Functional and Structural Dissection of the SWI/SNF Chromatin Remodeling Complex: A Dissertation

Abstract

The yeast SWI/SNF complex is the prototype of a subfamily of ATP-dependent chromatin remodeling complexes. It consists of eleven stoichiometric subunits including Swi2p/Snf2p, Swi1p, Snf5p, Swi3p, Swp82p, Swp73p, Arp7p, Arp9p, Snf6p, Snf11p, and Swp29p, with a molecular weight of 1.14 mega Daltons. Swi2p/Snf2p, the catalytic subunit of SWI/SNF, is evolutionally conserved from yeast to human cells. Genetic evidence suggests that SWI/SNF is required for the transcriptional regulation of a subset of genes, especially inducible genes. SWI/SNF can be recruited to target promotors by gene specific activators, and in some cases, SWI/SNF facilitates activator binding. Biochemical studies have demonstrated that purified SWI/SNF complex can hydrolyze ATP, and it can use the energy from ATP hydrolysis to generate superhelical torsion, mobilize mononucleosomes, enhance the accessibility of endonucleases to nucleosomal DNA, displace H2A/H2B dimers, induce dinucleosome and altosome formation, or evict nucleosomes. A human homolog of Swi2p/Snf2p, BRG1, is the catalytic subunit of the human SWI/SNF complex. Interestingly, isolated BRG1 alone is able to remodel a mononucleosome substrate. Importantly, mutations in mammalian SWI/SNF core subunits are implicated in tumorigenesis. Therefore, it remains interesting to characterize the role(s) of each subunit for SWI/SNF function. In this thesis project, I dissected SWI/SNF chromatin remodeling function by investigating the role of the SANT domain of the Swi3p subunit. Swi3p is one of the core components of SWI/SNF complex, and it contains an uncharacterized SANT domain that has been found in many chromatin regulatory proteins. Earlier studies suggested that the SANT domain of Ada2p may serve as the histone tail recognition module. For Swi3p, a small deletion of eleven amino acids from the SANT domain caused a growth phenotype similar to that of other swi/snf mutants. In chapter I, I have reviewed recent findings in the function of chromatin remodeling complexes and discuss the molecular mechanism of their action. In chapter II, I characterized the role of the SANT domain of Swi3p. I found that deletion of the SANT domain caused a defect in a genome-wide transcriptional profile, SWI/SNF recruitment, and more interestingly impairment of the SANT domain caused the dissociation of SWI/SNF into several subcomplexes: 1) Swi2p/Arp7p/Arp9p, 2) Swi3p/Swp73p/Snf6p, 3) Snf5p, and 4) Swi1p. Artificial tethering of SWI/SNF onto a LacZ reporter promoter failed to activate the reporter gene in the absence of the SANT domain, although Swi2p can be recruited to the LacZ promoter. We thus demonstrated that the Swi3p SANT domain is critical for Swi3p function and serves as a protein scaffold to integrate these subcomplexes into an intact SWI/SNF complex. In Chapter III, I first characterized the enzymatic activity of the subcomplexes, especially the minimal complex of Swi2p/Arp7p/Arp9p. We found that this minimal subcomplex is fully functional for chromatin remodeling in assays…