Multiple roles for PARP1 in ALC1-dependent nucleosome remodeling

The SNF2 family ATPase Amplified in Liver Cancer 1 (ALC1) is the only chromatin remodeling enzyme with a poly(ADP-ribose) (PAR) binding macrodomain. ALC1 functions together with poly(ADP-ribose) polymerase PARP1 to remodel nucleosomes. Activation of ALC1 cryptic ATPase activity and the subsequent nucleosome remodeling requires binding of its macrodomain to PAR chains synthesized by PARP1 and NAD⁺. A key question is whether PARP1 has a role(s) in ALC1-dependent nucleosome remodeling beyond simply synthesizing the PAR chains needed to activate the ALC1 ATPase. Here, we identify PARP1 separation-of-function mutants that activate ALC1 ATPase but do not support nucleosome remodeling by ALC1. Investigation of these mutants has revealed multiple functions for PARP1 in ALC1-dependent nucleosome remodeling and provides insights into its multifaceted role in chromatin remodeling.

The human ALC1 (Amplified in Liver Cancer 1) protein (also referred to as CHD1L or Chromodomain-Helicase-DNA-binding protein 1-Like) is a SNF2 family chromatin remodeling enzyme that functions together with the poly(ADP-ribose) polymerase PARP1 to catalyze ATP- and NAD⁺-dependent nucleosome remodeling. The ALC1 gene is amplified in a subset of hepatocellular carcinomas, and overexpression of the ALC1 protein leads to transformation of cultured cells and appearance of spontaneous tumors in mice (1, 2). Although the precise mechanism(s) by which ALC1 overexpression contributes to tumorigenesis remains unknown, ALC1 has been implicated in multiple DNA damage repair pathways (3–6). Several recent studies have shown that ALC1 overexpression confers resistance to PARP inhibitors used in treatment of DNA repair–deficient tumors, while loss or reduction of ALC1 expression renders cells exquisitely sensitive to these drugs (7–10). Hence, understanding the functional relationships between ALC1 and PARP1 is of considerable interest.We and others initially demonstrated that ALC1 has cryptic DNA-dependent ATPase and ATP-dependent nucleosome sliding activities that are strongly activated in the presence of PARP1 and NAD⁺ (3, 11), which PARP1 and other PARPs use as substrate for synthesis of poly(ADP-ribose) (PAR) (12). ALC1 is unique among SNF2 family members in containing a macrodomain. The macrodomain, located at the enzyme’s C terminus, binds PAR chains containing three or more ADP-ribose residues (3, 11, 13–15). ALC1 macrodomain mutations that abolish PAR binding block ALC1 ATPase and nucleosome remodeling, indicating that PAR binding by the macrodomain is important for ALC1 activation. Recent studies have led to a working model for how binding of PAR to ALC1 macrodomain contributes to nucleosome remodeling. According to this model, ALC1 SNF2 ATPase domain interacts with and is held in an inactive state by the macrodomain. Upon binding of PAR to the macrodomain, this interaction is released, leading to structural changes in the ALC1 ATPase domain that relieve autoinhibition (13, 16). In subsequent steps, nucleosome binding by ALC1 stabilizes the catalytically active conformation of the ATPase, and a linker region between the ATPase and macrodomains contacts an acidic patch on nucleosomes to couple ATP hydrolysis to nucleosome sliding (17).Although it is well established that one key role of PARP1 in ALC1-dependent nucleosome remodeling is to produce PAR, it is less clear whether it makes additional contributions. Recent findings indicating that free tri-ADP ribose is sufficient to activate ALC1 ATPase activity in the absence of PARP1 (13) suggest that the role of PARP1 might be limited merely to synthesizing PAR chains. In this case, PARP1 might act simply as a bystander in ALC1-dependent nucleosome remodeling. On the other hand, PARP1 possesses both nucleosome binding and histone chaperone activities (18). This, together with our previous evidence that ALC1 and PARP1 bind cooperatively to nucleosomes to form an ALC1–PARP1–nucleosome intermediate prior to remodeling (14), makes it tempting to speculate that PARP1 might play a more active role.In the course of experiments investigating the mechanism(s) by which PARP1 contributes to ALC1-dependent nucleosome remodeling, we identified PARP1 mutants capable of activating ALC1 ATPase, but defective in supporting ALC1-catalyzed nucleosome remodeling. By investigating the properties of these and additional PARP1 mutants, we show that both the PARP1 C-terminal ADP-ribosyl transferase domain and its N-terminal region, which contains nucleosome binding activity, play important roles in ALC1-dependent nucleosome remodeling. We report these findings, which bring to light a role for PARP1 in chromatin remodeling.