Anita Corbett - US grants

The broad long term objective of this proposal is to understand how the Ran GTPase drives bi-directional movement of macromolecules across the nuclear envelope. There is evidence to suggest that Ran cooperates with a number of accessory proteins to move proteins into and out of the nucleus. The specific aims of this proposal are: 1) to examine the in vivo interactions that are essential for Ran function; 2) to test the hypothesis that the essential cellular role of the Ran-GDP binding protein, nuclear transport factor 2 (NTF2) is to concentrate Ran-GDP at the nuclear pore, where Ran is required to initiate nuclear import; and 3) to use a genetic approach to define the site(s) on the nuclear pore that represent(s) the docking site for NTF2 and most probably the NTF2- Ran complex. The proposed studies use the budding yeast Saccharomyces cerevisiae as a model for in vivo genetic and cell biological experiments and extend to biochemical studies, in vivo functional studies, and cell biological experiments and extend to biochemical studies, in vivo functional studies, and structural studies of both the yeast proteins and their highly conserved human counterparts. The health-relatedness of this proposal is two-fold. First, activated signal transduction pathways send a signal to the nucleus in order to respond to stimuli and activate transcription. This is most often accomplished by the movement of a protein into the nucleus. This aspect of signaling is often ignored or trivialized, yet it may represent an unexploited targeted for blocking specific cellular signals as well as the unregulated signals that arise in transformed cells. Second, viruses that infect human cells exploit the endogenous nuclear transport machinery both to gain entry to the nucleus and later to rapidly export their own replicated genetic material. A more detailed understanding of the machinery that mediates nuclear transport may provide novel targets for anti-viral therapies.

[unreadable] DESCRIPTION (provided by applicant): The absolute compartmentalization of the genetic material within the nucleus of the eukaryotic cell creates a critical control point for intracellular signaling. Whenever expression of specific genes is regulated in response to an intra- or extraceullar signals, information is transferred across the nuclear envelope. In many cases this information is transmitted as specific proteins that enter the nucleus to elicit changes in gene expression. Despite the biological importance of this process, the mechanism of this signal dependent nuclear targeting and translocation is not understood at the molecular level. In order for these events to serve as therapeutic targets the detailed molecular mechanism must be delineated. [unreadable] [unreadable] The broad long-term objective of this proposal is to understand how soluble transport factors cooperate with the nuclear pore complex to mediate bidirectional nuclear transport. This study combines in vivo analyses of the highly conserved transport factors in the budding yeast, S. cerevisiae, with quantitative analysis of protein-protein interactions and microinjection into Xenopus oocytes to learn how cargoes are targeted to and delivered into the nucleus. The specific aims of this proposal are to: 1) Analyze molecular interactions between NTF2 and the nuclear pores that are required to translocate NTF2 through pores and exploit this analysis to distinguish between current models for transport through the nuclear pore complex; 2) Examine the mechanism of NLS cargo delivery into the nucleus; and 3) Investigate how phosphorylation within NLS sequences modulates protein import. Results from these experiments will provide novel insights into the mechanism of nucleocytoplasmic transport. [unreadable] [unreadable] The health-relatedness of this proposal is two-fold. First, activated signal transduction pathways send signals to the nucleus in order to respond to stimuli and activate transcription. This transport step may represent an unexploited target for blocking specific cellular signals as well as the unregulated signals that arise in transformed cells. Second, viruses that infect human cells exploit the endogenous nuclear transport machinery to gain entry to the nucleus. A more detailed understanding of the machinery that mediates nuclear transport may provide novel targets for anti-viral therapies.

The objective of this project is to develop advanced single molecule spectroscopy methods for quantitative analysis of protein-nucleic acid interactions, as well as in their application to understand the physical mechanisms by which the zinc finger protein Nab2 binds to nucleic acids with sequence specificity and discriminates between single stranded RNA and DNA. Protein-nucleic acid interactions control all phases of gene expression from maintenance of the genome to RNA expression to translation and protein synthesis. This project has two broad research aims: advancing fluorescence fluctuation spectroscopy (FFS) methods as quantitative tools to investigate molecular interactions; and applying these methods to investigate the biophysical mechanisms underlying molecular recognition in the interaction of an essential mRNA processing and export protein, Saccharomyces cerevisiae Nuclear Abundant Poly(A) Binding Protein (Nab2) with nucleic acids. The methods developed will be widely useful for molecular interaction studies. The projects described here are the collaborative effort of the Berland (Physics Department, Emory College) and Corbett (Biochemistry Department, Emory School of Medicine) laboratories.

This project will have a broad impact through teaching and training opportunities that enable undergraduate and graduate students to work at the interface between physics and biochemistry. Importantly, these studies will set the stage for interdisciplinary learning both in the laboratory and in the classroom. This work also affords opportunities for interfacing with the community beyond Emory as both PIs laboratories have a history of interactions with high schools in Atlanta through hosting both students and teachers in the lab.

