MECHANISMS OF CHROMATIN REMODELING BY ISWI FAMILY OF REMODELERS: A FUNCTIONAL AND STRUCTURAL INSIGHT INTO THE ROLE OF THE Itc1 SUBUNIT OF ISW2 REMODELING COMPLEX

ISWI type remodelers mobilize and space nucleosomes. These ATP-dependent remodeling complexes have a relatively small number of subunits (2-4) as compared to other classes of remodelers such as SWI/SNF, RSC and INO80/SWR-C. The accessory subunits of some of the ISWI remodelers from yeast have been shown to contact extensively extranucleosomal or linker DNA and appear to be involved in regulating the movement of nucleosomes along DNA. In the ISW2 complex, the Itc1 (accessory) and Isw2 (catalytic) subunits make up the minimal active complex. ISW2 moves mononucleosomes to the center of DNA as a function of the length of extranucleosomal DNA. This same property is also responsible for the nucleosome spacing activity of ISW2 observed in nucleosomal arrays. The Itc1 subunit has been shown to contact the linker DNA starting at the entry site of the nucleosome and extending over at least 59 bp of linker DNA. The role of the Itc1 subunit in regulating the remodeling activity of the ISW2 complex was investigated by deleting different regions of Itc1 and monitoring the effects on complex assembly, ATPase activity and nucleosome mobilization activities of ISW2. A key finding was that a domain of 322 amino acids at the C-terminus of Itc1 was crucial for regulating nucleosome movement. Deletion of this domain causes ISW2 to move nucleosomes from one end to the other of DNA without pausing or stopping at a central position unlike wild type (WT) ISW2. The missing domain appears to be responsible for sensing linker DNA length to stall or stop remodeling when linker DNA is shortened to certain lengths. Loss of another region of 122 amino acids near the C-terminus was found to adversely affect the processivity of the ISW2 complex. The regions of Itc1 contacting the different parts of linker DNA were mapped by site-directed DNA cross-linking and peptide mapping. The mapping data along with molecular modeling provided an idea of the spatial arrangement of Itc1 with linker DNA like that previously obtained for the Isw2 subunit. The domain of Itc1 that interacts with Isw2 and is required for complex assembly was also identified. Next, we have used an approach of arresting nucleosome movement by placing DNA gaps that block translocation to study the changes in contacts of Itc1 with linker DNA upon ATP hydrolysis and remodeling. Results from such experiments highlighted massive conformational changes in both Itc1 and Isw2, which cause bending of the extranucleosomal DNA and assist to pump it inside the nucleosome for DNA translocation. ISW1 complexes have a common catalytic subunit but different accessory subunits. Considering the vital role that accessory subunits play in modulating the catalytic activities of remodelers, a comparative analysis of the remodeling properties of ISW1a, ISW1b and ISW2 complexes with various nucleosomal substrates was done. The analysis revealed significant differences in substrate specificities and translocation mechanisms among these three remodelers. The most intriguing observation was that ISW1a requires two sites on DNA to initiate translocation, unlike other known remodeling complexes. One of the sites is at SHL2, signature of all remodelers characterized to date, whereas the other unique site is 10 bp from the nucleosome edge on the extranucleosomal DNA. These mechanistic differences exhibited by different complexes of the same family underscore the importance of auxiliary subunits as regulators of enzyme function