Vanderbilt Biological Sciences Retreat | 2008

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A special thanks to: Suraj Adhikary Dan Anderson Dan Carlin Wes Dulaney Jenifer Ferguson Eric Janson Roz Johnson Patrick Jones

Diane Kanter Jonas King Leslie Maxwell Jason Pitts Tessy Sebastian Nikki Shariat Robert Taylor Elizabeth Thatcher

and...

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#29 Tessy Sebastian

2008 Biological Sciences Retreat Schedule of Events

Graham Lab, 2nd Year Role of Apl4 and Rcy1 in the Recycling Pathway of Drs2p to the trans-Golgi Network The transport of newly synthesized proteins from the Golgi complex is a process essential to the health and viability of the cell. This trafficking is achieved by the formation of a variety of transport vesicles that bud off from the trans-Golgi network (TGN) and move to various organelles of the cell. In Saccharomyces cerevisiae, the formation of these vesicles is mediated by Drs2p, a P-type ATPase, found in the TGN and which has been shown to mediate the flipping of phospholipids. This flippase activity enables the formation of buds and, ultimately, vesicles.

Experimental data has suggested that the Drs2p is involved in clathrin-mediated protein transport from the TGN to the early endosomal compartments. Drs2p has also been proposed to enable the transport of proteins from the TGN to the plasma membrane. Normally, Drs2p is localized to the TGN and cycles to the plasma membrane and/or early endosome, and then back to the TGN as part of its normal pathway. However, the path of Drs2p recycling, from the early endosome or the plasma membrane back to the TGN, is not well characterized. There is evidence, nevertheless, that the yeast proteins Rcy1 and, possibly, AP-1 are involved in the retrieval of Drs2p. However, it is unclear whether these proteins act individually or in concert to recycle Drs2p back to the TGN. Here, we sought to better define the roles of Rcy1 and AP-1 in Drs2 trafficking.

Joe C. Davis YMCA Monday, 20 October 2008 8:30 - 9:00 a.m.

Breakfast

9:00 - 10:15 a.m.

Session I: Graduate Student Presentations Chair: Elizabeth Thatcher

9:00 9:15 9:30 9:45 10:00 10:15 - 10:30 a.m.

Break

10:30 - 12:00 p.m.

Session II: Graduate Student Presentations Chair: Dan Anderson

10:30 10:45 11:00 11:15 11:30 11:45

#30 Chunyao Wei Patton Lab, 2nd Year A Genetic Strategy to Identify miRNA Targets microRNAs (miRNAs) are small (~22 nucleotide) non-coding RNAs that play crucial roles in a variety of biological processes by regulating the expression of target genes at the post-transcriptional level, typically by inhibiting translation. Because miRNAs pair imperfectly with their target genes, computational programs to predict targets are often unreliable. Since individual miRNAs usually target multiple genes, a strategy to globally identify all possible targets is needed. Here, we propose a genetic strategy to identify miRNA targets. We constructed a pSuper retro-virus vector expressing HSV thymidine kinase (TK) fused to downstream 3’ UTR sequences with binding sites for specific miRNAs. When cells expressing TK are treated with ganciclovir, nucleotide metabolism is disrupted, DNA replication is blocked, and cell death ensues. In the absence of TK, ganciclovir is not toxic. Our strategy is to co-express miRNAs that bind to the 3’ UTR sequences downstream of TK which should therefore silence TK and allow survival in the presence of ganciclovir. For miRNA expression, the same vector expressing TK contains an H1 promoter downstream from which any miRNA sequence can be cloned. Initial experiments will be designed to test a single miRNA with known binding sites in the 3’ UTR sequence downstream of TK. If this succeeds, libraries of 3’ UTR cDNA sequences can placed downstream of the TK gene followed by co-transfection with specific miRNAs. In this way, individual clones can be selected and the 3’ UTR elements sequenced to determine all mRNA targets for every miRNA.

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#01. Robert Taylor #02. Gulfem Guler #03. Brian Robertson #04. Joshua Broussard #05. Morgan Sammons

#06. Jenifer Ferguson #07. Emily Rubinson #08. Daniel Carlin #09. Patrick Jones #10. Joshua Clanton #11. Weston Dulaney

12:00 - 1:30 p.m.

Lunch

1:30 - 3:00 p.m.

Poster Session #19-30

3:00 - 4:30 p.m.

Session III: Graduate Student Presentations Chair: Eric Janson

3:00 3:15 3:30 3:45 4:00

#12. Diane Kanter #13. Caleb Doll #14. Isi Tolliver #15. Kavitha Surendhram #16. Patrick Robertson

4:15 - 4:30 p.m.

Break

4:30 - 5:00 p.m.

Faculty Speaker #17. Antonis Rokas, Ph.D.

5:00 - 5:30 p.m.

Faculty Speaker #18. Seth Bordenstein, Ph.D.

5:30 - end

Dinner and Awards Drive home safely!

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#27 Haiting Ma Charles Singleton, Ph.D.

Solnica-Krezel Lab, 2nd Year Terence J. Van Raay and Robert J. Coffey

Professor of Biological Sciences Chair, Department of Biological Sciences Vanderbilt University

Characterization of potential protein-protein interaction of zebrafish Naked Cuticle ortholog (zNkd1) and b-catenin

To me, the graduate retreat is one of the highlights of all of the many Biological Sciences departmental events. I am sure this year will be no exception. It is a time that showcases the many talented graduate students we have in the department and the high quality and significant research that they carry out. I imagine the Gordon Conference administrators are highly jealous of our lineup of speakers and poster presenters. So sit back, listen, and enjoy learning about the many fascinating findings and results of our students' research efforts.

Douglas G. McMahon, Ph.D. Professor of Biological Sciences Director of Graduate Studies Vanderbilt University What’s unique about the BioSci Graduate Retreat is that it is truly a retreat of the students, by the students and for the students. Hats off to the students, and the staff, for offering up intriguing science and compelling interactions year after year. Lets get started!

The canonical Wnt signaling is a conserved pathway critical for both development and neoplasia in metazoans. Drosophila Naked Cuticle (Nkd) gene encodes a Wnt-inducible, cell-autonomous antagonist of Wnt signaling.

