Apoptosis is characterized by a series of well defined morphological and biochemical features that allow cells to initiate self-destruction in response to a variety of stimuli. CD4⁺CD8⁺ is a sub-population of immature thymocytes that are especially prone to the action of apoptosis-inducing agents and are sensitive to glucocorticoid-induced apoptosis, an event that plays a critical role in eliciting the antigen-specific thymocyte repertoire. Glucocorticoids induce apoptosis through activation of the GR, a ligand-induced transcription factor that transduces the hormonal signals into the regulated expression of target genes. While much is known about the structure and function of GR, key steroid-regulated genes believed to be required for thymocyte apoptosis have not been found. Based on the transcriptional-regulation of apoptosis by ecdysone-mediated induction of reaper gene expression in Drosophila, and p53-mediated transcriptional-activation of Bax gene expression in mammalian cells, our laboratory has set out to identify GR-regulated genes in a glucocorticoid-sensitive mouse thymocyte cell line called WEHI 7.2. Identification of such genes involved in this process, and elucidation of the underlying mechanisms by which they mediate apoptosis, have prime importance because malfunctioning of this system has been shown to cause severe human diseases and developmental abnormalities. I have isolated and characterized the full-length nucleotide sequence of mGIG1, a novel gene product that had previously been shown to be up-regulated in thymocytes undergoing glucocorticoid-induced apoptosis. The human homolog of the mouse GIG1 was also identified and mapped to chromosome 8, band q21. A peptide segment of GIG1 protein was expressed in bacteria and used to raise antibodies in rabbits in order to dissect its role in thymocyte apoptosis. Results of DNA sequence analyses suggest that mGIG1 may encode a DNA-binding protein that could function as a transcription factor in thymocytes. In a second project, I established a transient expression cloning system in mouse thymoma cells and isolated a cDNA sequence called mArg-2 on the basis of a functional cloning strategy. However, determination of the possible involvement of both GIG1 and Arg-2 in thymocyte apoptosis will require further experimentation. Finally, spatial and temporal coordination of abrupt changes in mismatch repair (MMR) gene expression with occurrence of apoptosis in mouse thymocytes prompted me to study apoptosis in MMR-deficient mice. My findings indicate that glucocorticoid and anti-Fas antibody-induced cell death machinery is intact and functional in MMR⁻/⁻ mice.

To investigate the regulation of calpastatin gene expression, bovine heart calpastatin cDNAs and 5' regions of the calpastatin gene were isolated. Analysis of 5' cDNA sequence identified a new translation initiation site that is in frame and 204 nucleotides upstream of the previously designated start site. Conceptual translation from this upstream AUG produces a protein containing 68 additional N-terminal amino acids. This "XL" region contains three potential PKA phosphorylation sites but shares no homology with other regions of calpastatin or with any known protein. Immunoblot studies demonstrated that heart and liver contain a calpastatin protein of 145 kDa on SDS PAGE that comigrates with full length bacterially-expressed calpastatin and calpastatin produced by coupled in vitro transcription-translation from the upstream AUG. An antibody raised against the XL region recognized the 145 kDa band, demonstrating that the upstream AUG is utilized and that the 145 kDa band represents full length calpastatin protein in vivo. The organization of the calpastatin 5' genomic region was determined by comparing calpastatin cDNA and genomic sequences. The region encompassing exon 1-4 contains large introns and spans at least 60 kb. Calpastatin promoter sequence analysis revealed that it belong to the family of "house keeping" genes which lack TATA box and are GC rich at the proximal promoter regions. Transient transfection assays demonstrated that sequence within 272 nucleotides upstream of transcription initiation of the calpastatin gene is sufficient to direct moderate level transcription. Promoter sequences further upstream act to inhibit and stimulate transcriptional activity. Exposure of transfected cells to dibutryl cAMP resulted in a seven to twenty fold increase in calpastatin promoter activity for constructs containing at least 272 nucleotides of upstream promoter sequence. Deletion and mutation analyses identified a cAMP responsive element at nt-76. These findings demonstrate that calpastatin gene and protein are both targets for cAMP-dependent kinase activity. beta-Agonist treatment can induce both calpastatin gene transcription and protein phosphorylation.