This project, which is at the interface of biology and physics, examines a very common macromolecular interaction domain, the zinc finger motif. Zinc finger proteins, which are among the most abundant proteins in eukaryotes, play critical functions in many biological processes. The researchers use a comprehensive approach to understand how an evolutionarily conserved family of zinc finger proteins including, the Saccharomyces cerevisiae Nab2 protein, which contains tandem (CCCH) zinc fingers, interacts with RNA. Nab2 is an essential yeast protein that plays critical roles in both mRNA processing and mRNA export from the nucleus to the cytoplasm. Previous studies have demonstrated that the Nab2 zinc finger domain is required for mRNA binding and the preliminary data suggests preferential binding to polyadenosine RNA. Three areas of research will be pursued: 1) determining whether the zinc finger motifs present in Nab2 and ZC3H14 do indeed confer sequence specific binding to poly(A) RNA; 2) defining how multiple zinc fingers contribute to binding specificity and/or high affinity nucleic acid binding thus providing insight into how tandem zinc fingers confer sequence-specific binding to poly(A) RNA; and 3) exploiting a zinc finger mutant of Nab2 (C437S) with documented decreased RNA binding to understand the requirement for RNA binding in vivo and to identify factors that regulate the interaction of Nab2 with mRNA transcripts. The Intellectual Merit of the research is two-fold: 1) the insights that will be gained into how a novel family of zinc finger proteins recognizes RNA; and 2) the development of biophysical methods not typically employed to study protein nucleic acid interactions. The researchers' approach employs Fluorescence Correlation Spectroscopy (FCS), biochemical approaches, and genetic studies in yeast. The studies described are the collaborative effort of the Corbett (Biochemistry Department, Emory School of Medicine) and the Berland (Physics Department, Emory College) laboratories. Thus, these studies lie at the interface of biology and physics. The Broader Impacts resulting from this research are significant contributions to training undergraduate students, graduate researchers, and postdoctoral fellows, including cross-disciplinary training through the focused interactions of trainees in physics and biology. Importantly, these studies also set the stage for the development of interdisciplinary learning in the classroom and afford enhanced opportunities for interface with the community beyond Emory. Both PI's laboratories have a long-standing history of interaction with students at all levels including those in high schools in the Atlanta area through hosting both students and teachers in the lab. The proposed studies would enhance interactions with students interested in Physics/Biophysics in and provide a strong illustration of the strength of interdisciplinary research.

The goal of this research is to understand how information within the genetic material of cells is actually read and used as a blueprint to create the building blocks needed to make and maintain cells. The researchers will study the messenger molecule, RNA, that moves the information from the cell nucleus out to the cell cytoplasm where the machinery is present to actually translate the genetic information. This process, called messenger RNA export is a critical step in gene expression or reading the genetic code. The work combines biochemistry and physics to approach this important question from a new direction and also to develop methods not previously used to study this question. Much of the work includes undergraduate students who work jointly between a Biological laboratory and a Physics laboratory. This interface between Biology and Physics also allows the researchers to develop new training methods including interdisciplinary courses and laboratories.

