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<p>The t(10;11) chromosomal translocation gives rise to the <italic>CALM-AF10</italic> fusion gene and is found in patients with aggressive and difficult-to-treat hematopoietic malignancies. <italic>CALM-AF10</italic>-driven leukemias are characterized by a perturbed epigenetic and transcriptional state. Specifically, the <italic>HOXA</italic> cluster genes are hypermethylated on Histone H3 lysine 79 (H3K79), which corresponds with their transcriptional upregulation. Conversely, <italic>CALM-AF10</italic> cells display global H3K79 hypomethylation. DOT1L, the H3K79 histone methyltransferase, interacts with the OM-LZ domain of AF10, and the AF10 OM-LZ domain has been shown to be necessary and sufficient for CALM-AF10-mediated transformation. These data have suggested a critical role for the AF10-DOT1L interaction in <italic>CALM-AF10</italic> leukemias. However, the mechanism(s) by which DOT1L-mediated epigenetics are perturbed and the precise role of CALM in leukemogenesis have remained unclear. </p><p>In this dissertation, we examine the contribution of CALM to CALM-AF10-mediated leukemogenesis. We determine that CALM contains a functional nuclear export signal (NES) that mediates steady-state cytoplasmic localization of CALM-AF10. An NES is a highly conserved leucine-rich amino acid sequence that is recognized by the nuclear export receptor, CRM1. Classically, CRM1 binds to NES-containing proteins and mediates their export from the nucleus to the cytoplasm through the nuclear pore complex. Through structure-function analyses, we determine that the CALM-derived NES is necessary and sufficient for CALM-AF10-dependent leukemogenesis. In addition, fusions of NES motifs from heterologous proteins (ABL1, Rev, PKIA, and APC) in-frame with AF10 are sufficient to immortalize murine hematopoietic progenitors <italic>in vitro</italic>. From these data, we conclude that a CRM1-dependent NES represents the functional contribution of CALM for CALM-AF10-mediated leukemogenesis. </p><p>In the second part of this dissertation, we examine the mechanism(s) by which the CALM NES imparts transformation potential to AF10. We determine that the CALM NES is essential for CALM-AF10-dependent <italic>Hoxa</italic> gene upregulation and aberrant H3K79 methylation. Using co-immunofluorescence microscopy, we observe increased cytoplasmic localization of DOT1L in the presence of CALM-AF10, suggesting that mislocalization of DOT1L may lead to a global loss of H3K79 methylation. In addition to mediating nuclear export, we find that the CALM-CRM1 interaction is critical for targeting CALM-AF10 to the <italic>Hoxa</italic> locus. Inhibition of CRM1 with Leptomycin B prevents transcription of <italic>Hoxa</italic> genes in <italic>CALM-AF10</italic> leukemia cells. These findings uncover a novel mechanism of leukemogenesis mediated by the nuclear export pathway and support further investigation of the utility of CRM1 inhibitors as therapeutic agents for patients with <italic>CALM-AF10</italic> leukemias.</p> / Dissertation

<p> Transforming Growth Factor Beta Induced Gene Human Clone 3, BIGH3, is an extra cellular matrix protein expressed by different cell types. BIGH3 promotes cell adhesion and has been recognized as a tumor suppressor protein in many studies, a function consistent with the finding that the expression of BIGH3 is reduced in various tumors and transformed cells when compared to healthy counterparts. </p><p> In the present study, we found that BIGH3 induces MG63 multi tumor spheroid (MTS) cells apoptosis and antagonized the development of tumor cells into large aggregate, supporting BIGH3 tumor suppressor role. MG63 spheroids were cultured in recombinant BIGH3 and vascular smooth muscle cells (VSMCs) conditioned medium . We have shown BIGH3 to be abundantly expressed by VSMCs. In addition, stimulation of BIGH3 gene by TGF-&beta;1 in MG63 cells resulted in overexpression of BIGH3 and subsequent increase in apoptosis by almost 3 fold. TUNEL assay was performed to detect apoptotic cells. Smaller and scattered tumor spheroids were observed in TGF-&beta;1 treated cells. Importantly, in-house developed anti- BIGH3 antibody reduced apoptosis percentage by almost one-half and antagonized the development of osteosarcoma cells into large aggregate spheroids. Within the formed spheroids, BIGH3 was immunologically detected in in the stroma and at cell bodies, suggesting a possible binding of BIGH3 to the cell surface. Collectively, these data suggest that BIGH3 plays a suppressive role in development of osteosarcoma tumor spheroids. </p><p> MG63 were cultured in agar-coated wells, where they developed into 3D aggregates. Unlike classical monolayer-based models (2D), multicellular tumor spheroid (MTS) cell culture system mimics the in vivo 3D structure of a solid tumor.</p>