Loss of function mutations in Drosophila Nkd gene lead to pronounced expansion of naked cuticles due to excess Wnt signaling. In comparison, vertebrate ortholog Nkd1 also attenuates Wnt signaling, although in a milder way. Epistasis and biochemistry experiments demonstrate that the function of Nkd depends on the binding of Nkd and Dvl. However, other potential mechanisms await characterization for better understanding of the function and evolution of Nkd family of Wnt signaling antagonists. Recent research in the Solnica-Krezel and Coffey groups suggests that zebrafish Nkd1 (zNkd1) may also interact with b-catenin, suggesting a potential mechanism whereby zNkd1 antagonize canonical Wnt signaling levels. Co-immunoprecipitation assay of zNkd1 and β-catenin is utilized to analyze zebrafish embryos injected with zNkd1-myc RNA and HEK293T cells transfected with a zNkd1-myc plasmid. In addition, we are purifying zebrafish β-catenin to perform pull down assays to further test the possibility. Besides the biochemical experiments, I will utilize sub-cellular localization of fusion and endogenous proteins in both HEK293T cells and zebrafish embryos using confocal microscopy to test the possibility of interaction between zNkd1 and β-catenin.

#28 Abigail Olena Patton Lab, 2nd Year Tissue Specific miRNA Expression Profiling in Zebrafish

Roz Johnson Educational Program Coordinator

Leslie Maxwell Administrative Assistant We would like to welcome each and every one of you to the 2008 Department of Biological Sciences annual retreat. We hope that you have wonderful learning and fun experience.

Jenifer Ferguson

Diane Kanter

2008 Retreat Coordinators We would like to welcome everyone to this year's BioSci retreat. We hope everyone has an enjoyable experience while learning about the excellent research going on in our very own department!

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MicroRNAs (miRNAs) are small, endogenous, non-coding RNAs that regulate gene expression by binding to target sites within the 3’ untranslated region (UTR) of mRNA. While miRNAs are important in overall development and disease, their exact role in heart development and in eye development and regeneration has not yet been elucidated. Here, we sought to identify miRNAs that function in the developing heart and eye of zebrafish (Danio rerio). Because the heart is highly evolutionarily conserved and because heart disease primarily stems from deregulation of heart growth, these studies will also provide insight into the role of miRNAs in heart disease in higher vertebrates. miRNA analysis of developing and regenerating retinas will afford greater understanding of the role of miRNAs in eye development and regeneration in vertebrates. Microarrays were done on heart tissue and eye tissue of wild type zebrafish embryos at varying stages in order to compare miRNA expression patterns across development in these tissues. Once miRNAs have been identified that function in heart and eye development, potential targets will be identified using targeting algorithms. These targets will be verified using EGFP fusion constructs containing miRNA targets in the 3’UTR, to give a clear picture of the role of miRNAs in gene regulation during vertebrate heart and eye development.

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#25 Jonas G. King Hillyer Lab, 2nd Year Possible involvement of AgSGS proteins during invasion of Anopheles salivary glands by Plasmodium sporozoites The invasion of the female mosquito’s salivary glands is a key interaction between Plasmodium and its mosquito host, the mechanics of which remain largely unknown. A salivary gland specific protein named aaSGS1 was recently shown to be important during the invasion of Aedes aegypti salivary glands by Plasmodium gallinaceum sporozoites. Four homologs were identified bioinformatically in the Anopheles gambiae genome and named AgSGS2, 3, 4 and 5. In the current study, RT-PCR and western blot analyses showed that the expression of AgSGS4 and AgSGS5 is salivary gland specific and occurs only in female mosquitoes. Western blots also showed that AgSGS4 and 5 are only present in adult mosquitoes more than one day old and that both proteins are upregulated following a blood meal.

These data suggest involvement in blood feeding activity. Immunohistochemistry showed that AgSGS4 and 5 localize on both the apical and basal surfaces of acinar salivary cells. Similarly, Western analyses showed that AgSGS4 and 5 are present in the saliva and are also released into the hemolymph following certain events. Salivary gland and hemolymph AgSGS differ in mass and further bioinformatic analyses predicted protease cleavage sites in both AgSGS4 and 5 that correspond to the mass of hemolymph fragments. The proteases predicted to cleave AgSGS are known to function in insect wound healing and innate immunity, suggesting possible roles for hemolymph AgSGS. Upcoming experiments examining the involvement of AgSGS during salivary gland invasion by Plasmodium berghei are outlined, along with further investigations into the role of hemolymph AgSGS.

#26 Yinzi Liu Solnica-Krezel Lab, 2nd Year with Adi Inbal Identification of Stat3 Targets in Zebrafish Convergence and Extension Gastrulation Movements Convergence and extension (C&E), which narrows the germ layers mediolaterally and elongates the embryo anteroposteriorly, is one of the evolutionarily conserved cell movements during zebrafish gastrulation. Noncanonical Wnt/ Planar Cell Polarity (PCP) pathway is implicated by more and more evidence in the regulation of cell polarization in C&E. However, what activates PCP signaling and what signal(s) attract cell migration toward the dorsal gastrula midline is still not clear.

It has been proposed that the zebrafish Signal transducer and activator of transcription 3 (Stat3) is a upstream activator of PCP pathway during C&E. Stat3 is activated in cells on the dorsal side shortly after the midblastula transition (MBT) by the maternal canonical Wnt/β-catenin pathway. Stat3 function is required for the anterior migration of the prechordal plate progenitor cell-autonomously and convergence of the paraxial mesendoderm noncell-autonomously. Therefore, the extracellular gradient of some unknown signaling molecules downstream of Stat3 is required for lateral mesendodermal cells to sense the direction of C&E. In order to identify the unknown Stat3 targets, we will purify Stat3-expressing prechordal plate cells and characterize the gene expression profile of the cells from both wild-type and Stat3-deficient embryos (injected with Stat3 antisense morpholino oligonucleotides). To isolate prechordal plate cells we generated Tg[gsc: Gal4-VP16] and Tg[UAS-CD8-GFP] transgenes, which afford expression of CD8 antigen under goosecoid gene promoter. I showed that progeny of Tg[gsc: Gal4-VP16] and Tg[UAS-CD8-GFP] transgenic fish, express CD8-GFP fusion protein in the prechordal plate. I will dissociate wild-type and Stat3-deficient gastrulae expressing CD8 and use magnetic beads coupled with anti-CD8 antibody for cell purification. A whole-genome microarray analysis will be carried out, followed by putative Stat3 targets identification.

