Thesis (Ph.D.)--Brown University, 2008. / Vita. Advisor : Albert E. Dahlberg. Includes bibliographical references.

Thesis (Ph.D.)--University of California, San Francisco, 2009. / Source: Dissertation Abstracts International, Volume: 70-04, Section: B, page: 2072. Adviser: Hiten D. Madhani.

<p> The vacuole in <i>Saccharomyces cerevisiae</i> serves as a model for the mammalian lysosome. In a genome wide screen for mutants with severe growth <u>h</u>ypersensitivity to <u>h</u>ygromycin B, our lab identified 14 <i>HHY</i> genes. Each of the <i> hhy</i> mutants is defective in vacuolar trafficking and/or function and also sensitive to rapamycin and caffeine, suggesting a compromised target of rapamycin (TOR) kinase pathway. My research divides the <i>hhy</i> mutants into two groups based on quantitative growth analyses in the presence of hygromycin B. (1) a super affected group (<i>s-hhy's</i>) and (2) a dose-dependent group (<i>d-hhy's</i>). The <i>s-HHY</i> genes include <i>CHC1, DRS2, SAC1, VPS1, VPS34, VPS45, VPS52,</i> and <i>VPS54</i>. Evaluation of the known functions of <i> s-HHY</i> gene products reveals vesicular trafficking function at the trans-Golgi and late endosome interface to be a common factor. In yeast, the TORC1 complex localizes to the vacuole. Because a compromised TORC1 complex signaling is suggested in <i>hhy</i> mutant strains due to their caffeine or rapamycin sensitivity, I hypothesized that compromised TORC1 signaling in <i>HHY's</i> may be due to defects in the vacuolar localization of Tor1 kinase. To assess Tor1 kinase localization, we utilized a strain expressing endogenously tagged Tor1-GFP and assayed localization to the vacuolar membrane in each of the s-hhy mutants using confocal microscopy. In wild-type cells, Tor1-GFP co-localizes with the vacuolar membrane marker FM4-64 while s-hhy deletion strains fail to localize Tor1-GFP to the vacuolar membrane when treated with hygromycin B. Our results implicate that Tor1p is transported to the vacuole membrane via the late endosome (CPY pathway) and not the ALP (Vam3) pathway. Additionally, <i>s-hhy</i> mutants are unable to recover growth after a 4-hour treatment with hygromycin B, similar to EG0 mutants, which fail to exit from G0 after treatment with the TORC1 inhibiting drug rapamycin. Based on our data, we propose a model in which the <i>s-HHY </i> gene functions in vesicular trafficking at the trans-Golgi/late endosome interface are involved in recruitment and subsequent transport of Tor1p to the vacuolar membrane, and that interface is hypersensitive to hygromycin B. We also propose that Tor1 kinase localization at the vacuole is essential for its cell cycle regulatory function.</p>

<p>The success of Drosophila as a system for genetic analysis is closely linked to its amenability to genetic manipulation. Part 1 of the dissertation elucidates a novel scheme for long-range targeted manipulation of genes. We integrated an 80-kb genomic fragment at its endogenous locus, utilizing a targeted attP attachment site for the phiC31 integrase. We achieved single-copy reduction of the resulting region duplication by inducing recombinational DNA repair. We showed that this two-step scheme of integration and reduction is efficient and useful for delivering modifications. We established a vector configuration that facilitates the recovery of modifications. The integrating genomic fragment allowed for delivery of a new attachment site at 70 kb from the existing <i>attP</i> into a new locus, making it susceptible to targeted mutagenesis. We extrapolate that with this scheme, only 1 200 lines bearing att-sites throughout the genome would suffice to render all Drosophila genes amenable to targeted mutagenesis. Excitingly, this method should be readily applicable to other systems. </p><p> In Part 2 of the dissertation, I explored the question of telomere organization in Drosophila. Telomeres demarcate the ends of linear chromosomes to distinguish them from broken ends. In yeast, they cluster at the periphery of the nucleus establishing a compartment of silent chromatin. To bring insight into telomere organization in a higher organism, we followed EGFPlabeled Drosophila telomeric protein HOAP <i>in vivo</i> and found that the 16 telomeres cluster into 4-6 foci per nucleus in somatic tissues. Interestingly, HOAP signal intensity in the clusters doubles in interphase, potentially due to loading of HOAP to newly replicated telomeres. We tested several predictions about rules governing clustering. First, by inspecting mutant embryos that develop as haploids, we found that clustering is not mediated by associations between homologs. Second, by demonstrating clustering capability for a telomere of novel sequence, we eliminated DNA sequence homology and identity as important factors. Third, by marking both ends of a chromosome, we ruled out predominance of intra-chromosomal interactions. We propose that clustering is indiscriminate of sequence and is likely maintained by a yet undetermined factor. </p>

