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.

The regulation of protein function through oligomerization is a common theme in biological systems. In this work, I have focused on the effects of the oligomeric states of the human Rad52 protein on activities related to DNA binding. HsRad52, a member of the RAD52 epistasis group, is thought to play an important and as yet undefined role in homologous recombination. HsRad52 preferentially binds to ssDNA over dsDNA and stimulates HsRad51-mediated strand exchange (Benson et al., 1998). In either the presence or absence of DNA, HsRad52 has been observed to form both 10 nm ring-like structures as well as higher order oligomers consisting of multiple 10 nm rings (Van Dyck et al., 1998; Van Dyck et al., 1999). Earlier protein-protein interaction studies mapped the domain responsible for HsRad52 self-association in the N-terminus (residues 85-159) (Shen et al., 1996). Data presented here identifies a novel self-association domain in the C-terminus of HsRad52 that is responsible for the formation of higher order oligomers. VanDyck et al. observed DNA ending binding complexes consisting of multiple rings (Van Dyck et al., 1999). They proposed that these higher order oligomers may be functionally relevant. In this work, we demonstrate that DNA binding depends on neither ring shaped oligomers nor higher order oligomers but that activities of HsRad52 that require simultaneous interaction with more than one DNA molecule depend on the formation of higher order oligomers consisting of multiple HsRad52 rings. Early studies of HsRad52 proposed that the DNA binding domain resides in the highly conserved N-terminus of the protein (Park et al., 1996). A series of studies using truncation mutants of HsRad52 have provided evidence that supports this hypothesis. For example, we demonstrated that a truncation mutant containing only the first 85 residues of the protein is still able to bind DNA (Lloyd, submitted 2002). In this study, we demonstrate that aromatic (Y65, F79 and Y81) and hydrophobic (L43, I52 and I66) residues within the N-terminus contribute to DNA binding by either directly contacting the DNA or by stabilizing the structure of the protein. In summary, through the work presented in this dissertation, we have determined that the formation of 10 nm rings is mediated by a self-association domain in the N-terminus and that the formation of higher order oligomers consisting of multiple HsRad52 rings is mediated by an additional self-association domain in the C-terminus. We have correlated the oligomeric properties of HsRad52 with its biochemical functions related to DNA binding. Additionally, we have demonstrated that aromatic and hydrophobic residues contribute to DNA binding. Further studies will differentiate between the contribution of these residues to the DNA binding by stabilizing the overall structure of the protein versus making specific DNA contacts. Additional studies will also address how the oligomeric state of HsRad52 contributes to its role in HsRad51-mediated strand exchange.

The photon receptor, rhodopsin, and the GTP-binding regulatory protein, transducin, belong to a family of G protein-coupled receptors. The activation process through which guanine nucleotide exchange of the G protein is accomplished was investigated utilizing these components of the visual transduction system. Rhodopsin, modelled as an enzyme in its interaction with substrates, transducin and guanine nucleotides, was characterized to catalyze the G protein's activation by a double-displacement mechanism. Remarkable allosteric behavior was observed in these kinetic studies. Equilibrium binding studies were performed to investigate the molecular basis of the positive cooperative behavior between transducin and rhodopsin. These experiments show that the origins of the allosterism must arise from oligomeric assemblies between receptor and G protein. The determined Hill coefficient, nH = 2, suggests that at least two transducin molecules are involved, and the Bmax parameter a1so indicates that multimeric assemblies of rhodopsin may participate in the positive cooperative interactiions. Physical studies of transducin in solution were performed and do not indicate the existence of a dimeric structure, in contrast to the kinetic and binding experiments which analyze interactions at the membrane surface. Since the latter environment represents the native surroundings in vivo, aspects of the allosteric behavior must be considered for a complete understanding of the signal transduction mechanism. The reported findings are interpreted in the context of homologies between other G protein-coupled receptor systems in order to develop a model for the molecular basis of the mechanism and regulation of this mode of signal transduction.

