GLUCAGON GENE EXPRESSION: ANALYSIS OF PREPROGLUCAGON

Glucagon is a 29 amino acid polypeptide hormone produced in the (alpha) cells of the pancreatic islets. The purpose of this research was to understand better the role of glucagon in the regulation of metabolic processes. As with other polypeptide hormones, the synthesis of glucagon is thought to involve a larger precursor, which is then enzymatically cleaved to the functional form. The specific research objectives were to obtain cloned copies of the messenger RNA (mRNA) for pancreatic glucagon, to determine their primary sequences, and from this coding information to deduce the amino acid sequence of the initial glucagon precursor. From this suggested preproglucagon sequence and prior information on possible proglucagon intermediate processing products, the overall objective of this research is to propose a possible pathway for the biosynthesis of pancreatic glucagon.^ Synthetic oligodeoxynucleotide probes of 14-nucleotides (14-mer) and 17-nucleotides (a 17-mer) complementary to codons specifying a unique sequence of mature glucagon were synthesized. The ('32)P-labeled-14-mer was hybridized with size-fractionated fetal bovine pancreatic poly(A('+))RNA bound to nitrocellulose. RNA fractions of (TURN)14S were found to hybridize specifically, resulting in an (TURN)10-fold enrichment for these sequences. These poly(A('+))RNAs were translated in a cell-free system and the products analyzed by gel electrophoresis. The translation products were found to be enriched for a protein of the putative size of mammalian preproglucagon ((TURN)21 kd). These enriched RNA fractions were used to construct a complementary DNA (cDNA) library is plasmid pBR322.^ Screening of duplicate colony filters with the ('32)P-labeled-17-mer and a ('32)P-labeled-17-mer-primed cDNA probe indicated 25 possible glucagon clones from 3100 colonies screened. Restriction mapping of 6 of these clones suggested that they represented a single mRNA species. Primary sequence analysis of one clone containing a 1200 base pair DNA insert revealed that it contained essentially a full-length copy of glucagon cDNA.^ Analaysis of the cDNA suggested that it encoded an initial translation product of 180 amino acids with an M(,r) = 21 kd. The first initiation codon (ATG, methionine) followed by the longest open reading frame of 540 nucleotides was preceded by a 5'-untranslated region of 90 nucleotides, and was followed by a longer 3'-untranslated region of 471 nucleotides, resulting in a total of 1101 nucleotides. . . . (Author's abstract exceeds stipulated maximum length. Discontinued here with permission of author.) UMI ^

Functional requirements of histone deacetylases in Tup1-Ssn6 repression

The corepressor complex Tup1-Ssn6 regulates many classes of genes in yeast including cell type specific, glucose repressible, and DNA damage inducible. Tup1 and Ssn6 are recruited to target promoters through their interactions with specific DNA binding proteins such as α2, Mig1, and Crt1. Most promoters that are repressed by this corepressor complex exhibit a high degree of nucleosomal organization. This chromatin domain occludes transcription factor access to the promoter element resulting in gene repression. Previous work indicated that Tup1 interacts with underacetylated isoforms of H3 and H4, and that mutation of these histones synergistically compromises repression. These studies predict that Tup1-hypoacetyalted histone interaction is important to the repression mechanism, and in vivo hyperacetylation might compromise the corepressors ability to repress target genes. ^ One way to alter histone acetylation levels in vivo is to alter the balance between histone acetyltransferases and histone deacetylases. To date five histone deacetylases (HDACs) have been identified in yeast Rpd3, Hos1, Hos2, Hos3 and Hda1. Deletion of single or double HDAC genes had little to no effect on Tup1-Ssn6 repression, but simultaneous deletion of three specific activities Rpd3, Hos1, and Hos2 abolished repression in vivo. Promoter regions of Tup1-Ssn6 target genes in these triple deacetylase mutant cells are dramatically hyperacetylated in both H3 and H4. Examination of bulk histone acetylation levels showed that this specific HDAC triple mutant combination (rpd3 hos1 hos2) caused a dramatic and concomitant hyperacetylation of both H3 and H4. The loss of repression in the rpd3 hos1 hos2 cells, but not in other mutants, is consistent with previous observations, which indicate that histones provide redundant functions in the repression mechanism and that high levels of acetylation are required to prevent Tup1 binding. Investigation into a potential direct interaction between the Tup1-Ssn6 corepressor complex and one or more HDAC activities showed that both Rpd3 and Hos2 interact with the corepressor complex in vivo. These findings indicate that Tup1-Ssn6 repression involves the recruitment of histone deacetylase activities to target promoters, where they locally deacetylate histone residues promoting Tup1-histone tail interaction to initiate and/or maintain the repressed state. ^