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Session I Chair: Elizabeth Thatcher Patton Lab, 5th Year

#23 John Gibbons Rokas Lab, 2nd Year Deciphering the Functional Diversity of Intragenic Tandem Repeats across Ten Aspergillus Proteomes

#01 Robert Taylor Gamse Lab, 4th Year leftover(kctd12.1) Misexpression Disrupts Asymmetry in the Zebrafish Epithalamus The paired habenular nuclei are a well-conserved relay center connecting the limbic system with effector nuclei in the midbrain, and are present in all vertebrates. In recent years, an array of asymmetries has been described between the left and right habenulae in zebrafish. These asymmetries include left-right differences in size, transcriptional activity, neuropil and synaptic density, and both afferent and efferent innervation. Thus, the zebrafish habenular complex is a valuable vertebrate model of both nervous system laterality and higher-order connectivity. leftover (lov), a potassium channel tetramerization domain-containing (kctd) gene, is asymmetrically expressed predominantly in the left habenulae, and encodes a small T1-oligomerization domain-containing protein of unknown function. Lov localizes to the cytosol and can be detected in habenular axons and dendrites, but is not required for normal habenular development. In the present study, we use the GAL4/ UAS system to drive bilateral habenular overexpression of Lov:Myc fusion protein. Surprisingly, overexpression of Lov:Myc inhibits left-sided habenular characteristics such as left-sided gene expression, neuropil density, and characteristic axon targeting. This result suggests that Lov:Myc may act to suppress endogenous Lov function in a dominant negative manner. Further genetic and functional analysis may reveal a novel role for KCTD proteins in the fine control of the neuronal development.

Intragenic tandem repeats (ITRs) are consecutive repeats of three or more nucleotides found in coding regions. ITRs are the underlying cause of several human genetic diseases, and have been associated with phenotypic variation, including pathogenesis, in several kingdoms. We have examined the evolution and functional role of ITRs in ten genomes spanning the fungal genus Aspergillus, a clade of relevance to medicine, agriculture, and industry. We identified several hundred ITRs in each of the species examined. ITR content varied extensively between species, with an average 89% of ITRs unique to a given species. For the fraction of conserved ITR regions, sequence comparisons within species and between close relatives revealed that they were highly polymorphic. ITR-containing proteins were evolutionarily less conserved, compositionally distinct than other proteins in the genome and overrepresented for domains associated with cell-surface localization and function. Furthermore, ITR-containing proteins were preferentially associated with the functional processes of transcription, cellular communication and cell-type differentiation and disassociated from metabolism and energy. Despite the evolutionary liability of ITR regions, their functional associations appear to be remarkably conserved across eukaryotes. Fungal ITRs are likely involved in a variety of developmental processes involving transcriptional regulation, such as control of self / non-self recognition, as well as to cell-surface associated functions such as cell adhesion. Thus, the contribution of ITRs to the fungal lifestyle may be more general than previously assumed.

#24 Charlene Hawkins

#02 Gulfem Dilek Guler Fanning Lab, 4th Year with Hanjian Liu, Jeannine Gerhardt, Sivaraja Vaithiyalingam and Walter Chazin Replication protein A-dependent recruitment of DNA helicase B to stalled replication forks

Friedman Lab, 2nd Year with Asmitha Sathiyakumar Characterizing Regions of Est1p That Contribute to Nuclear Localization and Telomerase Assembly

DNA repair mechanisms are highly integrated with DNA replication to ensure genomic stability, in part through dual function proteins that have roles in both DNA replication and DNA damage response. We suggest that human DNA helicase B (HDHB) helps to coordinate DNA replication with damage responses and repair.

DHB is a robust 5’-3’ DNA helicase of superfamily I. It is highly conserved among vertebrates and its activity is necessary for initiation of DNA replication in both mouse and human cells. Both human and mouse DHB has primosome activity, which is conserved in all kingdoms of life and is required for replication initiation, intra-S phase checkpoint signaling, and restart of stalled replication forks. In accordance with a role in preserving genomic stability, silencing of HDHB elevates the number of aphidicolininduced chromosomal breaks. HDHB is enriched in common fragile site FRA16D during unperturbed S phase.

HDHB accumulates on chromatin upon treatment of S phase cells with DNA damaging agents, most prominently with genotoxins that arrest replication forks. These findings suggest that HDHB is recruited to inactivated replication forks. HDHB interacts physically with N terminal domain of RPA70 subunit (RPA70N), a region previously identified as a recruitment dock for damage response proteins ATRIP and p53. Preliminary NMR and immunoprecipitation results indicate that HDHB binds to RPA70N in the same basic cleft as ATRIP and p53. Genotoxin-induced chromatin recruitment of HDHB is defective in RPA70 silenced cells. Our results suggest that HDHB is recruited to stalled replication forks through RPA70N and protects genomic stability.

Telomerase is a ribonucleoprotein complex that restores telomeric sequences lost with consecutive rounds of DNA replication in a cell cycle dependent manner. In Saccharomyces cerevisiae, telomerase contains a catalytic reverse transcriptase termed Est2p, an RNA template used for nucleotide addition, TLC1, and two additional subunits, Est1p and Est3p, that serve regulatory functions in the assembled complex. It has been shown previously that Est1p is both necessary and sufficient for Est3p’s recruitment to the telomerase complex. Here we have performed deletion analysis in Est1p to map regions required for complex assembly and Est3p recruitment. Results of these experiments indicate that the C-terminal 136 amino acids of Est1p are important for its Est3p recruitment function and that an N-terminal region of Est1p may contribute to its interaction with Est2p. Additionally, this analysis has led to the identification of a putative nuclear localization sequence (NLS) in Est1p.

Here we show that mutation of positively charged residues at the N-terminus (within residues 107123) of Est1p to alanine results in failure of the protein to localize to the nucleus and in shortened telomere lengths. Telomere shortening is a direct consequence of mislocalozation since attachment of the SV40 NLS to these mutants rescues both the nuclear localization and telomere length phenotypes. We are currently investigating which nuclear import pathway is required for Est1p localization.