Control of mRNA degradation is an important step in the regulation of gene expression. In Saccharomyces cerevisiae, pathways of mRNA decay have been determined and have provided a framework for understanding how mRNA decay is controlled. I have studied how the process of translation affects the decay mechanism of a yeast transcript and I have isolated and characterized yeast mutants that exhibit reduced rates of mRNA decay. The process of translation has been shown to affect mRNA decay rates in eukaryotes. However, using a MFA2 mRNA that cannot be translated due to insertion of secondary structure in its 5' untranslated region, I have determined that translation of the MFA2 mRNA is not required for its degradation. This observation demonstrates that translation of an mRNA, per se, is not required for the normal kinetics or mechanism of mRNA decay. Additionally, I have demonstrated that the translational inhibitor, cycloheximide, reduces the rate at which the MFA2 transcript is decapped. Inhibition of decapping occurs even on MFA2 transcripts that cannot be translated due to insertion of secondary structure. This result suggests that the general stabilizing effects of translational inhibitors on mRNAs may not be due to the inhibition of translation of these transcripts. The identification of mRNA decay pathways in yeast, deadenylation-dependent decapping and deadenylation-independent decapping, provided a basis by which gene products required for mRNA decay through these pathways could be identified. To this end, a screen of mutant yeast strains was undertaken. I have isolated and characterized two mutants, mrt1 and mrt3, that exhibit reduced rates of deadenylation-dependent decapping on several yeast transcripts. This result suggests that the MRT1 and MRT3 gene products promote deadenylation-dependent mRNA decapping. A third mutant, dcp1, was also isolated, and the wild-type DCP1 gene was identified. Characterization of dcp1/ mutants by myself and others revealed that the DCP1 gene encodes the decapping enzyme, or an essential component of the decapping enzyme, required for both deadenylation-dependent and deadenylation-independent mRNA decapping. This result demonstrates that the DCP1 gene product, Dcp1p, is required for all known mRNA decapping in yeast.

The process of mRNA turnover is a critical component of the regulation of gene expression. In the past few years, a discrete set of pathways for the degradation of polyadenylated mRNAs, in eukaryotic cells have been described. The major pathway of mRNA degradation in yeast occurs by deadenylation of the mRNA, which primarily leads to a decapping reaction, thereby exposing the mRNA to rapid 5' to 3' exonucleolytic degradation. A critical step in the primary pathway is decapping, since it effectively terminates the mRNA's existence and is the site of numerous control inputs. I discuss the properties of the decapping enzyme and how its activity is regulated to give rise to differential mRNA turnover. The major pathways of mRNA turnover in eukaryotic cells are initiated by shortening of the poly(A) tail. In this work, I demonstrate by several criteria that CCR4 and CAF1 encode critical components of the major cytoplasmic deadenylase in yeast. First, both Ccr4p and Caf1p are required for normal mRNA deadenylation in vivo. Second, both proteins localize to the cytoplasm. Third, Caf1p co-purifies with poly(A) specific exonuclease activity, and this activity is dependent on the presence of Ccr4p. Interestingly, because Ccr4p and Caf1p have been shown previously to interact with transcription factors, these results suggest an unexpected link between mRNA synthesis and turnover. Both the Ccr4 and Caf1 proteins have significant homology to known exonucleases, in this work I demonstrate by several criteria that CCR4 encodes the catalytic subunit of the deadenylase. First, over-expression of Ccr4p rescues the deadenylation defects of a caf1Δ strain, indicating that Caf1p is not essential for deadenylation. Second, purification of Ccr4p co-purifies with poly(A) specific exonuclease activity, and this activity is not dependent on the presence of Caf1p. Third, point mutants in predicted catalytic residues of the Ccr4p exonuclease domain result in deadenylation defects in vivo and in vitro. The strong conservation of Ccr4p and Caf1p in other eukaryotes suggests that they will function in the process of deadenylation in other organisms.

