activity of FACT by poly(ADP-ribosyl)ation

Jing-Yi Huang et al. Nucleic Acids Res. 2006.

Abstract

Chromatin-modifying factors play key roles in transcription, DNA replication and DNA repair. Post-translational modification of these proteins is largely responsible for regulating their activity. The FACT (facilitates chromatin transcription) complex, a heterodimer of hSpt16 and SSRP1, is a chromatin structure modulator whose involvement in transcription and DNA replication has been reported. Here we show that nucleosome binding activity of FACT complex is regulated by poly(ADP-ribosyl)ation. hSpt16, the large subunit of FACT, is poly(ADP-ribosyl)ated by poly(ADP-ribose) polymerase-1 (PARP-1) resulting from physical interaction between these two proteins. The level of hSpt16 poly(ADP-ribosyl)ation is elevated after genotoxic treatment and coincides with the activation of PARP-1. The enhanced hSpt16 poly(ADP-ribosyl)ation level correlates with the dissociation of FACT from chromatin in response to DNA damage. Our findings suggest that poly(ADP-ribosyl)ation of hSpt16 by PARP-1 play regulatory roles for FACT-mediated chromatin remodeling.

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Figures

Figure 1

hSpt16 is poly(ADP-ribosyl)ated in vivo. (A) Poly(ADP-ribosyl)ation status of endogenous FACT. HeLa cells were cultured in the absence or presence of 5 mM 3AB for 24 h. FACT heterodimer was immunoprecipitated from lysates and the status of poly(ADP-ribosyl)ation was determined using rabbit anti-poly(ADP-ribose) antibody (lanes 1–3). The same blot was probed sequentially with anti-FACT antibody (8D2 and 3E4, lanes 4–6). The levels of poly(ADP-ribose) (pADPr) of cell extracts were shown (lanes 7 and 8). The protein levels of hSpt16 and SSRP1 in lysates were also shown (lower panel, lanes 7 and 8). (B) FLAG-hSpt16 but not FLAG-SSRP1 is poly(ADP-ribosyl)ated in vivo. Whole cell extracts from 293T cells overexpressing FLAG-hSpt16 or FLAG-SSRP1 were immunoprecipitated with anti-FLAG M2 agarose followed by immunoblotting with monoclonal anti-poly(ADP-ribose) antibody (10H-2) (lanes 1–3). The same blot was reprobed with anti-FLAG antibody (lanes 4–6). The asterisk indicates bands that were recognized by 10H-2 antibody non-specifically.

Figure 2

Enhanced poly(ADP-ribosyl)ation of hSpt16 in response to DNA damage. (A) HeLa cells were mock exposed or exposed to 500 µM H2O2 in the presence or absence of PARP inhibitor 3AB as indicated. FACT immunoprecipitated from extracts derived from equal cell numbers by anti-hSpt16 antibody were analyzed by immunoblot analysis. Same blot was probed sequentially with rabbit anti-poly(ADP-ribose) antibody (lanes 1–7) and anti-FACT antibodies (8D2 and 3E4, lanes 8–14). Numbers on the left side indicate positions of protein molecular weight markers. Signals of poly(ADP-ribosyl)ated polypeptides (∼110 kDa) in lanes 1–3 should be poly(ADP-ribosyl)ated PARP-1. (B) HeLa cells were subjected to γ-irradiation (8 Gy) in the presence or absence of PARP inhibitor 3AB. FACT was immunoprecipitated with anti-SSRP1 antibody (3E4) from lysates prepared before or after irradiation at indicated time and the immunoprecipitates were analyzed by immunoblot analysis. Same blot was probed sequentially with anti-poly(ADP-ribose) antibody (lanes 1–9) and anti-FACT antibodies (lanes 10–18). The input lanes showed the loading of HeLa extracts from equal cells numbers (lanes 1–4 and 10–13). Numbers on the left side indicate positions of protein molecular weight markers.