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#21 Ryan Baldridge

#03 Brian Robertson

Graham Lab, 2nd Year

Johnson Lab, 4th Year

Substrate specificity of Saccharomyces cerevisiae P4 ATPases

Real-time Luminescence Monitoring of Cell-cycle and Respiratory Oscillations in Yeast

Membrane asymmetry is maintained in many cellular membranes by energy-dependent processes. Drs2 is a Saccharomyces cerevisiae P4 ATPase that is thought to help generate and maintain the asymmetry by flipping phosphatidylserine from the luminal leaflet of late Golgi cisternae to cytosolic leaflet. Dnf1 is another P4-ATPase that is present at the plasma membrane, and contributes to phosphatidylcholine and phosphatidylethanolamine flip at the plasma membrane. Current evidence indicating Drs2 and Dnf1 as flippases does not exclude the idea that Drs2 and Dnf1 drive phospholipid translocation by a secondary transport mechanism. The focus of this work was to determine if the phospholipid specificity of the transport process is conferred by the P4-ATPases, consequently supporting a primary transport mechanism. A potential substrate binding site was identified by sequence comparisons and this region was swapped between Drs2 and Dnf1. Plasma membrane NBD-lipid uptake analysis of the swapped protein yielded inconclusive results for alteration of lipid specificity. Complementation tests of the chimeric protein indicate it does not complement the cold sensitivity of a drs2Δ strain. The localization of the chimeric protein may help to shed light on these results.

#22 Daniel Erickson

The budding yeast Saccharomyces cerevisiae is a widely studied model organism for many eukaryotic processes and has significant commercial value as a workhorse for industrial fermentation and biological production of useful chemicals and enzymes. When grown in the presence of abundant nutrients (e.g. over-night, batch cultures in rich media) S. cerevisiae cells divide rapidly and asynchronously with others in the culture. However, when S. cerevisiae is grown in a starving, dense culture that is maintained by a continuous supply of slowly introduced nutrients, half of the cells in the culture divide synchronously, on a timeframe proportional to the rate of supplied nutrients (~ 8 hours). Under these growing conditions, S. cerevisiae establishes stable respiratory oscillations characterized by recurring events of intracellular hypoxia which have a period of about one half of the average cell division time of the culture (i.e. ~ 4 hours). I believe these recurring events of hypoxia serve to synchronize cell division such that 2 populations of synchronously dividing cells are formed, each population taking turns dividing every 4 hours. The cell division rate, once synchronized, serves as a “timer” for the next occurrence of hypoxia and thus serves as a timer and stabilizer for the oscillation. Using a luminescent reporter for cell division promoter activity, I show that there is a direct correlation between premature induction of cell division and respiratory oscillation phase resetting that occurs as a result of introducing substances like ethanol which may serve as a signal for respiration in the oscillating system.

#04 Joshua Broussard

Abbot Lab, 2nd Year The community context of secondary endosymbiosis in ant-hemipteran mutualisms

Webb Lab, 3rd Year The Function of an Adaptor Protein, APPL1, in Cell Migration

Many insects have mutualistic associations with intracellular bacterial endosymbionts. In the insect order Hemiptera, nutritional deficiencies connected to sap-feeding have favored the formation of highly derived obligate microbial mutualisms which supply essential nutrients lacking in the insect diet. Recent discoveries have described more loosely associating “secondary” endosymbionts that affect insect fitness by shaping interactions with host plants, parasitoids, and predators.

Most studies to date have examined the functional basis of these interactions in lab-raised clonal lines of the pea aphid. However, any mutualistic phenotype is dependent on both the biotic and abiotic environment of the mutualism. Many hemipterans participate in mutualisms with ants, in which the aphids produce honeydew to recruit ant protection from predators. In order to better understand the potential interactions between the two microbial and ant mutualisms, we collected hemipterans in two separate geographic locales in central Colorado. We then screened the hemipterans for 5 bacterial symbionts, in order to determine bacterial frequencies as function of aphid species, numbers, host plant, and distance from ant mound on prevalence of bacteria within aphid clones. We found a high frequency of Hamiltonella defensa and Serratia symbiotica within two aphid species, Obtusicauda frigidae, Aphis varians, respectively, and two undescribed rickettsial species in a membracid Publilia modesta. We have also begun to see correlations between bacteria prevalence and aphid numbers and distance from ant mounds, implying correlations between ant attendance and bacteria presence. In the future, we intend to study the effect the direct effect of these bacteria on ant attendance in the ant-hemipteran mutualisms.

Cell migration is multifaceted and is central to many biological processes, as well as pathological conditions such as cancer invasion and metastasis.

Cell migration is a response to a complex web of signaling events for which the underlying molecular mechanisms are still not well understood. Adaptor proteins, such as APPL1, play an important role in integrating components of a signaling cascade through the use of their multiple functional domains. Western blot analysis of endogenous APPL1 in cell lines revealed an inverse relationship between APPL1 expression and a cells ability to migrate, suggesting a potential role for APPL1 in cell migration. Our results indicate, for the first time, that APPL1 regulates cell migration. Increasing levels of APPL1 by exogenous expression decreases the average velocity of migrating cells. A potential mechanism by which APPL1 could regulate migration is through integrin trafficking. APPL1-GFP traffics with mCherry-a5-integrin as well as colocalizes with members of the Rab family of small GTPases that are known regulators of integrin trafficking. APPL1 also directly binds to proteins such as Akt, whose many downstream effectors regulate processes essential in cell migration. Our results show that Akt is involved in APPL1’s regulation of cell migration. Expression of constitutively active Akt increases the average velocity of cell migration, and APPL1 expression is able to abolish this effect.

We have also shown that APPL1 expression results in a decrease in the level of active Rac. Although the function of APPL1 is still not well understood, we hypothesize that APPL1 can regulate migration through its effects on Akt, integrin trafficking, and Rac.