Until now, CI tetramer cooperativity has been theorized to be critical for the wild type behavior of λ. Specifically, it was believed that a CI cooperativity mutant phage would not be able to grow lysogenically. Further, if it could be altered in some way to grow lysogenically, it was thought that the lysogen would not have the ability to switch to and/or complete lytic growth after induction. However, the work on which these theories were based was done with uncoupled systems. Then, inferences were drawn as to the role cooperativity played in the intact switch. While this research was groundbreaking, CI levels were unregulated since the regulatory network was disconnected from the action of CI. This is unrealistic since within the lysogen, CI levels are extensively coupled to CI activity via feedback. In this work, I study the role of CI tetramer cooperativity in an intact phage. In this way, CI levels remain coupled to the extensive regulation which controls them. The CI level is regulated by its own activity. To test the role of CI cooperativity in λ physiology, specifically in the genetic switch, three CI cooperativity deficient phage were made. None could form stable, single lysogens. Therefore, the CI Y210X mutations were combined with mutations in the O(R) region that should confer increased occupancy of O(R)2, allowing stable lysogeny. A CI cooperativity deficient phage was isolated that could successfully complete all three aspects of λ physiology: lytic growth, lysogenic growth, and a threshold response to the switch between the two. Therefore, CI cooperativity is not required for wild type λ physiology. In addition, a CI Y210F phage was isolated. CI Y210F has wild type cooperativity. However, phenylalanine cannot make the contacts described in the CI structure papers, indicating that the proposed details of the cooperativity contacts shown in the papers are likely to be unnecessary or possibly incorrect.

Our first aim was to generate transgenic mice to express beta-galactosidase reporter gene under the control of the two promoters. The model would allow us to study the temporal and spatial expression of PTH1R during the onset of embryonic endochondral ossification, and in the adult. Our transgenic animals would allow us to identify regulatory elements that are essential for tissue specific PTH1R expression. We have cloned 11 kilobases of mouse PTH1R gene promoter sequence containing four untranslated exons U1, U2, U3 and SS, and fused this to a Lac Z reporter gene, which was in turn fused to a 250 by fragment containing the A-rich polyadenylylation signal. Three additional constructs were made with deletion of transcription start sites in exon U1 (DeltaU1), U3 (DeltaU3) and both (DeltaU1DeltaU3). Both the control and transgenic adult littermates showed high levels of beta-galactosidase-like activity in epiphyseal growth plate and kidney medulla. However, beta-galactosidase activity was not observed for fetuses aged post coital 14.5 and 15.5 days. We were unable to show tissue specific reporter activity in our transgenic animals. / In other study, we found that P2 is the predominant promoter controlling PTH1R gene expression in both bone and cartilage. (Abstract shortened by UMI.)

Zinc cluster proteins represent a subclass of zinc finger proteins and function as transcriptional regulators. An in vivo genetic screening system was developed in yeast to identify DNA binding sites and specificities for these proteins. / An oligonucleotide library of 200 000 clones was constructed. Control screening trials with Hap1p and Gal4p demonstrated effectiveness in recovering binding sites. Sequencing of isolated clones showed correlation with published target sequences and binding was confirmed by electrophoretic mobility shift assay (EMSA). / Screening over 100 000 clones of the library with the YLR228c gene product allowed the isolation of 10 clones. Mutational EMSA studies were performed to identify nucleotides important for binding to derive a consensus sequence. A CGG triplet was found to be significant for binding. It can be hypothesized that Ylr228p may bind as a monomer to its targets.

A growing repertoire of proteins is involved in the molecular events surrounding clathrin-coated vesicle (CCV) formation, which is seminal to the regeneration of synaptic vesicles in nerve terminals, as well as to more universal membrane trafficking mechanisms. Amphiphysin-I and endophilin-I are two nerve terminal-enriched proteins thought to function in CCV formation. In the first study of this thesis, we identified a novel amphiphysin-I isoform (termed amphiphysin-II), through a homology search of the expressed sequence tag (EST) database. We demonstrated that like amphiphysin-I, amphiphysin-II is highly enriched in nerve terminals, where it interacts with dynamin-I and synaptojanin-I through its SH3 domain. Further, both amphiphysins were found to bind directly to the vesicle coat protein, clathrin, in an SH3 domain-independent manner. / In the next two studies, we cloned a number of amphiphysin-II splice variants that were found to display differential clathrin binding. The use of these splice variants, together with amphiphysin-II truncation and deletion mutants, allowed us to identify two distinct clathrin-binding domains that are also conserved in amphiphysin-I. Using these amphiphysin constructs we also determined that the amphiphysins are targeted to the plasma membrane through their N-termini, which also appears to be important in facilitating amphiphysin oligomerization. / In the final study of this thesis, we turned our attention to a related endocytic SH3 domain-containing protein, endophilin-I. In an attempt to further understand its complete role within the cell, we screened a brain expression library with its SH3 domain, and identified the rat germinal center kinase-like kinase (rGLK), a member of the germinal center kinase (GCK) family of c-jun N-terminal kinase (JNK) activating enzymes, as a novel endophilin-I-binding partner. We characterized the interaction both in vitro and in vivo and determined the sequence on rGLK that mediates the interaction. Importantly, overexpression of full-length endophilin-I was found to enhance rGLK-mediated JNK activation, whereas fragments of endophilin-I blocked JNK activation, suggesting a novel role for endophilin-I in JNK intracellular signaling. / Taken together, the data in this thesis not only adds to the growing list of endocytic accessory proteins, but also contributes to our understanding of their individual molecular interactions and functions within the cell.

