Accessory subunit of ISW2, Itc1 senses extranucleosomal DNA for regulating nucleosome remodeling

Abstract

AbstractATP‐dependent chromatin remodeling complexes utilize the energy from ATP hydrolysis to bring about changes in the chromatin structure and make DNA accessible to regulatory factors. One such ATP‐dependent chromatin remodeling enzyme from Saccharomyces cerevisiae is ISW2. ISW2, a four subunit complex, consists of Isw2, the catalytic subunit, Itc1, the largest accessory subunit and two histone fold subunits Dpb4 and Dls1. ISW2 complex is well characterized in terms of its function in vivo and in vitro, which makes it a model remodeling enzyme to study biological functions. This work aims at defining the role of Itc1 subunit in nucleosome remodeling by ISW2. Systematic C‐terminal truncations of Itc1 were created with FLAG epitope tag for affinity purification of native complexes. Nucleosome binding affinity was reduced with shortening of the C‐terminus indicating the role of Itc1 in stabilizing the interaction of ISW2 with nucleosomes. Remodeling activity of most of these mutants was unaffected except one which resulted in nucleosome positioning defect. This mutant ISW2 complex was named as DNP (Defect in Nucleosome Position) ISW2. The defect in nucleosome positioning is not due to the loss of complex integrity or change in stoichometry of subunits. Further characterization of DNP ISW2 by site‐directed mapping of nucleosome positions showed that the truncation of the C‐terminus of Itc1 resulted in loss of ability to sense extranucleosomal DNA to effectively remodel nucleosomes.This work was supported by Public Health Service grant GM 70864 from the National Institute of Health