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#05 Morgan Sammons

#19 Suraj Adhikay

Link Lab, 4th Year

Eichman Lab, 2nd Year

Examination of the role of Znf9 in cap-independent translation and myotonic dystrophy type 2

Structural and biochemical analysis of an alkylation damage specific DNA repair enzyme – Methylpurine DNA glycosylase II

Myotonic dystrophy is the most common form of adult-onset muscular dystrophy, affecting approximately 1 in every 8,000 individuals. Myotonic dystrophy type 2 (DM2) is part of a larger class of diseases known as nucleotide repeat expansion disorders, which include fragile X syndrome and Huntington’s disease. DM2 is caused by a large expansion of the tetranucleotide CCTG in the first intron of the ZNF9 gene, and the pathology of DM2 is believed to be due to an RNA-mediated gain-of -function caused by the large expansion of nucleotides. The role of the ZNF9 gene product in the mechanism of the disease is still uncharacterized.

Znf9 was initially identified as a single-strand nucleic acid binding protein, but a more detailed molecular function is unknown. Znf9 was identified in a proteomic screen to identify regulators of the process known as cap-independent translation. Preliminary evidence suggests that Znf9 functions as a trans-activating factor for cap-independent translation. Additionally, Znf9 interacts with actively translating ribosomes, further suggesting a role in translation. Finally, myoblasts from patients with myotonic dystrophy show a reduced level of cap-independent translation that may be explained by reduced function of Znf9.

The genome of living organisms is constantly challenged by various environmental and chemical agents that threaten the chemical integrity of DNA. For example, alkylating agents covalently modify nucleobases to generate mutagenic and cytotoxic lesions that can lead to various diseases including cancer. All organisms possess strategies to repair such DNA damage. Alkylation damage is repaired by the base excision repair pathway (BER), which is initiated by damage-specific DNA glycosylases. These enzymes hydrolyze the glycosylic bond linking the damaged base to the phosphoribose backbone, to liberate a free base and an abasic site that must be further repaired by BER proteins to restore the undamaged DNA. Methylpurine DNA glycosylase II (MpgII) is a ubiquitous bacterial DNA repair enzyme that excises several N-alkylpurine adducts, including 3methyladenine and 7-methylguanine, from DNA. Compared to other alkylpurine DNA glycosylase, MpgII recognizes a specific subset of DNA lesions. Structurally, MpgII is predicted to belong to the EndoIII family (along with MutY and MIG) of the Helix-Hairpin-Helix (HhH) superfamily of DNA repair glycosylases. The EndoIII family of proteins is characterized by presence of an iron-sulfur ([4Fe-4S]2+) cluster near the C-terminus, which is believed to aid in recognition of DNA lesions and enhance DNA binding. We plan to determine the crystal structure of MpgII from Aquifex aeolicus, which would help us understand the structural basis of DNA lesion recognition. This could provide a platform for rational design of DNA-glycosylase inhibitors which can be used in conjunction with chemotherapy to reduce the harmful effects of the procedure.

Session II Chair: Dan Anderson Johnson Lab, 5th Year

#20 Kimberly Anderson

#06 Jenifer Ferguson

Patton Lab, 2nd Year

Friedman Lab, 4th Year

Isolated Growth Hormone Deficiency Type II: Mechanisms and Therapies Multiple Degron Motifs Mediate S. cerevisiae Est1p Cell -cycle Regulated Degradation Telomeres, maintained by telomerase, protect chromosome termini from recognition as DNA breaks. In Saccharyomyces cerevisiae, Est2p, a reverse transcriptase, and the TLCI RNA template constitute the catalytic core of telomerase, while Est1p and Est3p are regulatory components. Est1 protein levels are reduced during G1 phase due to proteasome-dependent degradation.

Because Est1p is both necessary and sufficient for Est3p association, this cell cycle-regulated degradation is a key regulatory event in the assembly of the enzyme.

To gain further insight into Est1p’s degradation, I set out to identify the machinery that targets Est1p to the proteasome during G1 phase. The Anaphase Promoting Complex (APC), an E3 ubiquitin ligase, is active during G1 and is therefore a good candidate. Consistent with this idea, deletion of the gene encoding the APC-activating factor Cdh1p partially stabilizes Est1p during G1. Proteins directly targeted for ubiquitination by APCCdh1 contain specific degron sequences (termed Destruction or “D” boxes) that mediate interaction with Cdh1p and are required for substrate degradation. EST1 contains six matches to this sequence (RxxL), at least three of which significantly contribute to Est1p degradation during G1. These data suggest that APCC dh1p recognizes multiple D-box sequences within Est1p, resulting in instability. To test this hypothesis, Est1p degradation was monitored in a cdc23-1 strain wherein the APC is inactivated at the restrictive temperature. A known APC target, Clb2p, is fully stable in this background; however, Est1p is not. We conclude the APC contributes to Est1p degradation, but is not the sole mechanism targeting it to the proteasome in G1.

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Isolated Growth Hormone Deficiency Type II is a dominant negative disorder associated with aberrant splicing events in the human growth hormone gene that lead to exon skipping, production of a shortened isoform of growth hormone, and prevention of growth hormone release from pituitary somatotrophs. Patients with IGHD II present with low circulating growth hormone levels and short stature. Recombinant hGH can be used to treat IGHD II, but does not address both the anterior pituitary hypoplasia that may result from severe IGHD II, and subsequent pan-pituitary problems. Additionally, the mechanism of dominant negativity is poorly understood, slowing the development of alternative therapeutic options.

In order to explore the mechanism of IGHD II, a corticotrophic cell line will be used to create stably transfected inducible wild type and 17.5kD mutant growth hormone alleles. These cells lines will be used in vesicle trafficking and secretion studies to elucidate the point at which expression of the 17.5kD isoform prevents the release of wildtype GH secretion and the mechanism that leads to pituitary hypoplasia.

In addition, we have previously shown that a genetically delivered shRNA is able to rescue a mouse model of IGHD II. We are currently developing a delivery vehicle for siRNA directed against the 17.5kD GH mRNA in order to discover a clinically relevant means to treat IGHD II via RNAi.