Collagen I modulates growth and gene expression in prostate cancer cells

Prostate cancer is the second leading cause of male cancer-related deaths in the United States. Interestingly, prostate cancer preferentially metastasizes to skeletal tissue. Once in the bone microenvironment, advanced prostate cancer becomes highly resistant to therapeutic modalities. Several factors, such as extracellular matrix (ECM) components, have been implicated in the spread and propagation of prostatic carcinoma. In these studies, we have utilized the PC3 cell line, derived from a human bone metastasis, to investigate the influence of the predominant bone ECM protein, type I collagen, on prostate cancer cell proliferation and gene expression. We have also initiated the design and production of ribozymes to specific gene targets that may influence prostate cancer bone metastasis. ^ Our results demonstrate that PC3 cells rapidly adhere and spread on collagen I to a greater degree than on fibronectin (FN) or poly-L-lysine (PLL). Flow cytometry analysis reveals the presence of the α1, α2 and α3 collagen binding integrin subunits. The use of antibody function blocking studies reveals that PC3 cells can utilize α2β 1 and α3β1 integrins to adhere to collagen I. Once plated on collagen I, the cells exhibit increased rates of proliferation compared with cells plated on FN or tissue culture plastic. Additionally, cells plated on collagen I show increased expression of proteins associated with progression through G1 phase of the cell cycle. Inhibitor studies point to a role for phosphatidylinositol 3-kinase (PI3K), MAP kinase (MAPK), and p70 S6 kinase in collagen I-mediated PC3 cell proliferation and cyclin D1 expression. To further characterize the effect of type I collagen on prostate cancer bone metastasis, we utilized a cDNA microarray strategy to monitor type I collagen-mediated changes in gene expression. Results of this analysis revealed a gene expression profile reflecting the increased proliferation occurring on type I collagen. Microarray analysis also revealed differences in the expression of specific gene targets that may impact on prostate cancer metastasis to bone. ^ As a result of our studies on the interaction of prostate cancer cells and the skeletal ECM, we sought to develop novel molecular tools for future gene therapy of functional knockdown experiments. To this end, we developed a series of ribozymes directed against the α2 integrin and at osteopontin, a protein implicated in the metastasis of various cancers, including prostate. These ribozymes should facilitate the future study of the mechanism of prostate cancer cell proliferation, and disease progression occurring at sites of skeletal metastasis where a type I collagen-based environment predominates. ^ Together these studies demonstrate the involvement of bone ECM proteins on prostate cancer cell proliferation and suggest that they may play a significant role on the growth of prostate metastases once in the bone microenvironment. ^

From gene to protein: Investigating the disease etiology of retinitis pigmentosa

Retinitis pigmentosa (RP) is a name given to a group of inherited retinal dystrophies that lead to progressive photoreceptor degeneration, and thus, visual impairment. It is evident at both the clinical and the molecular level that these are heterogeneous disorders, with wide variation in severity, mode of inheritance, and phenotype. The genetics of RP are not simple; the disease can be inherited in dominant, recessive, X-linked, and digenic modes. Autosomal dominant RP (adRP) results from mutations in at least ten mapped loci, but there may be dozens of genetic loci where mutations can cause RP. To date, there are over a hundred genes known to cause retinal degenerative diseases, and less than half of these have been cloned (RetNet). Among the dozens of retinitis pigmentosa loci known to exist, only a few have been identified and the remainders are inferred from linkage studies. Today, the genes for seven of the twelve-adRP loci have been identified, and these are rhodopsin, peripherin/RDS, NRL, ROM1, CRX, RP13 and RP1. My research projects involved a combination of the continued search for genes involved in retinal dystrophies, as well the investigation into the role of peripherin/RDS and RP1 in the disease etiology of autosomal dominant RP. ^ Most of the mutations leading to inherited retinal disorders have been identified in predominately retina expressed genes like rhodopsin, peripherin/RDS, and RP1. Expressed sequence tags (ESTs) that were retina-specific were culled from sequence databases and, together with laboratory analysis, were analyzed as potential candidate genes for retinal dystrophies. Thirteen of the fifty-five identified retina-specific ESTs mapped to within candidate regions for inherited retinopathies. One of these is RP1L1, a homologue of RP1 and a potential cause of adRP. ^ Once a disease-associated gene has been identified, elucidating the role of that gene in the visual process is essential for understanding what happens when the process is defective as it is in adRP. My next projects involved investigating the role of a novel 5′ donor +3 splice site mutation on the mRNA of peripherin/RDS in adRP affected individuals, and comparative sequencing in RP1 to define conserved regions of the protein. Comparative sequencing is a powerful way to delineate critical regions of a sequence because different regions of a gene have different functions, and each region is subject to different levels of functional or structural constraints. Establishing a framework of conserved domains is beneficial not only for structural or functional studies, but can also aid in determining the potential effects of mutations. With the completion of sequencing of human genome, and other organisms such as Saccharomyces cerevisiae, Caenorhabditis elegans , and Drosophila, the facility of comparative sequencing will only increase in the future. Comparative sequencing has already become an established procedure for pinpointing conserved regions of a protein, and is an efficient way to target regions of a protein for experimental and/or evolutionary analysis. ^