Activation mechanism of the α-factor pheromone receptor of Saccharomyces cerevisiae was analyzed using biochemical and genetic techniques. An in vitro partial proteolysis assay was developed to determine the conformational change of the receptor that occurs upon binding of agonist. The activation specific cleavages were established by comparing cleavage products with antagonist versus agonist occupied receptor. Of the changes in peptide pattern that were revealed by trypsinization, the fragment resulting from the exposure of the third loop to the protease was found to be agonist specific and to be G-protein independent. A low-affinity binding receptor mutant was isolated which failed to undergo this agonist induced conformational change. Four intra-allelic suppressors of this receptor mutant were isolated and all were mapped to the ends of transmembrane helices 4, 5, 6 and 7; all were found to be replacements of non-polar residues by polar ones. The role of the suppressor mutations in conformational change was analyzed.

Cells as dynamic, interactive, and self contained units of life have a need for molecular motors that can create physical forces to move cargoes within the cell. Cytoplasmic dynein 1 is one such molecular motor that has many functions in the cell. The number and variety of functions that involve cytoplasmic dynein 1 suggest that there are a number of different binding sites on dynein for different proteins. Cytoplasmic dynein 1 is a multiprotein complex made up of six different subunit families. The many different combinations of subunits that could be used to make up a cytoplasmic dynein 1 holocomplex provides the variety of different binding sites for cargoes that can be individually regulated. The following chapters flush out how light intermediate chain 1 (LIC1), a subunit of cytoplasmic dynein 1, is involved with multiple dynein functions involving the binding of different cargoes to the cytoplasmic dynein 1 holocomplex, and how the binding of these cargoes can be regulated. First, LIC1 is found to be involved in the spindle assembly checkpoint. LIC1 appears to facilitate the removal of Mad1-Mad2, a complex important in producing a wait anaphase signal, from kinetochores. Second, the involvement of LIC1 in the spindle assembly checkpoint requires the phosphorylation of LIC1 at a putative Cdk1 phosphorylation site. This site is located in a domain of LIC1 that binds various proteins suggesting that this phosphorylation could also regulate these interactions. Third, LIC1 is involved in the centrosomal assembly of pericentrin, an important centrosomal protein. From the data presented herein, LIC1 is shaping up as a multifunctional cargo binder for cytoplasmic dynein 1 that requires regulation of its various cargoes.