[unreadable] DESCRIPTION (provided by applicant): In eukaryotic cells, messenger RNAs are extensively processed and or modified both co- and post-transcriptionally. For example, the production of a mature mRNA requires addition of a 5'-methyl guanine cap, splicing out of introns, and coupled 3'-end cleavage/polyadenylation. These maturation steps, which must occur with precise accuracy to produce mature mRNA that can exit the nucleus and interface with the translation machinery in the cytoplasm, are mediated by numerous RNA binding proteins. mRNA maturation is essential for gene expression and hence cellular function. Furthermore, consistent with the critical importance of RNA maturation for proper cellular function, there are numerous examples where human disease is linked to alterations in the mRNA maturation/processing machinery or the cellular machinery that monitors the accuracy of these events. In fact, the major question that underlies quality control of any class of RNA is how cells distinguish properly processed RNAs from those that are incorrectly processed. Our long-term goal is to understand how the cell monitors mRNA export from the nucleus and how this process impacts human disease. This long-term goal will be addressed here by testing the hypothesis that mRNA binding proteins interact with cellular surveillance machinery to assure that properly processed mature mRNAs are preferentially exported to the cytoplasm. Four independent but complementary specific aims are proposed. Aim 1 seeks to understand how a novel class of zinc finger polyadenosine RNA binding proteins recognizes mature mRNA. Aim 2 is designed to understand how associated mRNA binding proteins target mature mRNAs to the nuclear pore for export. Aim 3 seeks to identify new mechanisms that the cell employs for surveillance of the export process. Finally, Aim 4 investigates how changes in the bound complement of mRNA binding proteins contribute to irreversible export of mRNA transcripts from the nucleus to the cytoplasm. These aims will be accomplished through a combination of yeast genetics, biochemical and biophysical methods, structural biology, and high throughput cell biological approaches. PUBLIC HEALTH RELEVANCE: The goal of this project is to understand how cells move information from the cell nucleus where the genetic material is located to the cytoplasm where that information can be decoded and translated into the proteins that mediate all the cellular functions. We are interested in how cells avoid sending incorrect information, in the form of immature RNA messages, to the cytoplasm to avoid various disease states that can arise. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): This application addresses broad Challenge Area (15) Translational Science and Specific Challenge Topic 15-OD (ORDR)-101. The Challenge: Oculopharyngeal muscular dystrophy (OPMD) is a rare autosomal dominant disease of late onset for which no cure exists. It is characterized by eyelid drooping, difficulties in swallowing and weakness in proximal limb muscles. Although polyalanine expansion in PABPN1, a ubiquitous mRNA binding protein, causes OPMD neither the function of PABPN1 in skeletal muscle nor how expression of mutant PABPN1 leads to muscle pathology in specific muscles is known. Scientific Gap in Knowledge to be addressed: The expanded polyalanine tract in PABPN1 leads to aggregation of the mutant protein in intranuclear inclusions in skeletal muscle. Mutant PABPN1 may lead to pathology by sequestering either specific mRNA transcripts or proteins such as wildtype PABPN1 in aggregates. We hypothesize that the muscle stem cells in the head and neck muscles are the key cells affected by alanine-expanded PABPN1 and are responsible for the muscle-specific pathology observed in OPMD. The Approach: To analyze the role of mutant PABPN1 in muscle stem cells, experiments are proposed to identify mRNA transcripts associated specifically with alanine-expanded PABPN1 in muscle stem cells (Specific Aim 1), to analyze molecular defects in mRNA biogenesis linked to expression of alanine-expanded PABPN1 or depletion of wildtype PABPN1 (Specific Aim 2), and to determine whether alanine-expanded PABPN1 expressed specifically in muscle stem cells results in muscle pathology in novel transgenic mouse models (Specific Aim 3). Importantly, the Specific Aims are designed to compare the role of PABPN1 and the functional consequences of alanine-expanded PABPN1 expression in muscles affected in OPMD. Significance: Studies targeting muscle stem cells may lead to a paradigm shift in our understanding of the pathology of OPMD and afford new therapeutic strategies that target the appropriate molecular pathways altered in affected muscles. In addition, the information gathered in this proposal could also extend to other nuclear inclusion diseases in which a mutant protein forms aggregates. 7. PROJECT NARRATIVE The goal of these studies is to understand why people with mutations in the PABPN1 protein develop a disease called oculopharyngeal muscular dystrophy (OMPD) where eyelid and pharyngeal muscles are primarily affected. Our experiments will study the role of PABPN1 in the muscle cells that are affected in the disease. Understanding the role of PABPN1 specifically in these muscle cells will lead to a greater understanding of the pathogenesis of OPMD as well as possible new therapeutic strategies for this disease.

PROJECT SUMMARY The nuclear poly(A) binding protein 1 (PABPN1) is a ubiquitously expressed protein that plays critical roles at multiple steps in post-transcriptional regulation of gene expression. Short expansions of the polyalanine tract in the N-terminus of PABPN1 lead to Oculopharyngeal Muscular Dystrophy (OPMD). Patients who suffer from OPMD have progressive weakening of specific muscles most notably those of the pharynx. Defects in pharyngeal muscle function can cause choking and regurgitation leading to pneumonia or sudden death. There is no current treatment for OPMD. Much is still unknown regarding the mechanism by which ubiquitous expression of this alanine-expanded PABPN1 leads to muscle-specific pathology. We have discovered that the steady-state levels of both PABPN1 mRNA and protein are drastically lower in mouse and human skeletal muscle, particularly those impacted in OPMD, compared to other tissues. The low levels of PABPN1 in skeletal muscle could predispose this tissue to the deleterious effects of alanine-expanded PABPN1. The low level of Pabpn1 transcript present in muscle indicates tissue-specific regulatory mechanisms at either the transcriptional or post-transcriptional level or possibly both. We find that Pabpn1 expression in different tissues is in part regulated by a post-transcriptional mechanism that modulates transcript stability but further studies are needed to fully elucidate how PABPN1 expression is controlled. The goal of this proposal is to test our working hypothesis that low levels of PABPN1 in skeletal muscle predispose this tissue to the deleterious effects of alanine-expanded PABPN1. To define the mechanisms that lead to the low levels of expression of PABPN1 in muscle, we will map the cis-elements responsible for modulating the stability of the Pabpn1 transcript in skeletal muscle (Aim 1) and identify the cellular factors (RNA binding proteins and miRNAs) that bind to the PABPN1 transcript to modulate transcript stability in a tissue-specific manner (Aim 2). In addition, we will determine whether transcriptional mechanisms contribute to the low levels of PABPN1 transcript in skeletal muscle (Aim 3). Of particular importance to OPMD, we will exploit two novel mouse models (both a PABPN1 Knockout mouse and an alanine-expanded PABPN1 Knockin mouse) that we have create to directly test whether a decrease in the level of PABPN1 exacerbates the pathology induced by alanine-expanded PABPN1 (Aim 4). The long-term goal of our studies is to understand why ubiquitous expression of mutant PABPN1 leads to a muscle-specific disease. These studies are significant as they will identify pathways that could be manipulated to improve the quality of life for patients that suffer from OPMD. Furthermore, these studies could lay the groundwork for understanding tissue-specific pathology in other diseases.