Functional analysis of BMP4 signaling in mouse neural development

During early mouse neural development, bone morphogenetic protein (BMP) signaling patterns the dorsal neural tube and defines distinct neural progenitor cell domains along the dorsoventral axis. Unlike the ventral signaling molecule Sonic hedgehog, which has long-range activity by establishing a concentration gradient in the ventral neural tube, these dorsally expressed BMPs appear to have a limited domain of action. This raises questions as to how BMP activity is restricted locally and how restricted BMP signaling directs dorsal neural patterning and differentiation. I hypothesize that BMPs are restricted in the dorsal neural tube for correct dorsoventral patterning. ^ Previous studies have shown that the positively charged basic amino acids located at the N-terminus of several BMPs are essential for heparin binding and diffusion. This provides a novel tool to address these questions. Here I adapted a UAS/GAL4 bigenic mouse system to control the ectopic expression of BMP4 and a mutant form of BMP4 that lacks a subset of the N-terminal basic amino acids. The target genes, UAS-Bmp4 and UAS-mBmp4 , were introduced into the Hprt locus by gene targeting in mouse embryonic stem cells. The expression of the GAL4 transactivator was driven by a roof plate specific Wnt1 promoter. ^ The bigenic mouse embryos exhibit phenotype variations, ranging from mid/hindbrain defects, hemorrhage, and eye abnormalities to vasculture formation. Embryonic death starts around E11.5 because of severe hemorrhage. The different expression levels of the activated transgene may account for the phenotype variation. Further marker analysis reveals that mutant BMP4 induces ectopic expression of the dorsal markers MSX1/2 and PAX7 in the ventral neural tube. In addition, the expression of the ventral neural marker NKX2.2 is affected by the expanded BMP4 activity, indicating that ectopic BMP signaling can antagonize ventral signaling. Comparison of the phenotypes of the Wnt1/ Bmp4 and Wnt1/mBmp4 bigenic embryos that express transgenes at the same level, respectively, shows that mutant BMP4 causes the expansion of dorsal neural fates ventrally while wild type BMP4 does not, suggesting that mutant BMP4 acts farther than wild type BMP4. Together, these data suggest that the N-terminus basic amino acid core controls BMP4 long-range activity in neural development, and that BMP signaling patterns the dorsal neural tube through a secondary signaling pathway that involves homeodomain transcription factors MSX1/2 and PAX7. ^

Towards the identification of a tumor suppressor gene on chromosome 3p12: Physical mapping and candidate gene screening