Thioredoxin-1 (Trx-1) is a small redox protein that is overexpressed in a number of human cancers. Elevated levels of Trx-1 in tumors is associated with increased cell proliferation, decreased apoptosis, and decreased patient survival. However, the mechanism(s) for the growth stimulating and anti-apoptosis effects of Trx-1 are unknown. We used DNA microarray technology to identify genes whose expression was altered in MCF-7 breast cancer cells stably transfected with wild-type Trx-1 (MCF-7/Trx 9) or a redox inactive mutant Trx-1 (MCF-7/SerB 4) compared to empty-vector transfected cells (MCF-7/neo). The expression of cytochrome P450 1B1 (CYP1B1) mRNA and protein is increased by Trx-1 transfection of MCF-7 human breast cancer cells and decreased by a redox inactive mutant Trx-1. CYP1B1 is a tumor specific CYP which converts 17β-estradiol (E₂) to the carcinogenic 4-hydroxyestradiol (4-OHE₂). The expression of peroxiredoxin 1 (PRDX1) mRNA is increased as a result of Trx-1 overexpression in MCF-7 cells. The peroxiredoxins belong to a conserved family of antioxidant proteins that use thiol groups as reducing equivalents to scavenge oxidants. Transfection of mouse WEHI7.2 thymoma cells with human PRDX1 protects cells from apoptosis induced by H₂O₂. Spermine/spermidine N'-acetyltransferase (SSAT) mRNA expression and enzyme activity is decreased by Trx-1 transfection of MCF-7 human breast cancer cells. SSAT is an important enzyme in the polyamine catabolic pathway. The inhibition of SSAT enzyme activity is associated with decreased putrescine levels in the Trx-1 transfected cells. Therefore, it appears as if the modification of cellular redox signaling brought about by the overexpression of Trx-1 in breast cancer cells induces changes in gene expression that contribute to the transformed phenotype. Trx-1 redirects estrogen metabolism in a more toxic pathway due to the induction of CYP1B1, provides resistance to apoptosis induced by reactive oxygen species via the upregulation of PRDX1, and alters polyamine metabolism by inhibiting the expression of SSAT.

The biological actions of 1α,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) are mediated by the nuclear vitamin D receptor (VDR), which functions as a ligand-dependent transcriptional regulator. We have developed a six-stage molecular model summarizing the VDR transcriptional activation-life cycle, and tested this model using a variety of experimental approaches, including pull-down assays with GST-fusion proteins, as well as assays of the functional activity of VDR and its putative coactivators in transiently transfected mammalian cells. The six stages of the VDR life cycle are: (1) unoccupied VDR binds to a transcriptional corepressor which serves as a chaperone, maintaining the main protein players in close contact in an inactive complex; (2) VDR becomes occupied by 1,25(OH)₂D₃ ligand, enabling the receptor to heterodimerize strongly with a retinoid X receptor (RXR), leading to high affinity DNA binding and recruitment of coactivators with histone acetyl transferase (HAT) activity; (3) coactivator HAT activity promotes chromatin remodeling, rendering the gene promoter free to interact with the transcription preinitiation complex (PIC); (4) dissociation of VDR from the HAT coactivators, followed by association of a second set of coactivators that promote formation of the preinitiation complex (PIC); (5) transcriptional recycling of the liganded receptor and heteropartner to initiate additional rounds of transcription; and (6) ubiquitination and eventual degradation of VDR. Phosphorylation of VDR may influence all six stages. A testable conclusion from our model is that the role of the 1,25(OH)₂D₃ hormonal ligand would be primarily in the transition from stage 1 to stage 2, but the continued presence of 1,25(OH)₂D₃ appears to be necessary also for the progression from stages 3 through 6 to VDR degradation, or alternatively for recycling via stage 5. This characterization of the macromolecular cofactors that transduce the signal of the 1,25(OH)₂D₃ hormone to promote gene expression in vitamin D target tissues should add to our understanding of endocrine control of bone mineral remodeling and of epithelial cell differentiation. The present work also identifies new protein players that are candidates for mutation or dysregulation in the pathophysiology of vitamin D resistant bone disorders (osteoporosis), and in hyperproliferative diseases of vitamin D regulated epithelial tissues such as skin.