Intellectual Merit
The 19th Annual Southeastern Regional Yeast Meeting (SERYM) will be held on the campus of Emory
University from February 24-26, 2012. This annual meeting provides a unique opportunity for yeast researchers from states in the Southeastern region of the U.S. to come together to share their preliminary research findings and discuss new research tools and emerging technologies. The proposed meeting on Yeast is unique in that it is a regional meeting designed to increase the participation of undergraduates, graduate students and underrepresented groups and because it is aimed at researchers investigating a variety of yeast species not limited to the more common model organisims S. cerevisiae and S. pombe. A key strength of this meeting is the opportunity for both undergraduate and graduate students to present their research in platform and/or poster formats. The regional nature of this meeting provides opportunities for students to discuss their research work with leading scientists, laying the foundation for future scientific development and collaborations.

Broader Impacts
One of the goals of the meeting is to promote student attendance and participation and the requested funding will support the attendance of 16 students. Students had a strong presence at the 2011 meeting, with undergraduate and graduate students comprising 62% of the total meeting attendees and giving 62% of the platform and 66% of the poster presentations. Students from underrepresented groups will be given priority participation support. To increase the participation of students from underrepresented groups, we have invited students and mentors from Spelman College, Morehouse College, and Clayton State University to participate in SERYM 2012.

DESCRIPTION (provided by applicant): Oculopharyngeal muscular dystrophy (OPMD) is an adult onset disease characterized by eyelid drooping and difficulties in swallowing, with weakness also noted in proximal limb muscles. No cure exists for this disease. The mutation responsible for the disease is found within the polyadenylate-binding protein nuclear 1 (PABPN1) gene. The ubiquitously expressed PABPN1 protein regulates post-transcriptional gene expression through modulating 3'-end formation including poly(A) tail length and 3'-end cleavage/polyadenylation site selection. The autosomal dominant form of OPMD, which is the most common form of the disease, is characterized by a polyalanine expansion in the N-terminal domain of the protein from the normal 10 alanines to 12-17. Patients with autosomal dominant OPMD have one mutant allele of PABPN1 and one normal allele of PABPN1. However, current mouse models of OPMD are transgenic and thus contain two normal alleles of PABPN1 in addition to one mutant allele of PABPN1 leading to significant overexpression of PABPN1. Thus, these mouse models do not accurately reflect the genotype of patients afflicted with the disease. We have created a knock- in mouse model that expresses PABPN1 containing a polyalanine expansion of 17 alanines (Ala17PABPN1) in the presence of Cre-recombinase and is thus more closely aligned with the genetic changes in autosomal dominant OPMD patients. The goal of this proposal is to thoroughly understand the effects of Ala17PABPN1 on muscle pathology and function (Aim 1) as well as to begin to examine the impact on post-transcriptional processing of RNA (Aim 2) throughout the lifespan of these Ala17PABPN1 knock-in mice. We propose that this new mouse model will be invaluable for future studies in understanding the tissue-specific consequences of mutant PABPN1 and testing potential therapies.

DESCRIPTION (provided by applicant): Project Summary/Abstract The function and fate of any given cell is determined by the gene expression profile of that cell. While transcription plays a key role in determining gene expression, post-transcriptional regulatory events also are of vital importance in determining the spatial and temporal pattern of gene expression. One crucial post- transcriptional step is addition of a polyadenosine or poly(A) tail following 3'-end cleavage of mRNA. This poly(A) tail modulates numerous events including mRNA export from the nucleus, translation, transport and turnover, largely via the recruitment of polyadenosine RNA binding proteins (Pabs). Polyadenylation of additional classes of RNA has also been implicated in quality control. The critical importance of proper polyadenylation and Pab protein function is evident in the number of human diseases that arise due to mutations in genes encoding these proteins. In the previous funding cycle, we collaborated to identify mutations in the human ZC3H14 gene, which encodes a ubiquitously expressed, evolutionarily conserved, zinc finger, nuclear polyadenosine RNA binding protein (Pab), that cause an autosomal recessive form of non- syndromic intellectual disability (previously termed mental retardation). These patients have severely impaired brain function with IQs in the range of 35-50 as compared to an average adult IQ range of 90-110. Although little is known about the function of ZC3H14, studies of the budding yeast (Nab2) and Drosophila counterparts (dNab2) provide compelling evidence that this class of proteins plays an evolutionarily conserved role in regulating poly(A) tail length. The broad, long-term objective of this proposal is to define how poly(A) tail length is regulated by the zinc finger Pabs to understand both the critical role in post-transcriptional regulation of gene expression and the mechanisms underlying neuronal defects in affected patients. This proposal exploits exciting preliminary data collected through studies of this evolutionarily conserved class of proteins in budding yeast, Drosophila, and cultured neuronal cells capitalizing on established tools and models. These studies strongly implicate the complex of 3'-5 riboexonucleases known as the nuclear exosome in cooperating with Nab2 to regulate poly(A) tail length. Based on preliminary data, we will test the hypothesis that the Nab2 class of RNA binding proteins cooperates with the exosome to regulate poly(A) tail length of RNA thus ensuring proper neuronal function. This hypothesis will be tested through three complementary Specific Aims that seek to: 1) define a molecular mechanism for Nab2/ZC3H14 in regulating poly(A) tail length (Aim 1); extend these studies into our established Drosophila model to link molecular mechanisms to neuronal function (Aim 2); and finally determine whether specific RNAs or classes of RNAs accumulate extended poly(A) tails upon loss of Nab2/ZC3H14 function (Aim 3). Successful completion of these aims will provide insight into a critical point in post-transcriptional regulation of gene expression mediated by a recently described family of Pab proteins and also lend insight into the molecular defects underlying intellectual disability in patients.

