Background: ALC1 is a macrodomain-containing SNF2-like ATPase activated to catalyze ATP-dependent nucleosome remodeling by PARP1 and its substrate NAD ϩ .

The human ALC1 (Amplified in Liver Cancer 1) gene was originally identified as a gene present on a human chromosome 1q21 region amplified in a large fraction of hepatocellular carcinomas. Subsequent studies found that overexpression of the ALC1 protein was found to be oncogenic in human cells and mice, although the mechanism of action of ALC1 in oncogenesis is not known. The human ALC1 gene encodes a member of the SNF2 superfamily of ATPases. The ALC1 protein is unique among SNF2 family members because it includes a macrodomain that is capable of binding selectively to poly(ADP-ribose) (PAR). We and others have also previously demonstrated that ALC1 possesses DNA-dependent ATPase and ATP-dependent nucleosome remodeling activities that are strongly dependent on the presence of poly(ADP-ribose) polymerase PARP1 (or the closely related PARP2) and NAD+ within in vitro assays. Our preliminary investigation of the mechanism of PARP1- and NAD+-dependent activation of ALC1 DNA-dependent ATPase and nucleosome remodeling activities revealed (i) that both the ALC1 SNF2 ATPase domain and an intact macrodomain capable of binding PAR are essential for activation by PARP1 and NAD+, (ii) that activation of ALC1 in the presence of PARP1 and NAD+ results in increased affinity of ALC1 for DNA and nucleosomes, and (iii) that autoPARylated PARP1 is most likely the source of PAR required for activation of ALC1. These preliminary observations provided some insight into basic features of the function of the ALC1 protein, but they left unresolved major questions concerning the mechanism by which ALC1 is activated by PARP1 and NAD+ to remodel nucleosomes. In our recent studies, we extend our investigation of the mechanism by which ALC1 is “activated” to remodel nucleosomes by PARP1 and NAD+. Our findings argue that (i) ALC1 activation requires assembly of a stable ALC1.autoPARylated PARP1.nucleosome intermediate, (ii) that ALC1 regulates association of autoPARylated PARP1 with this intermediate, and (iii) stable binding of the ALC1 macrodomain to autoPARylated PARP1 is critical for ALC1 activation. Taken together, our findings are consistent with the model that a stable ALC1.autoPARylated PARP1 heterodimer is the active species in ALC1-dependent ATPase and nucleosome remodeling. These findings provide further important insight into the mechanism of action of this poorly understood oncogene. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-373. doi:1538-7445.AM2012-LB-373

Regulation of ALC1 (Amplified in Liver Cancer) involves interplay between the SNF2 ATPase domain, PAR binding macrodomain and other conserved elements Rushi D. Trivedi*1,2, Aaron J. Gottschalk1, Joan W. Conaway 1,2, Ronald C. Conaway 1,2 1Stowers Institute for Medical Research, Kansas City, MO; 2Department of Biochemistry, University of Kansas Medical Center, Kansas City, KS. ALC1, also known as CHD1L, was originally identified as a gene present on a human chromosome 1q21 region amplified in ~50% of human hepatocellular carcinomas. ALC1, a member of the SNF2 family of ATPases, has an N‐terminal SNF2‐like ATPase that is most closely related to that of ISWI, and a C‐terminal macrodomain that binds selectively to poly(ADP‐ribose) (PAR). Between the ATPase and the macrodomain is an evolutionarily conserved region with no clear homology to any known domains. This “linker” region can be further divided into three sub‐regions of greatest conservation. We have previously shown that wild type ALC1 possesses DNA‐dependent ATPase and ATP‐dependent nucleosome remodeling activities that are strongly activated by nucleosome bound PARylated PARP1. A point mutation in the ALC1 macrodomain that interferes with PAR binding prevents PARP1‐ and NAD‐dependent ALC1 activation. Our current line of studies are aimed at investigating the interplay between ALC1 ATPase and macrodomain in the context of nucleosome remodeling. Preliminary results suggest that the SNF2 ATPase of ALC1 is inherently inactive but is activated upon interaction with PARylated PARP. Upon performing an extensive mutagenesis study of the conserved regions within the “linker”, we have identified potential regulatory elements and a putative DNA binding domain that could facilitate the communication between the two domains. Furthermore, we are analyzing the interplay between the SNF2 ATPase, macrodomain, and “linker” by characterizing the activities of chimeric ALC1 constructs with macrodomains exchanged from different proteins. Grant Funding Source: Stowers Institute for Medical Research