<p> We have used the <i>C. elegans</i> primordial gonad to understand how stem cells assemble into a niche during development. The <i>C. elegans </i> primordial gonad contains two somatic gonad precursor cells (SGPs) and two primordial germ cells (PGCs). The primordial gonad assembles during embryogenesis when PGCs and SGPs come together adjacent the intestine. </p><p> As a first step in understanding niche assembly, we investigated how PGCs move to the site where the primordial gonad forms. PGCs and somatic cells move into the interior during gastrulation. Because somatic cells require transcription to ingress whereas PGCs are transcriptionally quiescent, we hypothesized that somatic cells might push or pull the PGCs into the embryo. We used videomicroscopy to identify cells that contact the PGCs, and used laser killing to determine if the contacting cells are required for PGC ingression. The PGCs are surrounding by adjacent mesodermal cells and internal endodermal cells. We found that the only contacting cells necessary for PGC ingression were the endodermal cells, which ingress into the embryo an hour before the PGCs. Killing or altering the fate of the endodermal cells prevented PGC ingression but not ingression of other somatic cells. Using fluorescent membrane markers and live imaging, we showed that PGCs and endodermal cells maintain contact throughout gastrulation, and that endodermal cells move dorsally as PGCs ingress form the ventral surface. PGCs express high levels of E-cadherin/HMR-1, and knocking down E-cadherin/HMR-1 caused PGCs to detach from endodermal cells and remain on the surface of the embryo. Finally, we show that the enrichment of HMR-1 protein in the PGCs is not due to transcriptional upregulation, but is instead due to an increase in protein expression mediated by the hmr-1 3' UTR. We propose that PGCs upregulate E-cadherin/HMR-1 to maintain tight adhesion with endodermal cells, which pull the PGCs into the embryo and position them at the site of primordial gonad assembly. Our results highlight the importance of germ cell - gut interactions during development and of E-cadherin-mediated adhesion in niche formation.</p>

<p> Human genes are split into regions that code for protein, exons, and regions that don't, introns. Upon transcription, the removal of these intervening introns is necessary if a usable mRNA molecule is to be translated. The process of intron removal and subsequent ligation of exons is called splicing and is carried out by a large complex called the spliceosome. This process is driven by sequence elements within the pre-mRNA itself and is the major contributor of diversity to the human transcriptome. Due to the ubiquitous nature of alternative splicing in almost every multi-exon gene, the regulation pathways of exon inclusion are a subject of wide study. </p><p> The different lengths of introns and exons as well as location of splice sites in a pre-mRNA molecule have been shown to have differing affects on the spliceosomes ability to recognize them. Using <i>in vitro</i> splicing and complex formation assays in parallel with cell transfection experiments, we determined that the distance between two splice sites across the intron or across the exon are strong predictors of splice site usage. Additionally, we found that two splice sites interact differently when placed at different lengths apart. Our findings suggest a mechanism for observed selection of specific intron/exon architectures. </p><p> Splice site recognition is also influenced by the presence of protein binding sequence elements in the pre-mRNA that alter spliceosomal recruitment. Previously, these proteins and sequence elements had been rigidly classified into splice enhancing or inhibiting categories. We show that this rigid classification is incorrect. We found that the location of these elements relative to the splice site determines their enhancing or silencing effect. That is, an enhancing element found upstream of a splice site imposes a silencing effect when relocated downstream of the splice site (and vice versa). </p><p> Spliceosomal proteins are conserved from yeast to humans. The sequence elements used in pre-mRNA sequences have been evolving over time but under pressure from multiple cellular processes, including splicing. To observe the effect of splicing on evolution, we took advantage of the synonymous mutation positions that are under the least amount of selective pressure from the genetic code. We mutated these positions and found that some caused a large decrease in exon inclusion. When we analyzed the comparative alignment data, we found that these specific nucleotide mutations were selected against across species in order to maintain exon inclusion. SNP analysis showed that this pattern of selection was broadly observable at synonymous positions throughout the human genome.</p>

<p> Between 8% and 10% of patients diagnosed with common variable immunodeficiency (CVID) have known mutations in the gene coding for transmembrane activator and calcium-modulating ligand interactor (TACI). Family members of CVID patients carrying the same mutations, but lacking clinical symptoms of CVID, have left the role of TACI in hypogammaglobulinemia unclear. There are two described isoforms of TACI observed at the mRNA and protein levels in humans, a long and a short form, identical save for the exclusion of the second cysteine-rich domain in the short form. The aim of this study was to determine which isoforms contain mutations in a subset of patients and their healthy relatives all known to carry at least one heterozygous mutation in TACI at the genomic level. All patients and healthy relatives carried TACI mutations within their long isoforms. However, in the cohort examined, mutations in the short isoform were only present in CVID patients and one healthy relative. These results may implicate the short isoform of TACI in CVID progression. Our lab has observed that the short isoform induces plasma cell differentiation more efficiently than the long, so this data also supports a more active role for the short isoform in B cell immunology. Mutation localization within additional CVID patients and mutation carrying relatives must be studied to determine if this trend is still significant.</p>