Figure 3

Interaction between FACT and PARP-1. (A) Co-immunoprecipitation of FACT and PARP-1 from HeLa cells extracts. HeLa whole-cell extracts were immunoprecipitated with control (non-specific), hSpt16 or SSRP1 antibody. The immunoprecipitates were examined for the presence of PARP-1 (middle panel) or FACT heterodimer (upper and lower panels) by immunoblotting. (B) FACT interacts with PARP-1 via hSpt16. M2-agarose bound FLAG-hSpt16 (left panel) or Ni-resin bound His-SSRP1 (right panel) was incubated with different set of purified proteins as indicated. After unbound proteins were removed by washing, the immunoprecipitates were subjected to immunoblot analysis using the indicated antibodies. M2-agarose beads or Ni-resin were used as controls.

Figure 4

In vitro poly(ADP-ribosyl)ation of hSpt16 by PARP-1. (A) hSpt16 is an in vitro substrate of PARP-1. In vitro poly(ADP-ribosyl)ation reactions were performed using the indicated sets of recombinant proteins. Proteins were separated on SDS–PAGE followed by immunoblot. The same blot was probed sequentially with monoclonal anti-poly(ADP-ribose) (pADPr) antibody (upper panel) and anti-FACT antibodies (hSpt16: 8D2 and SSRP-1: 3E4, lower panel). The band indicated by asterisk represent FLAG-hSpt16 that had been poly(ADP-ribosyl)ated. Arrows indicate the position of SSRP-1. PARP-1 was activated by adding fragmented DNA and NAD+. (B) Poly(ADP-ribosyl)ation disrupts the interaction between hSpt16 and PARP-1. In vitro poly(ADP-ribosyl)ation was carried out as described in (A). PARP-1 was precipitated by rabbit antiserum and the immunoprecipitates were subjected to immunoblot analysis as indicated.

Figure 5

Poly(ADP-ribosyl)ation of hSpt16 reduced its binding to nucleosome. (A) Stimulation of PARP-1 activity by purified HeLa mononucleosomes. Auto poly(ADP-ribosyl)ation of PARP-1 were performed using DNA, mononucleosome, and core histones activators as indicated. Poly(ADP-ribose) polymers were detected by monoclonal antibody 10H-2 (upper panel). The input of core histone and mononucleosome was shown using anti-H3 antibody (lower panel). (B) hSpt16 could be poly(ADP-ribosyl)ated when using mononucleosome to activate PARP-1. In vitro poly(ADP-ribosyl)ation was carried out as described in Figure 4A except that mononucleosome (not DNA) was used to activate PARP-1. The same blot was probed sequentially with monoclonal anti-poly(ADP-ribose) antibody (lanes 1 and 2) and anti-hSpt16 antibodies (lanes 3 and 4). (C) M2 agarose bound FLAG-hSpt16 was incubated with PARP-1 and mononucleosome in the presence or absence of NAD+. After incubation for 30 min at 37°C, unbound proteins were removed by washing and bound proteins were subject to immunoblot analysis using antibodies as indicated by the left side of each panel. (D) Endogenous FACT was immobilized on protein G beads using anti-hSpt16 antibody. FACT was either pre-modified or not by PARP-1 as described in Figure 4A. Mononucleosome binding assay was performed as in (C).

Figure 6

FACT is released from chromatin after poly(ADP-ribosyl)ation. (A) Differential release of FACT from chromatin by increasing concentration of NaCl. Control or H2O2-treated HeLa cells were sequentially extracted with extraction buffer containing the indicated concentration of NaCl. The released proteins and the final pellet were subject to immunoblot analysis using anti-FACT antibodies (upper panel). Tubulin α was used as a marker of soluble fraction (lower panel). (B) Poly(ADP-ribosyl)ation status of hSpt16 extracted with different salt concentrations. HeLa cells were treated with 500 µM H2O2 for 20 min and sequentially extracted with extraction buffer containing the indicated concentrations of NaCl. hSpt16 precipitated from different fractions were subject to immunoblot analysis using monoclonal anti-poly(ADP-ribose) (pADPr) antibody (upper panel). The same blot was reprobed with anti-hSpt16 antibody (lower panel). (C) ChIP analysis of chromatin binding of FACT. The associations of FACT or histone H3 with γ-actin gene under different conditions were analyzed. HeLa cells were subjected to different treatment before crosslinking with formaldehyde. Crosslinked chromatin was immunoprecipitated with the indicated antibodies (on the left side of each panel). Input, 1% of input chromatin was amplified as control.

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Modulation of nucleosome-binding activity of FACT by poly(ADP-ribosyl)ation - PubMed (original) (raw)