Synthesis of histone proteins occurs largely during the S phase of the cell cycle and coincides with DNA replication to provide adequate amounts of histones necessary to properly package newly replicated DNA. Controlling transcription from cell cycle dependent and proliferation specific genes, including histone H4, is an important level of regulation in the overall governance of the cell growth process. Coordination of histone gene transcription results from the cumulative effects of cell signaling pathways, dynamic chromatin structure and multiple transcription factor interactions. The research of this dissertation focused on the characterization and identification of transcription factors interacting on the human histone H4 gene FO108. I also focused on the elucidation of regulatory elements within the histone coding region. Our results suggest a possible mechanism by which a transcription factor facilitates reorganization of histone gene chromatin structure. The histone promoter region between -418 nt and -215 nt, Site III, was previously identified as both a positive and negative cis-regulatory element for transcription. Results of in vitroanalyses presented in this dissertation identified multiple transcription factors interacting at Site III. These factors include H4UA-1/YY1, AP-2, AP-2 like factor and distal factor (NF-1 like factor). Transient transfection experiments show that Site III does not confer significant influence on transcription; however, there may exist a physiological role for Site III which would not be detected in these assay systems. We analyzed the histone H4 gene sequences for additional transcription factor binding motifs and identified several putative YY1 binding sites. Using electrophoretic mobility shift assays (EMSA), we found that Site IV, Site I and two elements within the histone H4 coding region are capable of interacting with YY1. In transient transfection experiments using reporter constructs containing either Site III or one of the coding region elements as potential promoter regulatory elements, and an expression vector encoding YY1, we observed levels of expression up to 2.7 fold higher than from the reporters lacking these elements. Therefore, YY1 appears to interact at multiple regulatory sites of the histone gene and can influence transcription through these elements. Prior to this study, the role of the coding region in histone gene expression was not known. To determine if the coding region is involved in regulating transcription, I constructed and tested a series of heterologous reporter constructs containing various sequences of the histone coding region. Results from these experiments demonstrated that the histone coding region contains three repressor elements. Extensive in vitro analysis indicated that the three repressor elements interact with the repressor CDP/cut. Further analysis showed that CDP/cut interactions with the repressor elements are cell cycle regulated and proliferation specific. CDP/cut interactions increase during the cell cycle when histone transcription decreases. These observations are consistent with the hypothesis that CDP/cutis a cell cycle regulated repressor factor which influences transcription of the histone H4 gene as such. The proximal promoter region of the histone H4 gene between -70 nt and +190 nt is devoid of normal nucleosome structure. This same region contains multiple CDP/cut binding sites. We hypothesized that CDP/cut is involved with chromatin remodeling of the histone gene. DNase I footprinting and EMSA results show purified recombinant CDP/cut interacts specifically with the histone promoter reconstituted into nucleosome cores. Thus, CDP/cutmay facilitate the organization of chromatin of the histone gene. In conclusion, the research presented in this dissertation supports the hypothesis that expression from the human histone H4 gene FO108 is regulated by multiple cis-regulatory elements which interact with several proteins. CDP/cut interacts with Site II, the three repressor elements in the histone coding region and at Distal Site I. YY1 interacts at Site IV, Site III, Site I, and twice in the coding region. ATF/CREB interacts with Site IV and Site I. Distal factor interacts with Site III and within the histone coding region. IRF 2 interacts with Site II and Distal Site I. Thus, histone gene expression is probably regulated by transcription factors CDP/cut, YY1, IRF 2 and ATF/CREB interacting with multiple regulatory elements dispersed throughout its promoter and the coding region. Cell cycle regulation of these transcription factors may contribute to cell cycle dependent expression of the histone gene.

The nervous system requires rapid, efficient, and accurate transmission between cells for proper functioning. Synapses are the predominant structures through which such vital communication occurs. How synapses are formed, maintained, and eliminated are questions of fundamental importance. At the nerve-muscle synapse, formation of the postsynaptic apparatus is directed by agrin. The hallmark activity of agrin is the aggregation of acetylcholine receptors (AChRs) into dense clusters opposite the presynaptic nerve terminal. Early events in the agrin signal transduction cascade include activation of the receptor tyrosine kinase MuSK and tyrosine phosphorylation of AChRs, but how these events lead to AChR cluster formation is unknown. Using the calcium buffer BAPTA, we demonstrate that intracellular calcium fluxes are necessary for agrin-induced formation of AChR clusters. However, clamping calcium fluxes before agrin stimulation does not alter agrin-induced phosphorylation of either MuSK or AChRs, indicating that this calcium-dependent step occurs downstream of both MuSK and AChR phosphorylation. These results identify a new step in the agrin signaling pathway required for the formation of AChR clusters. We show that intracellular calcium fluxes also play an important role in stabilizing AChR clusters. Clamping intracellular calcium fluxes results in rapid dispersal of AChR clusters and dephosphorylation of both MuSK and AChRs, even if agrin is continually present. Furthermore, the protein tyrosine phosphatase inhibitor pervanadate inhibits both the dispersal and dephosphorylation, indicating a role for a tyrosine phosphatase in AChR cluster dispersal. Our data indicate that AChR clusters are maintained by agrin/MuSK-induced intracellular calcium fluxes that tonically inhibit a tyrosine phosphatase localized to AChR clusters. Our findings also show that distinct molecular mechanisms mediate the formation and the dispersal of agrin-induced AChR clusters. The work presented here expands our understanding of synaptic differentiation in several ways. First, I characterized a new, calcium-dependent step required for the formation of agrin-induced AChR clusters. Next, I showed that postsynaptic specializations must be actively maintained, and describe a molecular mechanism that stabilizes AChR clusters. Finally, dispersal and formation of AChR clusters occurs by distinct pathways. Our understanding of the mechanisms regulating the formation and modulation of synapses will help us to better understand how the nervous system develops and responds to the world around us.