Renal cell carcinoma (RCC) is the most common malignant tumor of the kidney. Characterization of RCC tumors indicates that the most frequent genetic event associated with the initiation of tumor formation involves a loss of heterozygosity or cytogenetic aberration on the short arm of human chromosome 3. A tumor suppressor locus Nonpapillary Renal Carcinoma-1 (NRC-1, OMIM ID 604442) has been previously mapped to a 5–7 cM region on chromosome 3p12 and shown to induce rapid tumor cell death in vivo, as demonstrated by functional complementation experiments. ^ To identify the gene that accounts for the tumor suppressor activities of NRC-1, fine-scale physical mapping was conducted with a novel real-time quantitative PCR based method developed in this study. As a result, NRC-1 was mapped within a 4.6-Mb region defined by two unique sequences within UniGene clusters Hs.41407 and Hs.371835 (78,545Kb–83,172Kb in the NCBI build 31 physical map). The involvement of a putative tumor suppressor gene Robo1/Dutt1 was excluded as a candidate for NRC-1. Furthermore, a transcript map containing eleven candidate genes was established for the 4.6-Mb region. Analyses of gene expression patterns with real-time quantitative RT-PCR assays showed that one of the eleven candidate genes in the interval (TSGc28) is down-regulated in 15 out of 20 tumor samples compared with matched normal samples. Three exons of this gene have been identified by RACE experiments, although additional exon(s) seem to exist. Further gene characterization and functional studies are required to confirm the gene as a true tumor suppressor gene. ^ To study the cellular functions of NRC-1, gene expression profiles of three tumor suppressive microcell hybrids, each containing a functional copy of NRC-1, were compared with those of the corresponding parental tumor cell lines using 16K oligonucleotide microarrays. Differentially expressed genes were identified. Analyses based on the Gene Ontology showed that introduction of NRC-1 into tumor cell lines activates genes in multiple cellular pathways, including cell cycle, signal transduction, cytokines and stress response. NRC-1 is likely to induce cell growth arrest indirectly through WEE1. ^

Gene regulation during placenta development: Murine adenosine deaminase as a model to study placenta specific expression

The formation of the placenta is one of the first and most important developmental events that occur in early mammalian embryogenesis. Even given this importance of the placenta, the academic community has largely ignored studying gene regulation during the development and maturation of the placenta. For this reason, an in-depth study of gene regulation in the trophoblast layer of the placenta using murine Adenosine Deaminase (Ada) as a model system has been undertaken. It has been determined that Ada is highly expressed in the placenta and is critical for embryo development. Dr. Kellems' laboratory has previously described a 1.8 kb fragment of the Ada 5 ′ flanking region that is capable of directing trophoblast specific expression in a transgenic model system. Preliminary studies have demonstrated several critical portions of this fragment that are necessary for the correct tissue specific expression in the placenta. My first specific aim was to elucidate the trans factor binding to one of these sequences, the FP3. Through electromobility shift assays (EMSA), the 30 bp FP3 was narrowed to a 5 bp sequence which computer databases predicted bound to Acute Myeloid Leukemia 1 (AML-1). This was confirmed by supershift analysis. The functional importance of this binding was demonstrated by a transgenic approach. A significant difference in expression of the reporter in the placenta was seen when the 5 bp sequence was mutated. This finding is a novel use for the AML-1 transcription factor which is the DNA binding portion of the heterodimer Core Binding Protein (CBP). The 5′ 240 bp region has also been demonstrated to contain functionally significant sequence. Through EMSA assays and computer predictions, the area has been narrowed to two pertinent regions that are predicted to contain GATA binding motifs. ^

Functional studies of insulin-like growth factor binding protein 2 pathway in glioma progression

Comparison of gene expressing profiles between gliomas with different grades revealed frequent overexpression of insulin-like growth factor binding protein 2 (IGFBP2) in glioblastomas (GBM), in which uncontrolled cell proliferation, angiogenesis, invasion and anti-apoptosis are hallmarks. Using the glia-specific gene transfer transgenic mouse and the stable LN229(BP2) GBM cell lines, we found that IGFBP2 by itself cannot transform cells in vitro and in vivo. IGFBP2 had growth inhibitory effects on mouse primary neural progenitors, but overexpression of IGFBP2 had no effect on GBM cells. ^ Although IGFBP2 does not initiate gliomagenesis, using tissue array technology, we observed strong correlation between IGFBP2 overexpression and VEGF up-regulation in human diffuse gliomas. Furthermore, overexpression of IGFBP2 in GBM cells not only enhanced VEGF expression but also increased the malignant potential of U87 MG cells in our angiogenesis xenograft animal model. ^ In parallel to these studies, using established stable SNB19 GBM cells that overexpress IGFBP2, we found that IGFBP2 significantly increased invasion by induction of matrix metalloproteinase-2 (MMP-2) as well as other invasion related genes, providing evidence that IGFBP2 contributes to glioma progression in part by enhancing MMP-2 gene transcription and in turn tumor cell invasion. ^ Finally, we found that primary filial cells infected with an anti-sense IGFBP2 construct have markedly increased sensitivity to γ irradiation and reduced Akt activation. On the other hand, SNB19(BP2) stable lines have consistently increased levels of Akt and NFkB activation, suggesting that one possible mechanism for anti-apoptosic function of IGFBP2 is through the activation of Akt and NFkB. Beside this, what is especially interesting is the finding that Akt protein was cleaved and inactivated during apoptosis by caspases, and IGFBP2 can prevent Akt cleavage, revealing another possible mechanism through it IGFBP2 exhibit strong antiapoptotic effects. Our data showed that IGFBP2 is a specific substrate for caspase-3, raising the possibility that IGFBP2 may inhibit apoptosis by a suicide mechanism. ^ In summary, using cellular, genomics, and molecular approaches, this thesis documented the potential roles of IGFBP2 in glioma progression. Our findings shed light on an important biological aspect of glioma progression and may provide new insights useful for the design of novel mechanism-based therapies for GBM. ^

