Poly(ADP-ribosyl)ation directs recruitment and activation of an ATP-dependent chromatin remodeler

Abstract

Posttranslational modifications play a key role in recruiting chromatin remodeling and modifying enzymes to specific regions of chromosomes to modulate chromatin structure. Alc1 (amplified in liver cancer 1), a member of the SNF2 ATPase superfamily with a carboxy-terminal macrodomain, is encoded by an oncogene implicated in the pathogenesis of hepatocellular carcinoma. Here we show that Alc1 interacts transiently with chromatin-associated proteins, including histones and the poly(ADP-ribose) polymerase Parp1. Alc1 ATPase and chromatin remodeling activities are strongly activated by Parp1 and its substrate NAD and require an intact macrodomain capable of binding poly(ADP-ribose). Alc1 is rapidly recruited to nucleosomes in vitro and to chromatin in cells when Parp1 catalyzes PAR synthesis. We propose that poly(ADP-ribosyl)ation of chromatin-associated Parp1 serves as a mechanism for targeting a SNF2 family remodeler to chromatin.

Fig. 1.

Alc1 binds poly(ADP-ribose). (A) Alc1 domain structure. Sequences below diagram show amino acid changes in catalytically inactive Alc1 mutant E175Q and macrodomain mutant D723A. The mutated macrodomain region is compared to the homologous sequence from the AF1521 macrodomain. Snf2N, SNF2 family N-terminal domain; HelicC, Helicase superfamily C-terminal domain, associated with DEXDc-, DEAD-, and DEAH-box proteins; macro, macrodomain. (B) Approximately 100 ng of each protein was incubated with ³²P-labeled PAR in buffer with the indicated NaCl concentrations. PAR binding was detected with a nitrocellulose filter binding assay. (C) Approximately 100 ng (1×) or 200 ng (2×) wild-type or mutant Alc1 was incubated with PAR in buffer containing 0.15 M NaCl. PAR binding was measured as in panel B. F-Alc1, Flag epitope-tagged Alc1; rec, recombinant.

Fig. 2.

Alc1 has Parp1- and NAD-dependent ATPase. (A) ATPase assays performed with ≈100 ng wild-type or mutant recombinant (rec) F-Alc1 or F-Alc1 from HEK 293/FRT (293FRT) cells and 150 ng HeLa cell oligonucleosomes, with or without Parp1 and NAD. (B) ATPase assays performed with recombinant F-Alc1, with or without Parp1 and NAD, in the presence of DNA or an equimolar amount of mononucleosomes assembled on the same DNA with HeLa cell histones. (C) ATPase assays performed as in panel B with the indicated combinations of recombinant F-Alc1, Parp1, NAD, and oligonucleosomes.

Fig. 3.

Alc1 has Parp1- and NAD-dependent nucleosome remodeling and binding activities. (A) Schematic showing location of positioned nucleosome (nuc) and length of HhaI and XhoI cleavage products. Asterisk, ³²P-labeled DNA end. (B) DNA or nucleosomes reconstituted with HeLa cell histones were monitored for restriction enzyme accessibility after incubation with ATP (lanes 3–6) or ATPγS (lane 7) and Alc1, Parp1, and NAD as indicated. (C) DNA or nucleosomes reconstituted with recombinant histones were monitored for restriction enzyme accessibility after incubation with ATP (lanes 3, 5, 7–13) or ATPγS (lane 4) and wild-type or mutant Alc1, Parp1, NAD, or 2 mM benzamide. (D) Mononucleosomes reconstituted with HeLa cell histones on biotinylated DNA and immobilized on streptavidin beads were incubated for the indicated times with recombinant F-Alc1, with or without Parp1 and NAD. Bound fractions were analyzed by anti-Flag western blotting. (E) Whole cell lysates from HEK 293/FRT cells expressing wild-type or mutant F-Alc1 were immunoprecipitated with anti-FLAG (M2) agarose. Precipitated proteins were analyzed by western blotting.

Fig. 4.

Alc1 recruitment after microirradiation depends on its macrodomain and on PARP1 activity. Microirradiated HeLa cells were imaged for recruitment of EYFP-Alc1 wild-type or EYFP-Alc1Δmacrodomain (Δmacro) and PARP1-mCherry. (A) Recruitment of EYFP-Alc1 and PARP1-mCherry to site of microirradiation (between arrows). (B) Loss of Alc1's macrodomain abrogates PARylation-induced recruitment of Alc1 to chromatin. The background in Alc1 images is lower because the integration time of the CCD camera was lower to allow accurate quantitation of the recruitment kinetics. (C) Kinetics of recruitment (n ≥ 6) to microirradiated sites of wild-type (black) and D723A macrodomain mutant (blue) Alc1, or recruitment of wild-type Alc1 after Parp1 knockdown (red).