GSBS Dissertations and Theses

Approval Date

May 2007

Document Type

Doctoral Dissertation

Department

Graduate School of Biomedical Sciences, Interdisciplinary Graduate Program, Program in Molecular Medicine

Subjects

Chromatin Assembly and Disassembly; Chromosomal Proteins, Non-Histone; Transcription Factors; Academic Dissertations; Dissertations, UMMS

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 including cruciform formation, restriction enzyme accessibility in mononucleosomal and nucleosomal array substrates, and mononucleosome mobility shift. However, it is defective in ATP-dependent removal of H2A/H2B dimers. Moreover, we found that Swi3p and the N-terminal acidic domain of Swi3p strongly interact with GST-H2A and H2B but not GST-H3 or H4 tails. We purified a SWI/SNF mutant (SWI/SNF-Δ2N) that lacks 200 amino acids within the N-terminal acidic domain of Swi3p. Intriguingly, SWI/SNF-Δ2N failed to catalyze ATP-dependent dimer loss, although this mutant SWI/SNF contains all the subunits and has intact ATP-dependent activity in enhancing restriction enzyme accessibility. These data help to further understand the molecular mechanism of SWI/SNF, and show that H2A/H2B dimer loss is not an obligatory consequence of ATP-dependent DNA translocation, but requires the histone chaperone function of the Swi3p subunit. Based on these findings, we proposed a new model of the structural and functional organization of the SWI/SNF chromatin remodeling machinery: SWI/SNF contains at least four distinct modules that function at distinct stages of the chromatin remodeling process. 1) Swi1p and Snf5p modules directly interact with gene specific activators and function as the recruiter; 2) Swi2p/Arp7p/Arp9p generates energy from ATP hydrolysis and disrupts histone/DNA interactions; and 3) Swi3p/Swp73p/Snf6p may play dual roles by integrating each module into a large remodeling complex, as well as functioning as a histone H2A/H2B chaperone to remove dimers from remodeled nucleosomes.

Chapter IV is a perspective from current work in this project. I first discuss the interest in further characterizing the essential role of Snf6p, based on its activation of LacZ reporter on its own. Using in vitro translated protein and co-IP studies, I tried to pinpoint the requirement of the SANT domain for SWI/SNF assembly. I found that Swi3p directly interacts with Swp73p, but not with other subunits. When Swi3p is first incubated with Swp73p, Swi3p also interacts with Snf6p, indicating that Swi3p indirectly interacts with Snf6p, therefore forming a subcomplex of Swi3p/Swp73p/Snf6p. This subcomplex can also be reconstituted using in vitro co-translation. Consistent with the TAP preparation of this subcomplex, partial deletion of the SANT domain of Swi3p does not affect the assembly of Swi3p/Swp73p/Snf6p in vitro. However, the assembly of SWI/SNF complex was not detected in the presence of eight essential in vitro translated subunits or from co-translation of all the subunits. I have discussed the interest in further characterizing the histone chaperone role of the Swi3p N-terminal acidic domain and the role of other core subunits of SWI/SNF such as Snf6p for transcriptional regulation.

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