The extracellular matrix (ECM) is defined as the many different proteins and secreted substances between cells. The ECM plays a major role in the signaling pathways that stimulate cells to perform many varied functions, ranging from control of gene expression at the cellular level to differentiation and development of tissues, organs, and ultimately the entire organism. A portion of this work describes the identification of the division abnormally delayed gene, which encodes a proteoglycan, that is involved in growth factor reception with important developmental consequences in Drosophila melanogaster. The remainder of this work deals with three Drosophila homologs of vertebrate proteins that may interact with integrins, a family of cell surface receptors for extracellular matrix ligands. The three integrin-interacting proteins are referred to by their vertebrate names, and include CD81, a member of the tetraspanin family, ILK, integrin-linked kinase, and CD98hc, a type II transmembrane glycoprotein which is the heavy chain of a multi-protein complex. In this work, the mutant phenotype of CD98hc is larval lethal and not temperature sensitive. Clonal analyses of CD98hc mutants show no phenotype of mutant clones in the eye. Genetic interactions in adult tissues or interactions affecting larval lethality between CD98hc and Drosophila integrin mutants were not observed, and it remains unclear whether CD98hc physically interacts with Drosophila integrins in tissue culture cells. Since no correlation was seen between the interactions of CD98hc and integrins in vertebrate cells and similar putative interactions in flies, this raises the question as to what role, if any, does CD98hc play as an integrin modulator in this organism.

In the course of thymocyte development, progenitor T cells from the bone marrow migrate to the thymus where they are positively or negatively selected depending on specific molecular interactions. This selection process takes place at a developmental stage where immature thymocytes are exquisitely sensitive to glucocorticoid exposure and they readily succumb in a process referred to as apoptosis. Glucocorticoid-induced thymocyte apoptosis has been shown to require RNA and protein synthesis, which indicates an active death-inducing process. One molecular event that occurs in immature thymocytes during this process is the rapid and high induction of a glucocorticoid-induced gene called GIG18. Within this dissertation, I identify a GIG18 open reading frame (ORF), that encodes a &sim;50KDa protein, that is 93% conserved between mouse and human. By expressing this ORF with a 6X-Histidine tag, I was able to purify the GIG18 protein on a nickel affinity column to generate a rabbit polyclonal antibody. The western blot showed glucocorticoid-regulated induction of two protein bands, a mildly induced band at the predicted size (50KDa) and a highly induced band at &sim;68KDa. Further analysis of mouse and human expressed sequence tags (ESTs) indicated that alternate transcripts produced from this gene, are likely account for the 68KDa band. The mouse GIG18 ORF identified in our lab maps to human chromosome 7 and contains 8 exons covering &sim;250 Kb. This region of human chromosome 7 corresponds to homologous sequence on mouse chromosomes 5 and 6. I also show that GIG18 belongs to family of three genes encoded in the human genome with two extensive regions of conserved sequence. Expression analysis of the GIG18 ORF in thymocytes and HeLa cells did not reveal any obvious cellular morphological variations or functions induced by this gene. I conclude this dissertation with a discussion of potential roles that GIG18 may play within the context of thymocyte development.

Herpes Simplex Virus type 1 is an important organism not only because it is a member of a family of disease-causing organisms, but it also serves as a model organism for the study of eukaryotic DNA replication. Here I use HSV-1 to investigate two aspects of DNA replication: initiation and proofreading. Cellular factors have been shown to be involved in DNA replication, and especially in initiation, in a variety of viral systems. Previous studies have identified the first cellular factor implicated in initiation of HSV-1 replication, OF-1. In this study, I have purified OF-1 and investigated its composition, binding properties and interactions with the viral origin binding protein UL9. I show that OF-1 is composed of two subunits, one of which contains DNA binding activity. I also found that OF-1 binds specifically to both single- and double-stranded origin DNA, that OF-1 binds most tightly to single-stranded DNA, and that OF-1 shows a preference for which strand is bound. I have demonstrated that, in the presence of UL9, OF-1 exhibits a higher affinity for its target DNA and that OF-1 inhibits the ATPase activity of UL9. I propose that UL9 binds to the origin of replication, loads OF-1 to the origin, and then is displaced by OF-1. Further implications for this model are discussed. I go on to investigate several aspects of error control in the wild type and a 3'-5' proofreading exonuclease mutant DNA polymerase from HSV-1. Proofreading is a primary factor influencing the fidelity of DNA replication. Previous studies in our lab have shown that exonuclease deficient polymerases are incapable of supporting viral growth in vivo. In these studies, I have expressed and purified both wild type and mutant polymerases and investigated their biochemical properties as well as the mechanism of lethality of the mutant. I have found that the mutant polymerase exhibits substantially elevated rates of nucleotide misincorporation as compared to the wild type. In addition, the mutant polymerase is seen to stall at a misincorporation, exhibiting a reduced ability to replicate past a mismatch. Based on these findings, I suggest that the inability of the mutant polymerase to replicate past a misinsertion is the primary cause of the reduced viability of viruses carrying the mutant enzyme.

