Thesis (Ph. D.)--University of Virginia, 2008. / Title from title page. Includes bibliographical references. Also available online through Digital Dissertations.

Thesis (Ph.D. in Biochemistry & Molecular Genetics) -- University of Colorado at Denver and Health Sciences Center, 2006. / Typescript. Includes bibliographical references (leaves 169-185). Free to UCD Anschutz Medical Campus. Online version available via ProQuest Digital Dissertations;

Thesis (Ph. D.)--University of Alabama at Birmingham, 2008. / Title from first page of PDF file (viewed Feb. 13, 2009). Includes bibliographical references (p. 73-93).

Dissertation (Ph.D.)--University of Toledo, 2010. / "Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Biomedical Sciences." Title from title page of PDF document. "A Dissertation entitled"--at head of title. Bibliography: p. 63-71, 126-140.

Thesis (Ph. D.)--Case Western Reserve University, 2006. / [School of Medicine] Department of Pathology. Includes bibliographical references. Available online via OhioLINK's ETD Center.

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.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Thesis (Ph.D. in Biochemistry) -- University of Colorado, 2006. / Typescript. Includes bibliographical references (leaves 153-171). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;

Chromatin remodeling is crucial to transcriptional regulation in vivo and a number of protein complexes capable of altering genomic architecture in the budding yeast Saccaromyces cerevisiaehave been identified. Among these, the SWI/SNF complex, a 2 MDa, eleven subunit protein assembly, has been the most extensively characterized. The SWI/SNF complex is required for the proper expression of a number of genes in yeast, although it is completely dispensable for the expression of others. Likewise, some, but not all, transcriptional activator proteins require SWI/SNF activity in order to function in vivo. The goal of this thesis work was to identify those components of the transcription process which dictate this dependence on SWI/SNF activity. Using the well characterized UASGALsystem, we have determined that one of these components is the nucleosome state of activator binding sites within a promoter. We find that while SWI/SNF activity is not required for the GAL4 activator to bind to and activate transcription from nucleosome-free binding sites, the complex is required for GAL4 to bind and function at low affinity, nucleosomal binding sites in vivo. The SWI/SNF -dependence of these nucleosomal binding sites can be overcome by 1) replacing the low affinity sites with higher affinity, consensus GAL4 binding sequences, or 2) placing the low affinity sites into a nucleosme-free region. These results provide the first in vivo evidence that the SWI/SNF complex can regulate gene expression by modulating the DNA binding of a transcriptional activator protein. To determine whether specific components of the GAL4 protein are necessary in order for the SWI/SNF complex to modulate binding to nucleosomal sites in our model system, we tested the SWI/SNF-dependent DNA binding of various derivative GAL4 proteins. We find that a functional activation domain is not required for SWI/SNF to modulate GAL4 binding in vivo. Interestingly, like the full length protein, GAL4 derivatives in which the activation domain has been mutated are able to partially occupy nucleosomal sites in the absence of SWI/SNF (binding in the absence of SWI/SNF is at least forty percent lower than in the presence of SWI/SNF), indicating the activation domain is also not required for SWI/SNF-independent DNA binding. These results support a model in which the SWI/SNF-dependence of a gene reflects the nucleosomal context of its important regulatory sequences, e.g. binding sites for transcriptional regulatory proteins. Although nucleosomal promoter regions have been correlated with SWI/SNF-dependence in the past, there has of yet been no gene at which nucleosome location has correlated with a specific genetic function. In the final part of this thesis work, we initiated a search for an endogenous SWI/SNF-dependent gene for which the nucleosome state of activator binding sites could be determined.

Chromatin structure is intricately involved in the mechanisms of eukaryotic gene regulation. In general, the compact nature of chromatin blocks DNA accessibility such that components of the transcriptional machinery are unable to access regulatory sequences and gene activation is repressed. These repressive effects can be overcome or augmented by the actions of chromatin remodeling enzymes. Numerous studies highlight two classes of these enzymes: those that covalently modify nucleosomal histones and those that utilize energy derived from ATP hydrolysis to destabilize the histone-DNA contacts within the nucleosome (13, 14, 92). Members of each of these groups of chromatin remodeling enzymes play pivotal roles in modulating chromatin structure and in facilitating or blocking the binding of transcription factors. Mutations in genes encoding these enzymes can result in transcriptional deregulation and improper protein expression. Therefore, the regulation of chromatin structure is critical for precise regulation of almost all aspects of gene expression. Consequently, enzymes regulating chromatin structure are important modulators of cellular processes such as cell viability, growth, and differentiation. There remain many uncharacterized members of the ATP-dependent class of remodeling enzymes; characterization of these proteins will further elucidate the cellular functions these enzymes control. Here, we focus primarily on the ATP-dependent remodeling complexes, specifically the chromodomain helicase DNA-binding (CHD) family. The CHD proteins are distinguished from other ATP-dependent complexes by the presence of two N-terminal chromodomains that function as interaction surfaces for a variety of chromatin components. These proteins also contain a SNF2-like ATPase motif and are further classified based on the presence or absence of additional domains. Genetic, biochemical, and structural studies demonstrate that CHD proteins are important regulators of transcription and play critical roles during developmental processes. Numerous CHD proteins have also been implicated in human disease. The first CHD family member, mChd1, was identified in 1993 in a search for DNA-binding proteins with an affinity for immunoglobin promoters. Since then, additional CHD genes have been identified based on sequence and structural homology to mChd1. Despite an increase in the number of studies relating to CHD proteins, the function of most remains unknown or poorly characterized. Using embryonic stem (ES) cells containing an insertional mutation in the murine Chd2 locus, we generated a Chd2-mutant mouse model to address the biological effects of Chd2 in development and disease. The targeted Chd2 allele resulted in a stable Chd2-βgeo fusion protein that contained the tandem chromodomains, the SNF2-like ATPase motif, but lacked the C-terminal portion of the DNA-binding domain. We demonstrated that the mutation in Chd2 resulted in a general growth delay in homozygous mutants late in embryogenesis as well as perinatal lethality. Similarly, heterozygous mice showed a decreased neonatal viability. Moreover, the surviving heterozygous mice showed a general growth delay during the neonatal period and increased susceptibility to non-neoplastic lesions affecting multiple organs, most notably the kidneys. We further examined the connection between Chd2 and kidney disease in this murine model. Our findings revealed that the kidney phenotype observed in Chd2 mutant mice led to the development of membranous glomerulopathy, proteinuria, and ultimately to impaired kidney function. Additionally, serum analysis revealed decreased hematocrit levels in the Chd2-mutant mice, suggesting that the membranous glomerulopathy observed in these mice is associated with anemia. Lastly, we investigated whether the type of anemia observed in the Chd2-mutant mice. Red blood cell (RBC) indices and morphological examination of the RBCs indicated that the anemia seen in the Chd2-mutant mice can be classified as normocytic and normochromic. Further analyses have been initiated to determine if the anemia is due to an intrinsic effect in erythropoiesis or a secondary consequence of the glomerular disease. In summary, our findings have contributed to our understanding of the putative chromatin remodeling enzyme Chd2. Although much remains to be studied, these findings demonstrate a role for Chd2 in mammalian development and have revealed a link between Chd2 and disease.