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#07 Emily Rubinson Eichman Lab, 3rd Year A New Protein Architecture for Processing Alkylation Damaged DNA: The Crystal Structure of DNA Glycosylase AlkD DNA glycosylases safeguard the genome by locating and excising chemically modified bases from DNA. AlkD is a recently discovered bacterial DNA glycosylase that removes positively charged methylpurines from DNA, and was predicted to adopt a protein fold distinct from other DNA repair proteins. The crystal structure of Bacillus cereus AlkD shows that the protein is composed exclusively of helical HEAT-like repeats, which form a solenoid perfectly shaped to accommodate a DNA duplex on the concave surface. Structural analysis of the variant HEAT repeats in AlkD provides a rationale for how this protein scaffolding motif has been modified to bind DNA. Mutational analysis of 7mG excision and DNA binding activities has identified catalytically essential residues. Comparison of this novel glycosylase fold with existing alkylpurine DNA glycosylase structures provides important insight into the requirements for alkylation repair within DNA. This data suggests that AlkD utilizes a novel strategy to manipulate DNA in its search for alkylpurine bases.

#08 Daniel Carlin Solnica-Krezel Lab, 4th Year with Adi Inbal, Seok-Hyung Kim, Vandana Grover, Michael Cooper Six3 Cooperates with Hedgehog Signaling to Pattern the Ventral Telencephalon The telencephalon develops at the anterior end of the central nervous system and gives rise to the cerebral cortex and basal ganglia. During development the telencephalon is patterned into several progenitor regions along the dorsoventral axis. Specifically, ventral telencephalic progenitors will give rise to a number of cell types, including cholinergic interneurons of the stiatum, oligodendrocytes, and GABAergic cortical interneurons. The molecular programming involved in generating these cell types is not well understood. We have found that the transcription factor Six3 is required for proper generation of ventral telencephalic progenitor domains in zebrafish.

Embryos with reduced function of Six3 show an expansion of dorsal telencephalic domains at the expense of ventral domains, notably similar to reduction of Hedgehog (Hh) signaling. We sought to identify an interaction between these two pathways. Whereas Hh signaling is required for nk2.1b expression in the most ventral region of the telencephalon, embryos with a partial loss of Six3 function coupled with loss of Hh signaling show a synergistic reduction of more dorsally located isl1-positive cells. Epistasis experiments show that Six3 can promote generation of isl1-positive but not nk2.1b-positive cells in the absence of Hh signaling. However, excessive Hh signaling is not sufficient to restore nk2.1b expression in embryos with reduced Six3 function, thus revealing a complex interaction between these pathways. Current work is aimed at generating tools and assays to identify the cellular mechanisms involved in reduction of the ventral telencephalic domains in embryos with reduced Six3 function. As Six3 and Hh signaling have both previously been linked to generation of holoprosencephaly—a congenital defect with complex inheritance in which the forebrain does not properly divide into two hemispheres—an understanding the nature of the interaction between Six3 and Hh signaling and the cellular basis of how this phenotype is generated are of particular interest.

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#09 Patrick L. Jones Zwiebel Lab, 3rd Year with Jonathan D. Bohbot, Guirong Wang Structure-Function Analysis of Mosquito Odorant Receptors Genes encoding two odorant receptors (OR2 and OR10) display a remarkable level of sequence conservation between and within the disease vector mosquitoes, Anopheles gambiae and Aedes aegypti. Microsyntenic relationships, gene structure, and expression pattern in olfactory tissues suggest these genes are the products of a gene duplication event pre-dating the separation of the Anopheline and Culicine lineages. We have utilized a Xenopus laevis oocyte heterologous expression system coupled with two-electrode voltage-clamp electrophysiology to functionally characterize OR2 and OR10 from An. gambiae (AgOR2/AgOR10) and Ae. aegypti (AaOR2/AaOR10). Here we report that a cognate odorant of An. gambiae and Ae. aegypti evokes responses in all OR2/OR10 proteins from both species. Based on our sensitivity criterion (EC50), OR2 and OR10 represent two distinct functional groups. The OR2 gene lineage is approximately 10-fold more sensitive than the OR10 gene lineage. Homologous gene lineages retain similar function, whereas paralogous genes, OR2 versus OR10, display diverging function. Ae aegypti possesses an additional paralog in AaOR9, which based on genomic structure and sequence similarity would be predicted to be functionally closer to AaOR10 than to AaOR2. We see a direct correlation between gene phylogeny and sensitivity to odorant. Additionally, we created reciprocal chimeric ORs, which reveal important aspects of OR evolution. We are proceeding with mutational analyses to determine which regions are responsible for odorant-sensitivity within this clade and provide new insight into the functionality of this protein family.

#10 Joshua A. Clanton Gamse Lab, 3rd Year Fibroblast growth factor signaling is required for parapineal formation The zebrafish epithalamus is a region of the forebrain consisting of the pineal organ, an accessory parapineal, and two bilaterally opposed habenular nuclei. The parapineal emerges from the left, anterior part of the pineal complex anlage beginning at approximately 30 hours post fertilization (hpf) and migrates leftward and posteriorly to lay adjacent to the left habenula. The migration of the parapineal is concomitant with the initiation of asymmetry in the habenulae, with the left habenula being greater in size, cell number, and gene expression than the right habenula. Little is currently known about what factors control parapineal morphogenesis.

One candidate is Fibroblast growth factor 8a. fgf8a is expressed within the developing epiphysis beginning at approximately 20hpf and persisting past 36hpf, overlapping with the appearance and migration of the nascent parapineal. In addition, fgf receptor 4 is expressed in migrating parapineal cells. acerebellar x15 (acex15) , an fgf8a mutant, has fewer parapineal cells than wild-type siblings. Treatment of embryos with SU5402, a small molecule inhibitor of Fgf receptors, from 24 to 30hpf also results in a paucity of parapineal cells. In acex15 mutants, the reduction in parapineal cell number may correlate with an increase in the number of ZPR1-positive cone cells, which are typically found only in the pineal anlage. These data suggest that Fgf signaling could be required for the maintenance of parapineal cell fate.

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#18 Seth Bordenstein, Ph.D. Assistant Professor of Biological Sciences Wolbachia pipientis: The Global Heritable Pandemic Many bacteria establish chronic interactions with the cells of multicellular eukaryotes. These obligate intracellular bacteria possess the extraordinary ability to invade, replicate, and persist inside host cells. They can range from harmless symbionts to the etiologic agents of devastating diseases.