DESCRIPTION (provided by applicant): The Training Program in Biochemistry, Cell and Molecular Biology provides the predoctoral candidate with multidisciplinary training in the broad area of cell structure and function. The Training Faculty constitute the interdepartmental Graduate Program in Biochemistry, Cell and Developmental Biology (BCDB), one of eight interdisciplinary training programs in the Graduate Division of Biological and Biomedical Sciences (GDBBS) at Emory. The 54 Training Faculty hold their primary academic appointments in diverse departments including, Biochemistry, Biology, Cell Biology, Chemistry, Human Genetics, Medicine (Digestive Diseases, Nephrology, Pulmonary Medicine), Ophthalmology, Pathology, Pharmacology, Physiology, Surgery, and the Winship Cancer Institute. Their research programs are clustered into four main overlapping themes: biochemistry/structural biology, cancer biology, cell biology and developmental biology. Concomitant with this range of biological problems, the trainee is able to take advantage of diverse model systems and state- of-the-art technical resources. The 7 stipends support students in their second year of training, although some exceptional students are supported for a portion of their third year as well. Trainees are selected for support from the larger pool of students matriculating in the parent BCDB Program, which currently has 64 trainees. All trainees take a core curriculum that emphasizes the foundations of biochemistry, molecular biology, biophysics, cell biology, genetics and developmental biology. Additional courses in scientific writing, seminar presentation, and ethics are also required. Advanced electives are available in the full range of biomedical disciplines to give the student in-depth and individualized preparation for their research careers. The training environment is enriched by the BCMB-sponsored, trainee-organized annual Symposium, weekly journal clubs and seminars. The successful graduate will be well suited, after appropriate postdoctoral training, to pursue an independent research career. In this regard, most graduates have moved on to top tier postdoctoral positions, and some now hold tenure-track, or tenured, positions in top academic institutions.

? DESCRIPTION (provided by applicant): Defining molecular mechanisms that ensure proper patterns of cell: cell connectivity in the developing nervous system has relied in part on genetic studies in vertebrate and invertebrate models, and in part on mapping loci responsible for heritable forms of brain dysfunction in humans. These efforts have identified several RNA binding proteins, whose individual loss alters neuronal morphology and connectivity, suggesting that post- transcriptional mechanisms play an important role in neurodevelopment. We co-discovered a form of heritable intellectual disability caused by mutations in the gene encoding a polyadenosine RNA binding protein termed ZC3H14. Our analysis of a D. melanogaster model of this disease created by deletion of the sole invertebrate ZC3H14 homolog, dNab2, has revealed cell-autonomous defects in neuronal projection in the mushroom bodies (MBs), twin neuropil structures in the brain involved in learning and memory. Axons from wildtype MB neurons project towards the midline of the adult brain, while those from dNab2-deficient MB neurons misproject through the midline and into the contralateral brain hemisphere. Given the shared molecular role of dNab2/ZC3H14 as RNA binding proteins, we hypothesize that the axon misprojection and cognitive defects resulting from loss of functional dNab2/ZC3H14 are the result of defects in post-transcriptional control of target RNAs. Our published finding that human ZC3H14 partially compensates for dNab2 loss when expressed in fly neurons indicates that at least some of these RNA targets are shared. This proposal focuses on identification of dNab2-bound RNAs from fly brain neurons that encode factors involved in axononogenesis. In Aim 1 we propose to use a validated approach, RNA Tagging, to specifically recover dNab2-bound RNAs from Drosophila neurons in their natural context in situ. A pilot application of this technique has already yielded two RNAs encoding protein with established roles in neuronal development. Aim 2 will pursue the hypothesis that dNab2 loss alters a key aspect of the post-transcriptional regulation of these dNab2-bound mRNAs, with ultimate effects on expression of their cognate proteins. Aim 3 completes the cycle by applying genetic tools to assess the in vivo role of each of these factors to wt brain development and their roles in dNab2 null phenotypes in the developing mushroom bodies. These Aims leverage the strength of our Drosophila dNab2 model to support our long-term goal of defining the mechanistic basis of ZC3H14-associated neuronal defects in vertebrates. Our approach does not discount roles for dNab2 in other neuronal processes or cell types, but rather allows us to focus on one novel function of dNab2 (axonogenesis) in an experimentally accessible group of neurons (MB cells). Insights into molecular roles for dNab2 in MB neurons could be relevant to molecular defects in the neurons of human patients lacking ZC3H14.