Capsule gene regulation in Bacillus anthracis and implications for virulence

The poly-D-glutamic acid capsule of Bacillus anthracis is considered essential for lethal anthrax disease. Yet investigations of capsule function have been limited primarily to attenuated B. anthracis strains lacking certain genetic elements. In work presented in this thesis, I constructed and characterized a genetically complete (pXO1 + pXO2+) B. anthracis strain (UT500) and isogenic mutants deleted for two previously identified capsule gene regulators, atxA and acpA, and a newly-identified regulator, acpB. Results of transcriptional analysis and microscopy revealed that atxA controls expression of the first gene of the capsule biosynthesis operon, capB, via positive transcriptional regulation of acpA and acpB. acpA and acpB appear to be partial functional homologs. Deletion of either gene alone has little effect on capsule synthesis. However, a mutant deleted for both acpA and acpB is noncapsulated. Thus, in contrast to previously published models, my results suggest that atxA is the master regulator of cap gene expression in a genetically complete strain. A detailed transcriptional analysis of capB and the regulatory genes was performed to establish the effects of the regulators and CO2/bicarbonate on specific mRNAs of target genes. CO2/bicarbonate is a well-established signal for B. anthracis capsule synthesis in culture. Taqman RT-PCR results indicated that growth in the presence of elevated CO2 greatly increased expression of acpA, acpB and capB but not atxA. 5′ end mapping of capB and acpA revealed atxA-regulated and atxA-independent transcriptional start sites for both genes. All atxA-regulated start sites were also CO2-regulated. A single atxA-independent start site was identified 5 ′ of acpB. However, RT-PCR analysis indicated that capD and acpB are co-transcribed. Thus, it is likely that atxA-mediated control of acpB expression occurs via transcriptional activation of the atxA-regulated start sites of capB. Finally, I examined the contribution of the B. anthracis capsule to virulence. The virulence of the parent strain, mutants deleted for the capsule biosynthesis genes ( capBCAD), and mutants missing the capsule regulator genes was compared using a mouse model for inhalation anthrax. The data indicate that in this model, capsule is essential for virulence. Mice survived infection with the noncapsulated capBCAD and acpA acpB mutants. These mutants initiated germination in the lung, but did not disseminate to the spleen. The acpA mutant had an LD50 value similar to the parent strain and was able to disseminate and cause lethal infection. Unexpectedly, the acpB mutant had a higher LD 50 and a reduced ability to disseminate. During in vitro culture, the acpB single mutant produces capsule and toxin similar to the parent strain. It is likely that acpB regulates the expression of downstream genes that contribute to the virulence of B. anthracis. ^

Characterization of cytochrome P450 4F subfamily: Functional role and response in inflammation

