The HAND and SANT Domains Regulate the In vitro and In vivo Activity of the ISW2 Chromatin Remodeling Complex

The catalytic subunit of ISW2 chromatin remodeling complex consists of the conserved DEXD/HELIC domain and HAND, SANT and SLIDE domain. The role of HAND and SANT domain were analyzed by specific deletion of the entire domain or mutation of key amino acid residues. HAND and SANT domain are found to be dispensable for ISW2 complex integrity and binding to nucleosomal substrate however are critical for efficient nucleosome remodeling. SANT domain was found to be required for efficient ATP hydrolysis and remodeling of equally spaced dinucleosomes. Deletion of HAND domain affected a subset Isw2 regulated genes; however, SANT domain affected a larger set of gene characteristic of a dominant negative phenotype. Further the role of these domains were investigated by mutating conserved ISWI specific, surface accessible basic amino acids, forming a basic path and predicted to interact with extranucleosomal DNA, to alanine. Multiple mutations in HAND or SANT domain did not affect complex assembly or binding but did affect the rate of nucleosome remodeling. In vivo de‐repression of transcription was observed at several Isw2 specific loci with either deletion or mutations in HAND or SANT domains. Consistently nucleosome position changes were observed upon domain deletion at STE6 promoter region indicating that conserved ISWI specific basic amino acid residues in these domains regulate ISW2 activity and gene expression through changes in nucleosome position. This work was supported by Public Health Service Grant GM70864