REGULATION OF ALC1'S SNF2 ATPASE: INTERPLAY BETWEEN THE SNF2 DOMAIN, THE MACRODOMAIN AND OTHER CONSERVED ELEMENTS
Issue Date
2015-05-31Author
Trivedi, Rushi
Publisher
University of Kansas
Format
158 pages
Type
Dissertation
Degree Level
Ph.D.
Discipline
Biochemistry & Molecular Biology
Rights
Copyright held by the author.
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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 (HCC). ALC1 overexpressing cells form tumors in nude mice, and transgenic mice overexpressing ALC1 develop several types of spontaneous tumors. ALC1 has also been proposed as a novel candidate gene for congenital anomalies of the kidneys and urinary tract (CAKUT). 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. It was previously shown that wild type 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 its substrate NAD+. Importantly, a point mutation in the ALC1 macrodomain that interferes with PAR binding prevents PARP1- and NAD-dependent ALC1 activation. In this work, we dissected the mechanism by which PARP1 and NAD+ activate ALC1 nucleosome remodeling. We demonstrate that ALC1 activation depends on the formation of a stable ALC1·PARylated PARP1·nucleosome intermediate. In addition, by exploiting a novel PAR footprinting assay, we obtained evidence that the ALC1 macrodomain remains stably associated with PAR on autoPARylated PARP1 during the course of nucleosome remodeling reactions. Results of biochemical experiments described here argue (i) that stable binding of the ALC1 macrodomain to autoPARylated PARP1 is critical for ALC1 activation and (ii) that activation of ALC1 depends on formation of a stable ALC1-autoPARylated PARP1-nucleosome intermediate. In the course of this study, we also find that apart from being regulated by PARP and NAD+, ALC1 possesses and additional mode of intradomain control via conserved domains in the linker region and the macrodomain. First, we identified a region, NMAC (N-terminal to Macrodomain ATPase Coupling) domain needed to couple ATP hydrolysis to nucleosome remodeling. Deleting NMAC led to a robust PARP and NAD+-dependent ATPase, which lacked appreciable remodeling activity. In addition, we identified an additional mode of control via the macrodomain when we replaced ALC1's PAR binding macrodomain with another macrodomain from the variant histone macroH2A1.1 and discovered this chimeric protein was constitutively active independent of PARP and NAD+. Taken together our findings suggest a model of positive control of the SNF2 ATPase via the macrodomain and an additional level of control over its remodeling activities via the NMAC and other conserved linker elements.
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