Date of Award

12-1-2011

Degree Name

Doctor of Philosophy

Department

Molecular Biology, Microbiology and Biochemistry

First Advisor

Bartholomew, Blaine

Abstract

SWI/SNF, the founding member of ATP dependent chromatin remodelers and its paralog RSC in yeast perform similar yet distinct functions inside the cell. In vitro these complexes use ATP dependent DNA translocation to either mobilize or disassemble nucleosomes. However, how these complexes interact with nucleosomes and the mechanism by which chromatin remodeling is achieved is not fully understood. Further, it is not understood how they perform disparate roles in vivo despite their similar biochemical activities. To understand the fundamental differences between these complexes the substrate specificity of RSC and SWI/SNF and their interaction with different parts of the nucleosome were investigated. SWI/SNF and RSC exhibited almost identical nucleosome binding affinities (~7 nm) with a minimal requirement of 20 bp of extranucleosomal DNA for efficient binding. Hydroxical-radical footprinting of RSC-nucleosome complex showed that RSC, unlike SWI/SNF, interacts extensively with approximately 50 bp of extranucleosomal DNA near the nucleosome entry site. RSC also interacts, but not as strongly as SWI/SNF, with almost one gyre of nucleosomal DNA (SHL-2 to SHL-6) on the same side of the extranucleosomal DNA. Analogous to the previously observed SWI/SNF-footprint the second gyre of nucleosomal DNA had no protection and in fact enhanced cleavage was seen starting from 3-4 helical turns from the dyad axis up to the exit site where DNA leaves the nucleosome. The asymmetry of the DNA footprint pattern confirmed binding of RSC in one preferred orientation guided by the extranucleosomal DNA at one end of the nucleosome like ISW2 and also like SWI/SNF but only when recruited by transcription factors. DNA crosslinking revealed that most of the SWI/SNF contacts are with a small region spanning the DNA translocation start site near SHL2 and does not extend to the rest of the footprint. Further, the SWI/SNF contacts are primarily through its catalytic subunit Snf2 which is found to intercalate between the DNA gyre and the histone octamer at SHL2. Consistent with its DNA footprint, RSC however makes extensive contacts with both nucleosomal and extranucleosomal DNA through five major subunits Sth1, Rsc2, Rsc3, Rsc30 and Rsc4. Excepting the catalytic subunit Sth1 which is highly homologous to Snf2, the remaining four are unique to RSC. Sth1 contacts a much broader region in the nucleosomal DNA than Snf2 with the primary contact being at SHL2 where it wedges between the DNA and the histone octamer surface. The accessory subunits Rsc2, Rsc3 and Rsc30 mostly contribute to the extranucleosomal DNA contacts of RSC. These subunits also make a second major contact near the dyad, with those made by Rsc3 and Rsc30 being the strongest. The histone N-terminal tails that emanate out of the nucleosome structure are implicated in the regulation of chromatin remodeling, in general, and in the activation of several SWI/SNF dependent genes, in particular. Remodeling kinetics studies with tailless nucleosomes revealed that the histone H4 tail is required for nucleosome mobilization, H2A/H2B dimer displacement and nucleosome disassembly by both RSC and SWI/SNF. Further, the H4 tail modulates RSC and SWI/SNF remodeling without affecting ATP hydrolysis or nucleosome binding. These data suggest a similarity between SWI/SNF and ISWI class of chromatin remodelers based on their dependence on the H4 tail. Owing to the presence of acetyl-lysine binding bromodomains in these complexes and to a greater extent in RSC the differences in their remodeling activities, if any, were expected to be accentuated by histone acetylation. Studies with H3 and H4 tail acetylated nucleosomes provided evidence for two pathways that work synergistically to recruit SWI/SNF and RSC to chromatin. While one of the pathways involves transcription activators, the other pathway of SWI/SNF recruitment is dependent on covalent acetylation of histone H3 tail. Bromodomain mediated recognition of these acetyl marks not only facilitates SWI/SNF recruitment but also stimulates their catalytic activity to mobilize nucleosomes. Importantly, extensive conformational changes occur in SWI/SNF in response to H3 tail acetylation. Chromatin remodeling by SWI/SNF and RSC is also regulated to different degrees by H3 tail acetylation depending on the number of bromodomains. The higher responsiveness of RSC to H3 tail acetylation than SWI/SNF can provide additional regulatory mechanisms for RSC which might ultimately account for their different functional roles inside the cell. When these same acetyl marks are within the H3 globular core and reside near the dyad axis of symmetry they are found to act in synergy with RSC and SWI/SNF to facilitate nucleosome movement as well as nucleosome disassembly. Unlike H3 tail acetylation, the remodeling enhancement by H3 core acetylation occurs via an acetyl lysine-bromodomain recognition independent mechanism. Further, supporting this recognition-independent mechanism H3 core acetylation does not affect the recruitment of these complexes. These data illustrate how histone acetylation modulates RSC and SWI/SNF function, and provide a mechanistic insight into their collaborative efforts to remodel chromatin.

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