ACTIVATION OF ALC1 (CHD1L) NUCLEOSOME REMODELING BY PARP‐1‐DEPENDENT POLY(ADP‐RIBOSYL)ATION

ALC1, also known as CHD1L, is a macrodomain‐containing SNF2‐family ATPase that has been implicated in various cancers and has apparent roles in DNA repair. ALC1 displays robust ATP‐dependent nucleosome remodeling activity only in the presence of PARP‐1 and NAD+. However, the mechanism by which PARP‐1 stimulates ALC1 nucleosome remodeling activity through NAD+‐dependent poly(ADP‐ribosyl)ation (‘PARylation’) remains less well understood. PARP‐1 is a multi‐domain enzyme that catalyzes most protein PARylation in cells. PARP‐1 consists of an N‐terminal region that includes three zinc finger domains (Zn1, Zn2 and Zn3), a BRCA1 C‐terminal domain (BRCT), a tryptophan‐glycine‐arginine‐rich domain (WGR) and a C‐terminal catalytic domain (CAT) that is responsible for protein PARylation. Previous studies have shown that DNA or nucleosome binding by domains within the PARP‐1 N‐terminal region allosterically activates CAT for poly(ADP‐ribose) (‘PAR’) synthesis.To explore the functions of PARP‐1 domains in stimulating ALC1 nucleosome remodeling activity, we performed a structure‐function analysis of PARP‐1. As expected, the PARP‐1 CAT domain, which is essential for PAR synthesis, is also needed for ALC1‐dependent nucleosome remodeling. To determine whether CAT is sufficient to stimulate nucleosome remodeling by ALC1, we took advantage of a gain‐of‐function mutation in the PARP‐1 CAT domain that renders PARP‐1 PARylation activity independent of DNA binding. CAT with this gain‐of‐function mutation exhibits robust PARylation activity but is unable to activate ALC1 nucleosome remodeling activity, arguing that one or more PARP‐1 N‐terminal domains are needed in addition to CAT for maximal ALC1‐dependent nucleosome remodeling. In further experiments, we tested the abilities of PARP‐1 mutants lacking individual domains within the PARP‐1 N‐terminus to activate ALC1. Consistent with previous studies indicating that the Zn2 and BRCT domains of PARP‐1 are dispensable for protein PARylation, we found that these domains are also dispensable for ALC1 nucleosome remodeling activity. In addition, results of experiments performed with constitutively active PARP‐1 mutants lacking Zn1, Zn3 or WGR domains suggest that each of these domains contribute to ALC1‐dependent nucleosome remodeling. Studies exploring the mechanism(s) by which Zn1, Zn3, and WGR domains contribute to nucleosome remodeling are underway.