The homodimeric hemoglobin (HbI) from the blood clam Scapharca inaequivalvis binds oxygen cooperatively and thus offers a simple model system for studying communication between two chemically identical sites. Although the individual subunits of HbI have the same myoglobin-fold as mammalian hemoglobins, the quaternary assemblage is radically different. Upon oxygen binding by HbI, only small tertiary changes are seen at the subunit interface in contrast to the relatively large quaternary changes observed with mammalian hemoglobins. Analysis of structures of this hemoglobin in the liganded (02or CO) and unliganded states has provided a framework for understanding the role of individual amino acid side-chains in mediating cooperativity. The work presented in this dissertation has directly tested the central tenets of the proposed structural mechanism for cooperativity in HbI, illuminating the key roles played by residue Phe 97 and interface water molecules in intersubunit communication. Heterologous expression of Scapharca dimeric hemoglobin: A synthetic gene has been utilized to express recombinant RbI in Escherichia coli. The HbI apoprotein constitutes 5-10% of the total bacterial protein in this system. Addition of the heme precursor δ-aminolevulinic acid to the expression culture results in a ~3-fold increase in the production of soluble hemoglobin. Recombinant HbI has been successfully purified to homogeneity, resulting in a final yield of 80-100 mg of pure holoprotein from a 12 L expression culture. Analysis of recombinant HbI reveals its oxygen binding properties to be indistinguishable from native HbI. It was necessary to correct a protein sequence error by mutating residue Asn 56 to aspartate in order to obtain diffraction quality crystals, that are isomorphous to native HbI crystals. These recombinant HbI crystals diffract to high resolution, permitting the functional effects of mutant HbI proteins to be correlated with detailed structural analysis.