CYP4F subfamily comprises a group of enzymes that metabolize LTB4 to biologically less active metabolites. These inactive hydroxy products are incapable of chemotaxis and recruitment of inflammatory cells. This has led to a hypothesis that CYP4Fs may modulate inflammatory conditions serving as a signal of resolution. ^ We investigated the regulation of rat CYP4F gene expression under various inflammatory prompts including a bacterial lipopolysaccharide (LPS) treated model system, controlled traumatic brain injury (TBI) model as well as using direct cytokine challenges. CYP4Fs showed an isoform specific response to LPS. The pro-inflammatory cytokines IL-1β, IL-6 and TNF-α produced an overall inductive CYP4F response whereas IL-10, an anti-inflammatory cytokine, suppressed CYP4F gene expression in primary hepatocytes. The molecular mechanism behind IL-6 mediated CYP4F induction was partially STAT3 dependent. ^ An alternate avenue of triggering the inflammatory cascade is TBI, which is known to cause several secondary effects leading to multiorgan dysfunction syndrome. The results from this study elicited that trauma to the brain can produce acute inflammatory changes in organs distant from the injury site. Local production of LTB4 after CNS injury caused mobilization of inflammatory cells such as neutrophils to the lung. In the resolution phase, CYP4F expression increased with time along with the associated activity causing a decline in LTB4 concentration. This marked a significant reduction in neutrophil recruitment to the lung which led to subsequent recovery and repair. In addition, we showed that CYP4Fs are localized primarily in pulmonary endothelium. We speculate that the temporally regulated LTB4 clearance in the endothelium may be a novel target for treatment of pulmonary inflammation following injury. ^ In humans, several CYP4F isoforms have been identified and shown to metabolize LTB4 and other endogenous eicosanoids. However, the specific activity of the recently cloned human CYP4F11 is unknown. In the final part of this thesis, CYP4F11 protein was expressed in yeast in parallel to CYP4F3A. To our surprise, CYP4F11 displayed a different substrate profile than CYP4F3A. CYP4F3A metabolized eicosanoids while CYP4F11 was a better catalyst for therapeutic drugs. Thus, besides their endogenous function in clearing inflammation, CYP4Fs also may play a part in drug metabolism. ^

The role of functional tumor suppressor 15-lipoxygenase 2 (15-LOX2) in normal human prostate epithelial cells and prostate cancer development

15-Lipoxygenase 2 (15-LOX2) is a recently cloned human lipoxygenase that shows tissue-restricted expression in prostate, lung, skin, and cornea. The protein level and enzymatic activity of 15-LOX2 have been shown to be down-regulated in prostate cancers compared with normal and benign prostate tissues. We report the cloning and functional characterization of 15-LOX2 and its three splice variants (termed 15-LOX2sv-a, 15-LOX2sv-b, and 15-LOX2sv-c) from primary prostate epithelial (NHP) cells. Western blotting with multiple NHP cell strains and prostate cancer (PCa) cell lines reveals that the expression of 15-LOX2 is lost in all PCa cell lines, accompanied by decreased enzymatic activity. 15-LOX2 is expressed at multiple subcellular locations, including cytoplasm, cytoskeleton, cell-cell border, and nucleus. Surprisingly, the three splice variants of 15-LOX2 are mostly excluded from the nucleus. To elucidate the relationship between nuclear localization, enzymatic activity, and tumor suppressive functions, we established PCa cell clones stably expressing 15-LOX2 or 15-LOX2sv-b. The 15-LOX2 clones express 15-LOX2 in the nuclei and possess robust enzymatic activity, whereas 15-LOX2sv-b clones show neither nuclear protein localization nor arachidonic acid-metabolizing activity. Interestingly, both 15-LOX2- and 15-LOX2sv-b-stable clones proliferate much slower in vitro when compared with control clones. When orthotopically implanted in nude mouse prostate, both 15-LOX2 and 15-LOX2sv-b suppress PC3 tumor growth in vivo. Finally, cultured NHP cells lose the expression of putative stem/progenitor cell markers, slow down in proliferation, and enter senescence. Several pieces of evidence implicate 15-LOX2 plays a role in replicative senescence of NHP cells: (1) promoter activity and the mRNA and protein levels of 15-LOX2 and its splice variants are upregulated in serially passaged NHP cells, which precede replicative senescence and occur in a cell-autonomous manner; (2) PCa cells stably expressing 15-LOX2 or 15-LOX2sv-b show a passage-related senescence-like phenotype; (3) enforced expression of 15-LOX2 or 15-LOX2sv-b in young NHP cells induce partial cell-cycle arrest and senescence-like phenotypes. Together, these results suggest that 15-LOX2 suppress prostate tumor development and do not necessarily depend on arachidonic acid-metabolizing activity and nuclear localization. Also, 15-LOX2 may serve as an endogenous prostate senescence gene and its tumor-suppressing functions might be associated with its ability to induce cell senescence. ^