Once these bacteria invade host cells, they all face a similar challenge of avoiding host defenses and creating strategies to persist prior to being subverted by the hosts. One of the most prolific intracellular bacteria in the world - a pandemic to animal species - is Wolbachia pipientis. This bacterium infects the majority of filarial nematodes, up to 70% of arthropod species, and humans; and in the first two cases, they colonize host eggs for chronic, maternal transmission. W. pipientis cause an array of symptoms in their animal hosts. They are obligate mutualists of filarial nematodes by assisting host fertility and larval development. Conversely, W. pipientis can be released from the hypodermis of the helminth vector and cause the acute pathologies of human river blindness, elephantiasis, and dog heartworm. Filarial nematodes are also the only nematodes that have an obligate insect vector, and the most prevalent W. pipientis strains are ancient, reproductive parasites that distort sex ratios and sex determination in 70% of the world’s arthropod species.

Thus, millions of animal species are infected by W. pipientis and the varied symptoms the bacteria pose can have important evolutionary, ecological, and human health consequences. In this talk I will touch upon our lab’s key research questions: Do these inherited infections promote rapid speciation in arthropods? What is the ancestry of bacterial parasitism and mutualism? What are the rule or patterns that characterize mobile elements in intracellular bacteria? and What animal immune signals regulate interactions with this chronic infection?

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#11 Weston Dulaney Fanning Lab, 3rd Year Interactions of SV40 Large T Antigen with the Catalytic Subunit of DNA Polymerase Alpha-Primase

#17 Antonis Rokas, Ph.D. Assistant Professor of Biological Sciences Harnessing Genomics for Evolutionary Insights

SV40 is a DNA tumor virus that expresses large T antigen (Tag), a multifunctional replication protein that binds viral origin DNA, unwinds the parental DNA, and recruits host proteins to replicate the viral genome.

DNA polymerase alpha-primase (pol-prim) is an essential factor in the initiation of replication as it generates a RNA/DNA primer that is extended by DNA polymerase delta to continue replication. The catalytic subunit of pol-prim, p180, has been shown to interact with human Mcm10 in vitro and in vivo. Another p180-interacting protein, And-1/Ctf4, was characterized in S. cerevisiae as an essential factor in generating sister chromatid cohesion. Interestingly, in two contradictory reports, either Mcm10 or And-1 was shown to protect p180 from degradation in human cells. Unlike pol-prim, Mcm10 and And-1 are not required to replicate SV40 DNA in vitro, but might stabilize p180 in infected cells. It has been previously shown that p180 interacts with Tag and that preformed Tag-p53 complexes have lowered affinity for p180. Using the crystal structure of the Tag helicase domain as a guide, we are using site directed mutagenesis, yeast twohybrid analysis, and GST pull-down assays to identify a possible binding surface for p180 on Tag proximal to the p53 binding site. By investigating the interactions of these proteins with one another and with Tag, we hope to determine the mechanism by which polprim is passed from Mcm10/And-1 to viral replication centers.

Session III

Examination of the DNA record can yield important clues about organisms’ biological past, and their history of change and adaptation. I will discuss two projects from our lab that have taken advantage of genomic data to understand the genetic basis of adaptation: (i) Although many fungi have been domesticated for human food production, the molecular signature left by their domestication has rarely been examined. We tested the effect of domestication on the proteome of Aspergillus oryzae, a fungus employed in the fermentation of several traditional Japanese sauces and drinks for two millennia. A variety of population genetic analyses are inconsistent with selective relaxation in A. oryzae due to domestication, suggesting that the relaxation of selective constraints usually observed in human-mediated domestication of animals and plants may not be typical of domesticated fungi.

Chair: Eric Janson Abbot Lab, 5th Year

#12 Diane Kanter Kaplan Lab, 4th Year with Irina Bruck Mcm subunits can assemble into two different active unwinding complexes

(ii) How species maintain genetic variation is central to explaining how they evolve. Japanese strains of Saccharomyces kudriavzevii cannot utilize galactose because all seven galactose (GAL) utilization genes are inactive pseudogenes, in stark contrast to several recently isolated Portuguese strains of S. kudriavzevii that can utilize galactose. In collaboration with the Johnson lab at Washington University in St. Louis, we investigated the genetic basis and genome-wide impact of this variation by sequencing all 18 known S. kudriavzevii isolates using Solexa’s next-generation sequencing platform, covering approximately 80% of their genomes. Surprisingly, all GAL genes are functional in the Portuguese strains, and none have been acquired from other species. Laboratory crosses provide no evidence for reproductive barriers between Japanese and Portuguese strains, and the unlinked GAL genes segregate independently. The divergence at neutral sites within the GAL genes suggests that the multi-locus GAL network has been maintained in these two states for nearly the entire S. kudriavzevii lineage, despite recent gene flow throughout most of the genome.

This natural variation suggests that, in addition to better-understood single nucleotide and copy number polymorphisms, species are also capable of maintaining complex, multi-locus variation involving complete gene networks.

The replication fork helicase in eukaryotes is a large complex that is composed of Mcm2–7, Cdc45, and GINS. The Mcm2–7 proteins form a heterohexameric ring that hydrolyzes ATP and provide the motor function for this unwinding complex. A comprehensive study of how individual Mcm subunit biochemical activities relate to unwinding function has not been accomplished. We studied the mechanism of the Mcm4-Mcm6-Mcm7 complex, a useful model system because this complex has helicase activity in vitro. We separately purified each of three Mcm subunits until they were each nuclease-free, and we then examined the biochemical properties of different combinations of Mcm subunits. We found that Mcm4 and Mcm7 form an active unwinding assembly. The addition of Mcm6 to Mcm4/Mcm7 results in the formation of an active Mcm4/Mcm6/Mcm7 helicase assembly. The Mcm4-Mcm7 complex forms a ringed-shaped hexamer that unwinds DNA with 3_ to 5_ polarity by a steric exclusion mechanism, similar to Mcm4/Mcm6/Mcm7. The Mcm4-Mcm7 complex has a high level of ATPase activity that is further stimulated by DNA. The ability of different Mcm mixtures to form rings or exhibit DNA stimulation of ATPase activity correlates with the ability of these complexes to unwind DNA. The Mcm4/Mcm7 and Mcm4/Mcm6/Mcm7 assemblies can open to load onto circular DNA to initiate unwinding. We conclude that the Mcm subunits are surprisingly flexible and dynamic in their ability to interact with one another to form active unwinding complexes.