Unlike in somatic cells, cancer cells adopt an aberrant pattern of methylation as well as histone acetylation, and therefore distort the chromatin structure. Chapters 2--4 of this thesis look at mechanisms carried out by the recently cloned DNA demethylase, how its demethylation activity is closely linked with the semblance of acetylation of chromatin, and how this relationship can be skewed in cancer. The three intriguing mechanisms described provide attractive models by which to explain general genome wide demethylation, site specific demethylation of genes upon their activation, and the relationship between aberrant methylation and histone acetylation in cancer. The thesis begins by characterizing the mechanism of demethylation carried out by the bona fida DNA demethylase---an enzyme identified and cloned in our laboratory found to demethylate both hemi and double-stranded DNA in vitro. This enzyme manifests the removal of methyl groups from DNA without damaging the DNA and is therefore a candidate protein responsible for hypomethylation seen during development as well as in transformed cells. One essential property of an enzyme that removes methylation from wide regions of the genome could be processivity. Southern blot analysis and sodium bisulfite mapping experiments determine that purified demethylase demethylates DNA in a processive manner in vitro. Experiments in Chapter 3 demonstrate how an active demethylase enzyme is involved in shaping patterns of methylation relative to the state of histone acetylation. We present evidence suggesting demethylase activity is directed by the state of histone acetylation, therefore contrasting the accepted dogma, and suggesting that the local histone acetylation state determines the resulting methylation pattern. Aberrant DNA methylation and histone deacetylation are frequently associated with silencing of tumor suppressor genes in cancer and yet cannot simply be explained by the level of methyltransferase(s) enzyme(s)

The CDP/Cux transcription factor contains four DNA binding domains: three Cut repeats (CR1, CR2 and CR3) and the Cut homeodomain (HD). The CCAAT-displacement protein (CDP) was first identified as it represses transcription of certain promoters by competing for the occupancy of the CCAAT sequence. CDP was then found to be the ortholog of the Drosophila Cut protein (Cux: Cu t homeobox&barbelow;). The goal of my project was to define the modes of interactions of CDP/Cux with DNA. I demonstrated that high affinity DNA binding requires the cooperation between at least two of the CDP/Cux DNA binding domains. Among all combinations of domains, Cut repeats 1 and 2 (CR1CR2) and Cut repeat 3 and the Cut homeodomain (CR3HD) exhibited the highest DNA binding affinities, but with different kinetics and specificities. Whereas CR1CR2 bound with fast kinetics to dimers of CART or CGAT, CR3HD bound to ATCGAT with slow kinetics. CR1CR2 was shown to be responsible for the CCAAT displacement activity of CDP/Cux. Surprisingly, the full-length CDP/Cux protein, p200, exhibited DNA binding properties similar to that of CR1CR2, indicating that CR3HD is inactive in this context. However, an amino terminally truncated 110 KDa CDP/Cux isoform capable of stable interaction with DNA was identified in cellular extracts. The p110 isoform was shown to be generated by proteolytic cleavage of p200 at the G1/S transition. The p200 and p110 isoforms displayed different transcriptional activities in reporter assays, as only p110 was able to activate transcription from the DNA polymerase a gene promoter. Interestingly, expression of p110 was found to be increased in human uterine leiomyomas as compared to the adjacent normal myometrium, raising the possibility that proteolytic processing of CDP/Cux is activated in cancer cells. Finally, cyclin D/CDK4 was shown to interact with CDP/Cux, phosphorylate it on several serine residues and inhibit proteolytic processing as well as DNA binding by CR3HD. Repla