Date of Award

8-1-2017

Degree Name

Doctor of Philosophy

Department

Molecular Biology, Microbiology and Biochemistry

First Advisor

Bartholomew, Blaine

Abstract

Chromatin is highly regulated nucleoprotein complex facilitating the dynamic balance between genome packaging and accessibility. The central workhorses regulating the dynamic nature of chromatin are ATP-dependent chromatin remodelers- ISWI, SWI/SNF, INO80, and CHD/Mi2. All chromatin remodelers transduce the energy from ATP hydrolysis to translocate on DNA, break histone-DNA contacts, and mobilize nucleosomes. However, the final outcomes of nucleosome remodeling are diverse - nucleosome sliding, dimer exchange, nucleosome disassembly, and nucleosome conformation alteration. This study sheds light on how different chromatin remodelers catalyze various structural transformations. We provide novel insights into the nucleosome dynamics, the role of histone octamer dynamics on nucleosome remodeling by ISW2, mechanism of dimer exchange by INO80 and mechanism of nucleosome disassembly by the coordinated action of RSC and histone chaperone Nap1. We also provide insights on how aberrant SWI/SNF complexes affect fundamental enzymatic properties such as ATPase and processive nucleosome remodeling. ISW2 remodelers sense and respond to the length of linker DNA separating the nucleosome and centers nucleosome. Histone octamers are perceived as a mostly static structure whereas DNA deforms itself to fit nucleosome. We have found change in histone octamer conformation as a novel step in ISW2 mobilizing DNA through the nucleosome. We provide evidence for an induced fit mechanism where histone-histone and histone-DNA interactions change in respond to remodeler, and these changes promote DNA entry into the nucleosome. Our data supports a model in which DNA translocation causes the change in histone octamer conformation, followed by DNA entry into nucleosome and resetting of the histone octamer core. We also move a step ahead and show that SANT domain promotes the entry of DNA into nucleosome and resets the histone octamer core allowing processive nucleosome mobilization. INO80 nucleosome remodeling provides two outcomes- nucleosome centering and dimer exchange. INO80 exchanges H2A.Z-H2B dimer for H2A-H2B. We show that INO80 is incredibly slow at centering nucleosome compared to ISW2. We also provide evidence for a mechanism where INO80 persistently displaces DNA from the dimer interface, unlike ISW2, facilitating dimer exchange. In another instance, we show that kinetic step sizes are modulated by a combination of enzyme and DNA sequence properties, and are not hardwired into the enzyme. ISW2 has been previously shown to translocate DNA with a kinetic step sizes of ~7bp and ~3bp. We show that kinetic step sizes may vary depending on nucleosomal location where we monitor DNA movement. Next, we studied the mechanism of nucleosome disassembly by RSC in the presence of Nap1. We found that Nap1 promotes the disassembly of the distal nucleosome that RSC collides with rather than the proximal nucleosomes it mobilizes. SWI/SNF tops the list of the frequently mutated epigenetic factor in cancer with its subunits mutated in more than 20% of all cancers. Loss of hSnf5 is a driver mutation in pediatric rhabdoid tumors. Our lab has previously identified that the deletion of Snf5 causes yeast SWI/SNF to lose an entire module comprised of Snf5, Swp82, and Taf14. In this study, we establish the properties of aberrant SWI/SNF complex formed in the absence of Snf5 module. The deletion resulted in lower ATP hydrolysis and nucleosome mobilization activities of the mutant SWI/SNF. We found that Snf5 module is necessary to couple ATP hydrolysis with DNA translocation. We studied the role of accessory domain AT-hooks in the ATPase subunit of SWI/SNF and found similar results. Interestingly, AT hook and SnAC domains, and Snf5 subunit were found to communicate with the same region in ATPase domain physically. These studies provide valuable mechanistic insights into chromatin structure and function and highlight how different chromatin remodelers catalyze different chromatin remodeling outcomes. We also provide new insights on how the activity of the core ATPase motor is regulated either by accessory domains on the same subunit or by accessory subunits as a part of the larger complex.

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