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#13 Caleb Doll

#15 Kavitha Surendhran

Gamse Lab, 3rd Year

Graham Lab, 3rd Year

The Vertebrate Translocon sec61α1 is Required for Habenular Asymmetry

Role for Drs2p, a potential phospholipid translocase from Saccharomyces cerevisiae, in membrane curvature and vesicle generation

Brain asymmetry is a conserved feature of the vertebrate central nervous system. Most non-mammalian vertebrates, including the zebrafish Danio rerio, exhibit lateralized differences in epithalamic development. The epithalamus includes the pineal complex and paired habenular nuclei. Previous studies have demonstrated left/right differences in habenular size, volume of dense neuropil, and gene expression; these ultimately suggest a mechanism of specific, lateralized control of neurogenesis. For example, in wild type zebrafish the leftover gene transcript is expressed in more cells of the left habenula as compared to the right. In an in situ hybridization screen of mutagenized fish, a mutant, named big time, was identified in which leftover transcript expression is expanded in the right rostral habenula. We mapped this mutation to the major subunit of the vertebrate translocon, the gene sec61α1. This endoplasmic reticulum pore may mediate epithalamic asymmetry through processing of signaling proteins that regulate habenular neurogenesis. The components of the Notch pathway are promising candidates, as mind bomb mutants with deficient Notch signaling phenocopy the big time habenular phenotype, and Notch overexpression results in a drastic reduction of leftover-expressing neurons. Therefore, the interaction between an intracellular pore complex and the Notch pathway could provide insight on the regulation of asymmetric neurogenesis.

Drs2p is a P-type ATPase responsible for the phospholipid translocase (flippase) activity in the yeast trans-Golgi network (TGN) and is also required for budding clathrin coated vesicles from the TGN. The proposed role of flippases in vesicle budding is based on the bilayer-couple hypothesis proposed by Sheetz and Singer. Flippase-catalyzed phospholipid (PL) translocation from the luminal leaflet to the cytosolic leaflet of the Golgi will increase the surface area of the cytosolic leaflet while decreasing that of the luminal leaflet. Because the two leaflets are physically coupled, the imbalance of phospholipid number should facilitate the generation of tightly curved transport vesicles that bud into the cytosol. Unlike other TGN resident proteins, Drs2p does not traffic through the late endosomes (LE). We hypothesize that Drs2p is restricted form the LE because its flippase activity would inhibit the inward budding of vesicles to generate multivesicular bodies (MVBs). If PL translocation plays a general role in vesicle budding, then floppases that mediate outward directed (luminal) phospholipid floppase activity may support the budding of intraluminal vesicles during MVB biogenesis. Our ultimate goal is to provide in vivo evidence for the flipping activity for the so called flippases and floppases and to demonstrate that these are indeed required for the inward and outward vesicle budding phenomenon in the Golgi/Endosomal system.

#16 Patrick D. Robertson

#14 Isi Ero-Tolliver

Eichman Lab, 4th Year

Fanning Lab, 3rd Year The role of a SV40 Large Tag-CUL7 complex in the degradation of the MRN complex The DNA tumor virus SV40 is a model system for tumorigenesis, which requires the viral oncoprotein large T antigen (Tag). Tag binds to and inactivates key tumor suppressor proteins such as retinoblastoma family proteins and p53 to promote G1/S cell cycle progression and inhibit apoptosis (Ahuja et al., 2005). Tag binding to the E3 ubiquitin ligase cullin subunit CUL7 is also required for the oncogenic activity of Tag, but its mechanism of action in oncogenesis and in the viral life cycle is not understood (Kasper et al., 2005). Our lab recently found that the interaction of Tag and CUL7 in productively infected cells targets subunits of the checkpoint signaling complex Mre11Rad50-Nbs1 (MRN) for degradation (Zhao et al., 2008). Previous results show that Tag mutants defective in CUL7 binding are unable to decrease levels of Nbs1. To map the region of CUL7 that binds Tag, I am using site-directed mutagenesis of CUL7 and testing mutants for interaction in yeast two-hybrid assays. The future direction of my research is to define the mechanism of MRN degradation directed by Tag-CUL7 by testing two models. In one model, CUL7-Tag targets MRN for ubiquitination by Roc1-E2 through Skp1-Fbx29 bound to CUL7. In a second model, CUL7-Tag targets MRN through direct binding to Tag. Based on both models, Nbs1-ubiquitin conjugates should accumulate in infected cells in the presence of proteasome inhibitors, which I am currently testing.

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Structural and Biochemical Characterization of the C-terminal Domain of Mcm10 Eukaryotic DNA replication is tightly regulated at the initiation phase to ensure that the genome is copied only once and at the proper time during each cell cycle. During replication initiation, over twenty different proteins are recruited to origins of replication to denature the DNA duplex and assemble a functional replication fork. Mini-chromosome maintenance protein 10 (Mcm10) is a DNA binding protein that is recruited to origins in early S-phase and is required for the activation of Mcm2-7, the replicative DNA helicase. Importantly, Mcm10 is necessary for subsequent loading of downstream replication proteins, including cell division cycle 45 (Cdc45), replication protein A (RPA), and DNA polymerase α-primase (pol α), onto chromatin. Despite its importance in both replication fork assembly and progression, the precise role of Mcm10 remains undefined. In order to better understand the structure-function relationship of vertebrate Mcm10, we have characterized the individual domains of Xenopus laevis Mcm10 (XMcm10), which shares a high sequence homology with the human protein. XMcm10 contains three structured regions: a putative oligomerization domain at the N-terminus (NTD) and two independent DNA binding domains located in the internal (ID) and C-terminal (CTD) regions of the protein. XMcm10CTD is stabilized by two zinc ions and binds to both single- and double-stranded DNA and to the p180 polymerase subunit of pol α. Here we present our progress towards the 3-dimensional structure of XMcm10-CTD using both x-ray crystallography and NMR spectroscopy. We aim to elucidate the DNA and pol α binding sites in XMcm10-CTD, with the ultimate goal of determining how the ID and CTD coordinate DNA and pol a binding.

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