PROJECT SUMMARY This is a new application for a T32 to support predoctoral training in Biochemistry, Cell and Developmental Biology (BCDB) at Emory University. The award will replace the current T32 training program, which is in its 29th year, and has successfully trained a diverse cohort of students for the biomedical research workforce. We are requesting 7 positions and anticipate the duration of appointment will be 12-24 months. The mission of the BCDB training program is to provide an outstanding training environment through evidenced-based learning approaches and top caliber research mentors for development of our Ph.D. level workforce in academic-, industry- and government-based research careers as well as other biomedical research-related careers. This mission will be accomplished through a core curriculum that includes discussion-based, active learning approaches to the fundamental principles of biological processes at the biochemical and cellular level and development of oral and written presentation skills through classroom and seminar experiences. In addition, the importance of rigor and reproducibility in biomedical research is emphasized throughout the training with a required course as well as emphasizing this training as part of the courses, seminars and journal clubs. The objectives of the training program are to use these approaches to provide Ph.D. scientists with the necessary skills in critical thinking, experimental design and quantitative approaches as they acquire an understanding of how to conduct their work in a manner that is responsible, rigorous and reproducible. The program has an inclusive environment and is active in the recruitment and retention of students from diverse backgrounds. There is also an established focus on training the faculty mentors to provide the most outstanding training environment possible. The program also provides unique training opportunities to students supported by the proposed T32 training program through their responsibilities in running an innovative journal club as well as their role in organizing the two annual BCDB symposia; one which highlights career opportunities for Ph.D. graduates. We plan to continue our track record of training success while also introducing new and innovative aspects to the training together with mechanisms to evaluate both student and program outcomes.

Project Summary There are over 5 million people in the US currently living with Alzheimer's Disease (AD). Despite the loss of life, deterioration in quality of life, and social and financial costs of this devastating disease, there is still no cure or effective treatment to slow disease progression. A variety of approaches show promise in defining the key molecular changes that underlie pathology in AD. Many of these studies focus on the microtubule associated protein Tau and understanding how Tau contributes to neurodegeneration. A genetic screen for modulators of Tau pathology in C. elegans identified Suppressor of Tau 2 (sut-2). The sut-2 protein is a member of an evolutionarily-conserved family of nuclear, zinc finger polyadenosine RNA binding proteins. The human protein is termed Zinc Finger Cys3 His #14 (ZC3H14) or MSUT2. In our preliminary studies, we have identified ZC3H14 enriched in insoluble aggregates in AD brain. Furthermore, we have identified physical interactions between murine ZC3H14 and components of the RNA spliceosome including the U1-70K protein, which the Seyfried laboratory has linked to Tau pathology. Finally, ZC3H14 binds to the Mapt transcript encoding Tau suggesting a role for ZC3H14 in modulating post-transcriptional processing and/or expression of Tau. Based on our preliminary data and the discovery of sut-2 as a Tau modifier, we hypothesize that ZC3H14 interacts with critical RNA binding proteins and contributes to Tau-mediated pathology. Drawing extensively on resources available in the Alzheimer's Disease Research Center (ADRC) at Emory and our own preliminary data, we propose a collaborative multi-PI (Corbett and Seyfried) approach to test our hypothesis through the following specific aims: Aim 1) Define the spectrum of ZC3H14-interacting proteins in normal and AD brain; and Aim 2) Assess whether a decrease in ZC3H14 levels ameliorates Tau-induced pathology in a mouse model of Tau-mediated pathology and probe potential mechanisms. Our collaborative team is uniquely qualified to perform the proposed studies as 1) we have access to the required patient samples and expertise in proteomics provided by the Emory Proteomics core directed by MPI Seyfried; and 2) we have created the first Zc3h14 (MSUT2) knockout mouse. The homozygous Zc3h14 null (Zc3h14-/-) mice are viable and fertile and available to cross to Tau P301S transgenic mice (Line PS19) to assess whether ZC3H14 modifies Tau phenotypes in mammals. In addition, we will exploit these mice for preliminary analysis of how ZC3H14 regulates Tau expression. The goal of this exploratory proposal is to probe the mechanism by which ZC3H14 contributes to Tau-induced pathology. The long-term goal of our studies is to identify molecular pathways that could be targeted to modulate Tau-induced pathology.

