ISW1 was initially identified as a single complex. Four years after its identification it was reported to form two separate complexes, namely ISW1a and ISW1b. ISW1a is composed of Isw1 and Ioc3; ISW1b consist of Isw1, Ioc2 and Ioc4. While these complexes share the same catalytic subunit, these complexes differ in their catalytic properties and function in vivo. ISW1a was shown to be localized at the promoter region of genes, functioning in transcription repression. True to this function, it exhibits directional remodeling activity and the ability to space nucleosome arrays at ~175bp increments, promoting the formation of a repressive chromatin structure. ISW1b lacks both of these biochemical properties and was found localized within the coding regions of genes functioning in the regulation of transcription elongation and termination. These differences in activity are attributed to the differences in subunit composition and the domain organization of these two complexes. Our work has identified and characterized novel domains within the Ioc2 and Ioc3 subunits that modulate the distinct biochemical properties of these complexes. We found that the IRD (ISW1a Regulator of Directionality) domain of the Ioc3 subunit, functions as a regulatory motif that dictates the directionality preferences and spacing activity exhibited by ISW1a during remodeling. Deletion of this domain resulted in an unregulated ISW1a complex in vivo which severely altered growth under stress conditions, gene expression and nucleosome organization genome-wide. Our work also characterized two domains within the Ioc2 component of the ISW1b complex, namely Ioc2 458-551 and a PHD domain. We find that these domains contribute significantly to the affinity of ISW1b for nucleosome substrates, as well as the ability of this complex to stimulate ATPase activity and remodel. Strikingly we find that the PHD domain functions as a distinguishing feature that differentiates ISW1a from ISW1b. Loss of this domain resulted in a mutant complex that remodels similarly to ISW1a. In vivo we also find that both of these domains contribute to nucleosome positioning and the modulation of gene expression of ISW1b regulated genes genome-wide. Overall this study provides an examination of the distinguishing features of the ISW1a and ISW1b complexes both in vitro and in vivo.

The dynamic nature of eukaryotic chromatin enables the packaging of large amounts of genetic material in a small space. At the same time, it provides controlled access to genomic DNA for a variety of nuclear processes for example, transcription and DNA repair. The transition between open and closed chromatin states is largely governed by ATP-dependent chromatin remodeling complexes, which operate on nucleosomes in concert, to modulate chromatin structure and composition. Exchange of the canonical and variant forms of histones in nucleosomes, and altering the spacing between consecutive nucleosomes, are two major ways which regulate chromatin-based processes and chromatin higher-order organization. The evolutionarily conserved INO80 and ISW1a complexes mediate these two aspects of nucleosome remodeling, respectively. Despite sharing conserved domain architecture of the core remodeling machinery, chromatin remodelers differ significantly in their modes of interaction with nucleosomes, and how they alter histone-DNA contacts. In this study, we have used a site-specific photocrosslinking approach coupled with peptide mapping to determine the interactions of subunits and domains of the S. cerevisiae INO80 and ISW1a complexes with nucleosomes. We find that specific interactions of remodelers with different regions of the nucleosome largely dictate their specialized functions and mechanisms. The ATP-dependent helicase-like (ATPase) domains of remodelers belonging to the ISWI and SWI/SNF families translocate along DNA close to the center of nucleosomes in order to mobilize, space or disassemble nucleosomes. In contrast, we observed that INO80 has a strikingly distinct mechanism, which is different even from its paralog SWR1. INO80 mobilizes nucleosomes as well as catalyzes the exchange of histone variant H2A.Z for the canonical histone H2A, while SWR1 mediates the reverse exchange of H2A for H2A.Z, without being able to mobilize nucleosomes. We have found that INO80, in order to promote H2A-H2B dimer exchange, translocates along DNA at the H2A-H2B interface close to the edge of nucleosomes and persistently displace DNA from H2A-H2B. Blocking either DNA translocation or the accumulation of DNA torsions close to the edge of the nucleosome interferes with this dimer exchange by INO80. SWR1 and other SWI/SNF and ISWI remodeling complexes translocate along DNA at the H3-H4 interface and do not persistently displace DNA from the histone octamer as does INO80. This study shows for the first time an ATP-dependent chromatin remodeler that invades nucleosomes at the DNA entry site instead of the center − a more logical approach for the displacement of H2A-H2B. We also investigated nucleosomal DNA interactions of other INO80 subunits and domains to understand the architecture of INO80 bound to nucleosomes. We found that the HSA (helicase-SANT-associated) domain of Ino80 along with actin-related protein (Arp) subunits Arp8 and Arp4 bind to the extranucleosomal DNA and is potentially involved in a coupling mechanism with the ATPase domain to regulate its activity. We also mapped the DNA binding regions of Arp8 and Arp4, which might be involved in recruiting INO80 to genomic sites. The ISWI remodeler ISW1a regulates the distance (spacing) between nucleosomes in an array by simultaneously interacting with two nucleosomes and directionally remodels one of them. We mapped DNA interactions of ISW1a subunits in mono- and di-nucleosomes. Our results show that the catalytic Isw1 subunit specifically interacts with the region of DNA translocation and DNA entry site of the asymmetrically positioned nucleosome in a di-nucleosome, which is preferentially mobilized. In contrast, the Ioc3 subunit interacts extensively with the linker DNA as well as the extranucleosomal DNA of the un-remodeled nucleosome. This bias in nucleosomal DNA interactions of ISW1a enables directional remodeling, which reveals the molecular basis of nucleosome spacing. We have identified a novel domain within the non-catalytic Ioc3 subunit of ISW1a that regulates nucleosome spacing. We found that when this domain is deleted, the catalytic Isw1 subunit loses its specificity and interacts with both the nucleosomes of a di-nucleosome substrate. This is consistent with the domain-deleted ISW1a mobilizing both nucleosomes efficiently, leading to the loss of its nucleosome spacing activity. In summary, this dissertation explores how different remodeling complexes have customized and regulated modes of nucleosome interaction in order to accomplish specialized remodeling outcomes. INO80 places its ATPase domain for translocation at the H2A-H2B dimer interface and persistently displaces DNA from its surface to promote H2A.Z exchange. Nucleosome spacing by ISW1a requires the catalytic Isw1 subunit to engage with and reposition one out of two consecutive nucleosomes in an array, while the Ioc3 subunit likely monitors the distance between them.