Proliferation and differentiation are essential processes for the growth and development of higher eukaryotic organisms. Regulation of gene expression is essential for control of cell division and differentiation. Normal eukaryotic cells have a limited proliferative capacity, and ultimately undergo cellular senescence and apoptosis. Terminal differentiation of cells is associated with loss of proliferative capacity and acquisition of specialized functions. Proliferation and differentiation are processes required for the creation and maintenance of diverse tissues both during embryonic development and postnatal life. The cell cycle is the process by which cells reproduce, and requires duplication and segregation of hereditary material. Loss of cell cycle control leads to genetic instability and cancer. Expression of replication-dependent histone genes is tightly coupled to DNA synthesis, thus making histone genes a good model for studying cell cycle regulation. The HiNF-D complex interacts with all five classes (H1, H2A, H2B, H3 and H4) of histone genes in a cell cycle-dependent manner. The CCAAT displacement protein (CDP)/Cux and the tumor suppressor pRB are key components of the HiNF-D complex. However, the molecular interactions that enable CDP/Cux and pRB to form a complex and thus convey cell growth regulatory information onto histone gene promoters are poorly understood. Transient transfection assays show that CDP/Cux represses the histone H4 promoter and that the pRB large pocket domain functions with CDP/Cux as a co-repressor. Direct interaction between CDP/Cux C-terminus and the pRB pocket domain was observed in GST pull-down assays. Furthermore, co-immunoprecipitation assays and immunofluorescence microscopy established that CDP/Cux and pRB form complexes in vivo and associate in situ. pRB interaction and co-repression with CDP/Cux is independent of pRB phosphosphorylation sites, as revealed by GST pull-down assays and transient transfection assays using a series of pRB mutant proteins. Thus, several converging lines of evidence indicate that complexes between CDP/Cux and pRB repress cell cycle-regulated histone gene promoters. CDP/Cux is regulated by phosphorylation and acetylation at the C-terminus, which contains two repressor domains and interacts with histone deacetylase HDAC1. In vivo function of the CDP/Cux C-terminus in development and gene regulation was assessed in genetically targeted mice (Cutl1tm2Ejn, referred to as Cutl1ΔC). The mice express a mutant CDP/Cux protein with a deletion of the C-terminus including the homeodomain. Indirect immunofluorescence microscopy showed that the mutant protein exhibited significantly reduced nuclear localization in comparison to the wildtype protein. Consistent with these data, DNA binding activity of HiNF-D was lost in nuclear extracts derived from mouse embryonic fibroblasts (MEFs) or adult tissues of homozygous mutant (Cutl1 ΔC -/-) mice, indicating the functional loss of CDP/Cux in the nucleus. No significant difference in growth characteristics or total histone H4 mRNA levels was observed between wildtype and Cutl1 ΔC -/- MEFs in culture. However, the histone H4.1 (murine FO108) gene containing CDP/Cux binding sites have reduced expression levels in homozygous mutant MEFs. Stringent control of growth and differentiation appears to be compromised in vivo. Homozygous mutant mice exhibit stunted growth (20-50% weight reduction), a high postnatal death rate of 60-70%, sparse abnormal coat hair and severely reduced fertility. Hair follicle deformities and severely diminished fertility in Cutl1 ΔC -/- mice suggest that CDP/Cux is required for normal development of dermal tissues and reproductive functions. Together the data presented in this dissertation provide new insight into the in vivo functions of CDP/Cux in the regulation of histone gene expression, growth control and differentiation.

An optimal antibody response to a thymus-dependent antigen requires cooperation between the B cell and an antigen-specific helper T cell. Major histocompatibility complex restriction of this interaction implies that the helper T cell recognizes antigen on the B cell surface in the context of MHC molecules, and that the antigen-specific B cell gets help by acting as an antigen presenting cell for the helper T cell. However, a number of studies have shown that normal resting B cells are ineffective as antigen presenting cells, implying that the B cell must leave the resting state before it can interact specifically with a helper T cell. On the contrary, other studies, including those using rabbit Ig as antigen, and rabbit globulin-specific mouse T cell lines and hybridomas, show that certain T cell lines can be efficiently stimulated by normal resting B cells. One possibility I considered was that small B cells are unable to process antigens, and that the rabbit Ig-specific T cell lines used above recognize native antigen on the B cell surface. In the majority of cases, experiments with B cell lines and macrophages have shown that antigen presentation requires antigen processing, a sequence of events which includes: internalization of antigen into an acid compartment, denaturation or digestion of antigen into fragments, and the return of processed antigen to the cell surface where it can then be recognized by the T cell in the context of class II molecules of the MHC. The experiments reported here show that the rabbit Ig-specific T cell lines do require an antigen processing step, and that small resting B cells, like other antigen presenting cells, process antigen before presenting it to T cells. Specifically, I show that an incubation of 2-8 hours is required after the antigen pulse before antigen presentation becomes resistant to fixation or irradiation. Shortly after the pulse, the antigen enters a pronase resistant compartment. Chloroquine, which raises the pH of endocytic vesicles, inhibits presentation. In addition, a large excess of antibody to native antigen fails to block presentation of antigen after a 2-8 hour incubation. Also, although membrane Ig, the antigen receptor on the B cell, is required for efficient presentation of antigen at low concentrations, antigen is no longer associated with the B cell receptor at the time of presentation to the T cell. Modulation of membrane Ig by anti-Ig blocks presentation before but not after the antigen pulse.