<p>In skeletal muscle, loss of neuronal nitric oxide synthase (nNOS) from the sarcolemma has been observed in a few muscular dystrophies and myopathies. However, the extent of this phenomenon, its mechanism, and its physiological impact are not well understood. Using immunofluorescent staining for nNOS, a survey of 161 patient biopsies found absent or reduced sarcolemmal nNOS in 43% of patients. Patient mobility and muscle functional status correlated with nNOS mislocalization from the sarcolemma. Mouse models of inherited and acquired myopathies showed similar loss of sarcolemmal nNOS and impaired mobility and muscle function. A proteomic approach, using mass spectrometry and differentially labeled control and steroid-induced myopathy (SIM) mouse samples, found novel nNOS binding proteins including alpha-actinin-3 (ACTN3), which exhibited decreased interaction with nNOS after steroid treatment. It revealed a potential explanation for impaired muscle function in SIM as nNOS interactions were lost at the sarcomere and gained at the sarcoplasmic reticulum impairing contractility. Treating nNOS-deficient mice with steroids demonstrated that loss of sarcolemmal nNOS reduces muscle contractility and strength in SIM through increased nitric oxide (NO) signaling. In SIM mice treated with a nitric oxide donor and steroids, nitric oxide partially protects the muscle from atrophy and improves muscle fatigability and recovery suggesting nNOS mislocalization also decreases NO availability. These findings show that loss of sarcolemmal nNOS is a common phenomenon that negatively impacts muscle function. Therapeutic strategies targeting nNOS or NO signaling need to allow for the complexity of local nitric oxide content and cellular context. </p>

<p> In this work, I describe my doctoral work studying the regulation of transcription with both computational and experimental methods on the natural genetic variation in a population. This works integrates an investigation of the consequences of polymorphisms at three stages of gene regulation in the developing fly embryo: the diversity at <i>cis</i>-regulatory modules, the integration of transcription factor binding into changes in chromatin state and the effects of these inputs on the final phenotype of embryonic gene expression.</p>

<p> Introduction: Pancreatic adenocarcinoma is anticipated to become the most fatal gastrointestinal neoplasm by 2017. Because patients who present with symptoms uniformly have advanced disease, 5-year survival is poor. Surveillance programs in the highest risk patients have produced disappointing results and use invasive tests that would not be safe or practical at the population level. Molecular markers, specifically aberrant DNA methylation, show promise in early studies. We aimed to: 1) measure the sensitivity and specificity of aberrant DNA methylation in stools as a minimally invasive way to detect pancreatic cancer; and, 2) identify novel methylation candidate markers highly sensitive and specific for pancreatic cancer. </p><p> Methods: To select candidate markers among those reported in the literature, DNA was extracted from unmatched tissue samples of pancreatic cancers and of normal colonic epithelia and assayed for aberrant methylation by methylation specific PCR. The most discriminant candidates and mutant <i>KRAS</i> were then assayed from matched archival stools of patients with pancreatic cancer in comparison to stools from healthy controls. Sensitivity and specificity for pancreatic cancer were determined from multivariate logistic regression models. To identify novel candidate markers, DNA was extracted from matched, archival tissues of pancreas cancer and two control groups (normal pancreas and normal colon) and sequenced using the reduced representation bisulfite technique. Among all mapped regions, those with the highest variance in methylation differences between cases and controls were filtered prior to analysis. Significant regions were then blindly assayed by methylation specific PCR in an independent, matched sample set, where sensitivity and specificity were measured using univariate logistic regression. </p><p> Results: In tissues, methylated <i>BMP3, EYA4, UCHL1</i> and <i> MDFI</i> were highly discriminant for pancreatic cancer in comparison to normal colon samples. However, when assayed in stools, only <i>BMP3 </i> remained significant. In combination with mutant <i>KRAS</i>, the area under the receiver operating characteristics curve for <i>BMP3 </i> in detection of pancreatic cancer, was 0.85, indicating strong association. Results were not significantly influenced by tumor location or stage. Reduced representation bisulfite sequencing identified over 500 differentially methylated regions which met a priori significance thresholds. The top 25 novel candidates were validated in independent samples, showing both strong association with cases, compared to controls and high signal to noise ratio. </p><p> Conclusions: We report the first demonstration of feasibility for the detection of pancreatic cancer using assay of aberrantly methylated DNA markers from stool. This is a critical first step in the long-term goal of developing a minimally invasive screening tool to curb the mortality rate of this devastating disease. Because the discriminant candidate markers, methylated <i>BMP3 </i> and mutant <i>KRAS</i>, are not unique to pancreatic cancer, we developed a marker discovery strategy which yielded dozens of highly discriminant, validated, novel candidates, many of which have never before been reported in association with cancer. Further studies are indicated to measure the site-specificity of these markers for pancreatic cancer, compared to other gastrointestinal neoplasms and to study the clinical utility of these novel candidates in distant biologic media, such as blood, stool or pancreatic juice.</p>