The Euglena gracilis chloroplast genome contains over 150 introns, including the only known examples of group III introns. Group III introns may belong to the intron super-family consisting of group II and nuclear pre-mRNA introns. It has been suggested that group III intron evolution may have paralleled that of nuclear pre-mRNA introns through loss of domains from a group II-like ancestor. Homologs of psbC14, a group III twintron containing the coding locus for the mat1 polypeptide, have been identified in 7 Euglenoids. All of the species examined contain both the group III twintron and the mat1 locus. As determined by comparative phylogenetic analysis, group III introns contain a structural homolog of group II intron domain VI. The mat1 loci encode peptide motifs characteristic of group II intron maturases. A group III intron-encoded protein whose predicted sequence is similar to group II intron-encoded maturases, and a bona fide domain VI within group III introns, are compelling evidence for a common ancestor of group II and group III introns. A novel mixed operon, psbK-ycf12-psaM-trnR, has been identified in the photosynthetic protist, E. gracilis. The tetracistronic transcripts are processed through endonucleolytic cleavage of the intergenic spacers and splicing of two group III introns and a group III twintron to produce three monocistronic mRNAs and a tRNA. The psbK operon was cloned by PCR amplification from 9 related Euglenoid species. In each species, gene order and content is conserved. The psbK operons contain phylogenetically conserved eubacterial promoter, translational, and 3' processing elements. Based on comparison of homologous intron content to phylogenetic analysis, group III intron evolution within the Euglenoid lineage is more complex than previously believed. Surprisingly, based on psbCi4 and psbK intron distribution data, group III introns may pre-date group II introns in the Euglenoid lineage. An aadA transformation cassette was adapted for expression in Euglena gracilis chloroplasts. Transgenic DNA was introduced into E. gracilis chloroplast by biolisitc transformation and stably maintained as an episomal element during continuous antibiotic selection. When a truncated E. stellata psbK operon was introduced into E. gracilis chloroplasts, transgenic DNA was transcribed and the resultant pre-mRNA accurately spliced.

Regulation of mRNA stability and mRNA translation are important components of gene expression within the eukaryotic cell. Multiple observations have suggested that the processes of translation and mRNA turnover are interrelated. Based on these observations, and the fact that the translation initiation machinery and the decapping enzyme both utilize the cap structure as a substrate, I hypothesized that these processes might be linked due to a competition at the cap between the cap binding complex and the decapping enzyme. Since disruption of translation using translational inhibitors or insertion of strong secondary structure within the 5' UTR affects the stability of mRNAs, I asked whether mutations within the translation initiation machinery itself would have a similar effect. I found that mutations in many different translation initiation factors led to an increase in the rate of mRNA turnover within the yeast cell. It was found that when the process of translation initiation is impaired in this manner that the rates of both deadenylation and decapping are increased. These results imply that the nature of the translation initiation complex bound to the 5' end of the mRNA is a critical component in determining mRNA half-life. One of the translation initiation factors, the cap binding protein eIF4E, is a logical candidate for a protein that might compete with the decapping enzyme for the cap structure. I purified the decapping enzyme and the cap binding protein from yeast and showed that addition of eIF4E could block decapping by the Dcp1p in an in vitro decapping assay. In addition, this inhibition was dependent on eIEF4E's ability to bind to the cap structure. This observation was then recapitulated in vivo by showing that an allele of eIF4E, which is unable to bind the cap structure, could suppress the decapping defect of the partially functional dcpl-1 allele. This same allele could not suppress the decapping defects of a lsm1Delta or pat1Delta, other mutations which affect decapping. These results argue that the translation initiation machinery acts as a physical block to the decapping enzyme and that decapping is composed of at least two genetically separable steps.