PROJECT SUMMARY Post-transcriptional processing of RNA is a critical regulatory step in gene expression. Many evolutionarily conserved RNA processing enzymes mediate these key post-transcriptional events. This proposal focuses on molecular mechanisms linked to PontoCerebellar Hypoplasia (PCH), which serves as a paradigm for a growing number of neurological diseases caused by mutations in genes encoding RNA processing factors. PCH is a group of autosomal recessive neurodegenerative diseases characterized by hypoplasia/atrophy of the cerebellum and pons that is often fatal within the first year of life. Mutations that cause PCH type 1 (PCH1) occur in genes that encode structural subunits of the RNA exosome (human Rrp40, 43, and 45), which plays critical roles in both RNA processing and degradation. Mutations that cause PCH types 2, 4, and 5 (PCH2/4/5) lie in genes that encode tRNA splicing endonuclease subunits (TSEN2, 15, 34, and 54). TSEN has a well-characterized role in tRNA processing but also other yet undefined functions. The subunits of these RNase complexes are all evolutionarily conserved and essential for viability. PCH1 mutations cause single amino acid substitutions that primarily occur in conserved residues. The discovery that mutations in multiple components of these complexes cause PCH strongly suggests that RNA processing dysfunction underlies PCH pathology. However, limited studies have assessed the functional consequences of these amino acid substitutions. Furthermore, given the common disease etiology, mutations in either the RNA exosome or TSEN complex could impair common RNA targets or classes of RNA targets, but the RNAs affected have not been systematically defined. These links to common biology strongly support our working hypothesis that mutations that cause PCH Types 1/2/4/5 impair the processing of a common set of RNA targets. Our previous collaborative efforts provide proof of principle that studies in model organisms can provide insight into how specific disease-causing amino acid substitutions impair RNA exosome function. Here we draw on our established collaboration and extensive preliminary data to perform a series of mechanistic studies in four aims. Aim 1 assesses the functional consequences of amino acid changes that occur in PCH using budding yeast; Aim 2 employs biochemical analysis in mouse cerebellum and cultured neuronal cells to define RNA exosome cofactors that could contribute to the tissue-specific nature of PCH; Aim 3 couples studies in budding yeast and cultured neuronal cells to identify common RNA targets of the TSEN and RNA exosome complexes; and, finally, Aim 4 employs tissue-specific RNAi in Drosophila to begin to assess the requirement for specific RNA exosome cofactors and TSEN subunits in neurons. The long-term goal of this work is to fully define the function of these evolutionarily conserved RNase complexes while providing insight into molecular mechanisms that could contribute to neurological dysfunction in PCH.

PROJECT SUMMARY The goal of this new Initiative to Maximize Student Diversity (IMSD) proposal is to build on an existing IMSD program at Emory University to increase the diversity of the scientific workforce. In spite of many years of increased focus on diversity, the rate of enrollment and graduation of under-represented (UR) students in undergraduate and graduate degree programs in the biological and behavioral sciences is markedly lower that of the general population. Emory University is a highly rated teaching and research institution with over 14,200 students that is located in Atlanta, Georgia. Emory?s record of recruiting, retaining and graduating diverse students is significant. Approximately 15% of each entering graduate class is underrepresented (UR); undergraduate classes are 20%, having doubled since the early 90?s. The biomedical and behavioral science departments at Emory awarded 1,667 BS, 805 MS and 149 PhD degrees to UR students in the most recent fifteen-year period. After receiving only three years of support in 2013, Emory?s Initiative for Maximizing Student Development (IMSD) program has made significant progress towards enhancing the mentoring environment and providing career development and support to undergraduate and graduate students. The long- term goal of the Emory IMSD is to increase the average number of UR students obtaining PhDs in the biomedical sciences by placing at least two-thirds of IMSD Undergraduate Scholars in graduate programs and graduating 90% of IMSD Graduate Fellows and placing them in strong postdoctoral positions. In this new IMSD proposal, we seek to continue our success and further enhance the impact of the Emory IMSD as we develop an inclusive community and a continuum of mentoring and career development programming via three specific aims and a structured plan to transition from the prior leadership to a new leadership team. Aim 1 will continue with efforts to build a learning community of IMSD Undergraduate Scholars. We will increase the number of Emory undergraduate UR students majoring in the biomedical/behavioral/quantitative sciences who receive a Ph.D. in a science-related field to 12 per year. This will more than double our ten-year average that existed when the Emory IMSD program was first proposed (4.8 per year from 2003-2012) and remove all demographic disparity. Aim 2 will enhance and build a learning community of IMSD Graduate Fellows. Through these efforts, we will recruit 40 additional UR PhD students over five years, with over 90% completing the PhD. Finally, Aim 3 will provide formal training in mentoring for both faculty and graduate students in the Emory community and the greater Atlanta area by continuing efforts to develop a community of mentors, the Atlanta Society of Mentors (ASOM), with transferable skills applicable at all levels and adept in issues of diversity. These aims will have significant impact by building on our prior success with creating an inclusive and integrated community of undergraduate scholars, graduate fellows and program faculty that enhances the culture of learning at Emory University and increases diversity within the scientific workforce.