Functional analysis of BLAP75, a BLM-associated protein

Bloom syndrome (BS) is an autosomal recessive disorder characterized by dwarfism, immunodeficiency, impaired fertility, and most importantly, early development of a broad range of cancers. The hallmark of BS cells is hyper-recombination, characterized by a drastically elevated frequency of sister chromatid exchange (SCE). BLM, the gene mutated in BS, encodes a DNA helicase of the RecQ protein family. BLM is thought to participate in several DNA transactions and to interact with many proteins involved in DNA replication, recombination, and repair. However, the precise function of BLM and the BLM-dependent anti-tumor mechanism remain obscure. ^ A novel protein, BLAP75 (BLM-associated polypeptide, 75KD), was identified to form an evolutionarily conserved complex with BLM and DNA topoisomerase IIIα (Topo IIIα). Our work demonstrates that loss of BLAP75 destabilized BLM and Topo IIIα proteins. BLAP75 colocalized with BLM in subnuclear foci in response to DNA damage and the recruitment of BLM to these foci was BLAP75-dependent. Moreover, depletion of BLAP75 by siRNA resulted in an elevated SCE rate similar to cells depleted of BLM by siRNA. In addition, RNAi-mediated silencing of BLAP75 greatly diminished cell viability. This cellular deficiency was rescued by expression of wild type BLAP75 but not BLAP75 with mutated conserved domain III, which abrogated the interaction between BLAP75, BLM and Topo IIIα, suggesting that the integrity of BLM-Topo IIIα-BLAP75 complex might be critical for cell survival. Finally, I found that BLAP75 was phosphorylated during mitosis and upon various DNA-damaging agents, implying that BLAP75 might also function in mitosis and DNA damage response. ^ Taken together, this study has defined BLAP75 as an integral component of the BLM complex to maintain genome stability. Our findings provide insights into the molecular mechanisms of the BLM helicase pathway and tumorigenesis process associated with these mechanisms. ^

Functional data analysis approaches for genotype-phenotype association studies from next-generation sequencing

Next-generation DNA sequencing platforms can effectively detect the entire spectrum of genomic variation and is emerging to be a major tool for systematic exploration of the universe of variants and interactions in the entire genome. However, the data produced by next-generation sequencing technologies will suffer from three basic problems: sequence errors, assembly errors, and missing data. Current statistical methods for genetic analysis are well suited for detecting the association of common variants, but are less suitable to rare variants. This raises great challenge for sequence-based genetic studies of complex diseases.^ This research dissertation utilized genome continuum model as a general principle, and stochastic calculus and functional data analysis as tools for developing novel and powerful statistical methods for next generation of association studies of both qualitative and quantitative traits in the context of sequencing data, which finally lead to shifting the paradigm of association analysis from the current locus-by-locus analysis to collectively analyzing genome regions.^ In this project, the functional principal component (FPC) methods coupled with high-dimensional data reduction techniques will be used to develop novel and powerful methods for testing the associations of the entire spectrum of genetic variation within a segment of genome or a gene regardless of whether the variants are common or rare.^ The classical quantitative genetics suffer from high type I error rates and low power for rare variants. To overcome these limitations for resequencing data, this project used functional linear models with scalar response to develop statistics for identifying quantitative trait loci (QTLs) for both common and rare variants. To illustrate their applications, the functional linear models were applied to five quantitative traits in Framingham heart studies. ^ This project proposed a novel concept of gene-gene co-association in which a gene or a genomic region is taken as a unit of association analysis and used stochastic calculus to develop a unified framework for testing the association of multiple genes or genomic regions for both common and rare alleles. The proposed methods were applied to gene-gene co-association analysis of psoriasis in two independent GWAS datasets which led to discovery of networks significantly associated with psoriasis.^

Functional Characterization of AtxA, the Bacillus anthracis Virulence Regulator

Coordinated expression of virulence genes in Bacillus anthracis occurs via a multi-faceted signal transduction pathway that is dependent upon the AtxA protein. Intricate control of atxA gene transcription and AtxA protein function have become apparent from studies of AtxA-induced synthesis of the anthrax toxin proteins and the poly-D-glutamic acid capsule, two factors with important roles in B. anthracis pathogenesis. The amino-terminal region of the AtxA protein contains winged-helix (WH) and helix-turn-helix (HTH) motifs, structural features associated with DNA-binding. Using filter binding assays, I determined that AtxA interacted non-specifically at a low nanomolar affinity with a target promoter (Plef) and AtxA-independent promoters. AtxA also contains motifs associated with phosphoenolpyruvate: sugar phosphotransferase system (PTS) regulation. These PTS-regulated domains, PRD1 and PRD2, are within the central amino acid sequence. Specific histidines in the PRDs serve as sites of phosphorylation (H199 and H379). Phosphorylation of H199 increases AtxA activity; whereas, H379 phosphorylation decreases AtxA function. For my dissertation, I hypothesized that AtxA binds target promoters to activate transcription and that DNA-binding activity is regulated via structural changes within the PRDs and a carboxy-terminal EIIB-like motif that are induced by phosphorylation and ligand binding. I determined that AtxA has one large protease-inaccessible domain containing the PRDs and the carboxy-terminal end of the protein. These results suggest that AtxA has a domain that is distinct from the putative DNA-binding region of the protein. My data indicate that AtxA activity is associated with AtxA multimerization. Oligomeric AtxA was detected when co-affinity purification, non-denaturing gel electrophoresis, and bis(maleimido)hexane (BMH) cross-linking techniques were employed. I exploited the specificity of BMH for cysteine residues to show that AtxA was cross-linked at C402, implicating the carboxy-terminal EIIB-like region in protein-protein interactions. In addition, higher amounts of the cross-linked dimeric form of AtxA were observed when cells were cultured in conditions that promote toxin gene expression. Based on the results, I propose that AtxA multimerization requires the EIIB-like motif and multimerization of AtxA positively impacts function. I investigated the role of the PTS in the function of AtxA and the impact of phosphomimetic residues on AtxA multimerization. B. anthracis Enzyme I (EI) and HPr did not facilitate phosphorylation of AtxA in vitro. Moreover, markerless deletion of ptsHI in B. anthracis did not perturb AtxA function. Taken together, these results suggest that proteins other than the PTS phosphorylate AtxA. Point mutations mimicking phosphohistidine (H to D) and non-phosphorylated histidine (H to A) were tested for an impact on AtxA activity and multimerization. AtxA H199D, AtxA H199A, and AtxA H379A displayed multimerization phenotypes similar to that of the native protein, whereas AtxA H379D was not susceptible to BMH cross-linking or co-affinity purification with AtxA-His. These data suggest that phosphorylation of H379 may decrease AtxA activity by preventing AtxA multimerization. Overall, my data support the following model of AtxA function. AtxA binds to target gene promoters in an oligomeric state. AtxA activity is increased in response to the host-related signal bicarbonate/CO2 because this signal enhances AtxA multimerization. In contrast, AtxA activity is decreased by phosphorylation at H379 because multimerization is inhibited. Future studies will address the interplay between bicarbonate/CO2 signaling and phosphorylation on AtxA function.

Systems Biology Approaches to Probe Gene Regulation in Bacteria

Mechanisms that allow pathogens to colonize the host are not the product of isolated genes, but instead emerge from the concerted operation of regulatory networks. Therefore, identifying components and the systemic behavior of networks is necessary to a better understanding of gene regulation and pathogenesis. To this end, I have developed systems biology approaches to study transcriptional and post-transcriptional gene regulation in bacteria, with an emphasis in the human pathogen Mycobacterium tuberculosis (Mtb). First, I developed a network response method to identify parts of the Mtb global transcriptional regulatory network utilized by the pathogen to counteract phagosomal stresses and survive within resting macrophages. As a result, the method unveiled transcriptional regulators and associated regulons utilized by Mtb to establish a successful infection of macrophages throughout the first 14 days of infection. Additionally, this network-based analysis identified the production of Fe-S proteins coupled to lipid metabolism through the alkane hydroxylase complex as a possible strategy employed by Mtb to survive in the host. Second, I developed a network inference method to infer the small non-coding RNA (sRNA) regulatory network in Mtb. The method identifies sRNA-mRNA interactions by integrating a priori knowledge of possible binding sites with structure-driven identification of binding sites. The reconstructed network was useful to predict functional roles for the multitude of sRNAs recently discovered in the pathogen, being that several sRNAs were postulated to be involved in virulence-related processes. Finally, I applied a combined experimental and computational approach to study post-transcriptional repression mediated by small non-coding RNAs in bacteria. Specifically, a probabilistic ranking methodology termed rank-conciliation was developed to infer sRNA-mRNA interactions based on multiple types of data. The method was shown to improve target prediction in Escherichia coli, and therefore is useful to prioritize candidate targets for experimental validation.