PROJECT SUMMARY In 2000, Emory University partnered with a consortium of Atlanta historically Black colleges and universities called the Atlanta University Center (AUC) to form our IRACDA program (Fellowships In Research and Science Teaching or FIRST). This partnership between Emory University, a research-intensive university, and these Minority Serving Institutions (MSI; Spelman College, Clark Atlanta University, Morehouse College, and Morehouse School of Medicine) has focused on providing our Scholars with outstanding research experiences at Emory combined with training and immersive experiences in teaching at the AUC schools. Thus, FIRST Scholars develop both their academic research credentials and teaching credentials, both of which will be important in their later academic life. FIRST also supports the AUC as FIRST Scholars develop new courses and laboratory modules as well as mentor undergraduates from underrepresented groups in research projects. With these additional educational opportunities through FIRST, these undergraduates that are women and/or from underrepresented groups are also exposed to enthusiastic, research-oriented teachers and role models ? many of whom are also women and/or from underrepresented groups ? who have been successful in graduate school and postdoctoral academic environments. Our Partner Institutions are also positively impacted as FIRST alumni, former Emory Postdoctoral Fellows, become AUC academic leaders and reinforce dynamic interactions among AUC faculty and Emory. Throughout its history, this program has been highly successful in recruiting and preparing a diverse pool of FIRST Scholars. Overall, 67% of our trainees are in highly competitive research positions and 15% in tenure-track posts at Minority-Serving Institutions (MSIs). In this current funding period, FIRST successfully filled six positions each year. A number of those recruited subsequently obtained extramural funding. Of the 82 FIRST Scholars, 76% were women and 62% were from underrepresented groups. These successes reflect our exceptional program. Our programmatic objectives in this competitive renewal are to build on these achievements as follows: 1) Provide Scholars with exemplary research and professional training that promotes their research, career, and leadership development; 2) At the AUC schools, expand evidenced-based teaching and biomedical research opportunities to engage early stage STEM students that are women and/or from underrepresented groups; 3) Increase the number of highly qualified STEM Postdoctoral Fellows from women and/or underrepresented groups entering competitive academic and biomedical careers. Through the FIRST Program, Emory is the only research-intensive institution in Georgia that provides comprehensive training in research and teaching for early career scientists. Continuation of FIRST will allow us to continue training and preparing STEM Postdoctoral Fellows that are sensitive to the MSI culture and, ultimately, will be in a position to serve as life-long guides and role models in academia and other research-related positions. By leveraging this training infrastructure, our goal is to expand the impact of FIRST to non-IRACDA funded Postdoctoral Fellows.

PROJECT SUMMARY/ABSTRACT Over 1.7 million individuals will be diagnosed with cancer and 600,000 cancer-associated deaths will occur in the US in 2020. Devoting resources to develop tools and reagents to define mechanisms of oncogenicity and applying this knowledge to the development of therapeutic agents and diagnostic tools is clearly required to improve patient outcome. Recent research has identified cancer-associated amino acid substitutions that occur in the evolutionarily conserved histone proteins, leading to the term ?oncohistones.? Examples of such changes linked to cancer include H3K27M, H3G34V/R/D, and H3K36M. While the mechanism of oncogenicity for these mutations varies, each amino acid change perturbs the histone methylation landscape, affecting transcriptional regulation. Understanding how oncohistones alter gene expression can provide critical therapeutic insight. Changes to the histone methylation landscape in H3K27M-expressing gliomas result in dopamine receptor D2 (DRD2) overexpression and these tumors respond to the DRD2 and/or CLPP antagonist ONC201. This example highlights how cancer-associated epigenetic changes can unmask potentially druggable targets. In our preliminary studies, we have identified a series of dominant H3 mutations, termed X to K (R42K, E50K, Q68K, E73K), in which the wildtype amino acid is changed to a lysine, in more than 30 patient tumors in breast and other cancers. Preliminary in vitro experiments suggest that X to K mutation promotes transformation; a major indicator that H3 X to K mutations produce bona-fide oncohistones. However, both how these X to K amino acid changes alter the function of these histones and the mechanism(s) by which X to K oncohistones produce oncogenic phenotypes is unclear. We hypothesize that H3 X to K changes confer oncogenic properties by (1) introducing localized structural changes that alter nucleosome integrity and/or function and/or (2) introducing a new substrate for chromatin modifiers, thereby supporting a novel, and potentially targetable, gene expression program. Drawing on the integrated environment at Emory/Winship, we propose a collaborative multi-PI approach to test our hypothesis through the following aims: Aim 1) Examine the impact of H3 X to K amino acid changes on histone function together with cell and tumor growth; and Aim 2) Define how X to K amino acid changes in histones alter gene expression. Importantly, the proposed studies lay the groundwork for defining both new cancer signatures and druggable targets. Our interdisciplinary team is uniquely qualified to perform the proposed preclinical studies, which are directly related to advancements in cancer treatment and diagnosis. The long-term goal of our studies is to develop diagnostic tools for the detection of oncohistone-associated tumors and identify a therapeutically actionable epigenetic signature in patient tumors characterized by these H3 X to K oncogenic mutations.