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External Awards for Research Training


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    Development and delivery of CRISPR-Cas9 nickases to correct the mutant Huntingtin (mHTT) gene

    CRISPR-Cas9 genome editing is a promising technology with the potential to treat genetic diseases by changing the DNA mutation that is the underlying cause of the disease. HD is caused by a CAG repeat expansion in Exon 1 of the Huntingtin gene. Our goal is to apply CRIRSPR-Cas9 genome editing tools to correct the mutant Huntingtin gene back to the wild type gene by reducing the number of CAG repeats to below the pathogenic threshold. We have very promising results in HD patient cells, and we are currently working on making the method work more efficiently in animal models. In one line of experiments, we are working on modulating cellular DNA repair pathways to probe the mechanism of induced repeat length contractions and identify reagents to improve this activity in the brain. Additionally, current approaches for delivery of these gene editing tools to the brain have challenges such as safety concerns due to continuous expression of these gene editing complexes. Therefore, we are concurrently, developing a new technique to deliver CRISPR-Cas9 gene editing tools to the brain in the form of ribonucleoprotein (RNP) complexes. This method will potentially reduce safety concerns due to short half-life of RNPs and promote uptake and distribution of editing throughout brain. If successful, a single treatment could revert the CAG repeat in mutant Huntingtin gene to the normal range. This approach will have therapeutic uses for HD and other neuropathological disorders associated with trinucleotide repeat expansions.

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    Activating Protein Expression Using Antisense Oligonucleotides

    Haploinsufficiency in diploid organisms is characterized by a working copy and nonfunctional copy of a gene, resulting in an insufficient amount of gene product (i.e., protein). This disrupts normal cell function, and can cause a myriad of diseases. Antisense oligonucleotides (ASOs) are small, predictable, and programmable tools that can be chemically engineered to directly control the stability, processing, and translation of RNA, making them useful for dissecting mechanisms of protein production. This proposal seeks to design and apply chemically-modified ASOs to systematically investigate endogenous protein repression mechanisms and identify key factors modulating full-length protein translation, using the NF1 gene as a model. NF1 is a tumor suppressor that inhibits Ras/MAPK signaling. NF1 haploinsufficiency causes neurofibromatosis type 1, a genetic disorder characterized by uncontrolled nerve cell proliferation and other complications. Steric blocking ASOs will be used to initiate translation at the primary start site in the NF1 5’-UTR and increase protein expression. ‘Gapmer’ ASOs will be used to target and degrade NF1 antisense transcripts and determine their effect on NF1 protein expression. Following sequencing of NF1 nascent RNA to identify cryptic splice sites, steric-blocking ASOs will be designed to mask nonproductive splice sites and improve pre-mRNA splicing efficiency. Synthesized ASOs will be screened in neuroblastoma cells and subsequently tested and optimized in NF1+/- haploinsufficient neurons and Schwann cells. Functionality of activated NF1 protein will be assessed by measuring Ras/MAPK activation. This project will increase our understanding of how protein expression is regulated, and may inform strategies to correct haploinsufficiency.

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    SAVI mutations in the adapter protein STING lead to intestinal inflammation and fibrosis in mice

    Studies in IBD patients and in mouse models have implicated STING activation as a regulator of intestinal inflammation. Murine studies in STING loss of function genetic models, have revealed both protective and detrimental roles for this pathway in the gut. In this study, we aim to explore the molecular mechanisms and consequences of STING mediating intestinal inflammation. Using a mouse model harboring a gain of function mutation in the gene encoding STING, Tmem173, we generated a new model of spontaneous colitis without chemical disruption. Asparagine to Serine (N153s) gain of function mutation, corresponds to the human N154s mutation in STING was recently reported in patients of STING-associated Vasculopathy with Onset in Infancy (SAVI)[1-5]. Patients suffering from SAVI exhibit an elevated Interferon Stimulated Gene (ISG) expression signature in peripheral cells as well as respiratory failure, skin rash and pulmonary fibrosis. Here we report that N153s mice also exhibit profound intestinal inflammation. We found that WT mice express low level of STING protein in the colon and that STING expression in the N153s mice increased over time and correlated with the onset of disease progression. Measurement of STING levels in colon tissue biopsies from IBD patients revealed stabilization of STING in areas of the colon with active disease compared to adjacent unaffected tissue, emphasising the potential link of STING expression in the gut to intestinal inflammation . Antibiotic treatment as well as replacement of the microbiota by WT fecal microbiota transplantation (FMT) alleviates intestinal inflammation and reduces protein levels of STING in the N153s mice colon, highlighting the microbiome as an essential contributing factor to disease. This proposal aims to build on these findings and define the molecular and cellular basis of chronic intestinal inflammation due to constitutive activation of STING. Specifically, we aim to: (1) assess dysbiosis and its effect on intestinal inflammation in N153s mice and (2) identify the cellular and molecular mechanism(s) initiating dysbiosis and colitis in N153s mice. Completion of this study will provide a new framework for understanding the progression of IBD. Furthermore, delineating STING function in the development of IBD may identify new therapeutic targets and improve treatment options for IBD and patient quality of life.

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    Alternative Splicing in White Blood Cells: A Biomarker for Fragile X Syndrome

    A long-sought goal in Fragile X Syndrome (FXS) research is the development of stable, robust, easily accessible, and quantifiable biomarkers for the disorder. Reliable biomarkers for FXS would address a number of issues that have hampered successful outcomes of some therapeutic trials. For example, several drugs used to treat FXS have been based on results from animal models, particularly one or two in-bred strains of mice. Although such therapeutics often strongly mitigate FXS-like pathophysiologies in mice, similar achievements have been noticeably less frequent in diverse human populations. These observations indicate that human-based biomarkers would more reliably predict outcomes of clinical trials. Human-based biomarkers could help stratify patient populations so that therapies could be targeted to certain individuals that would be most likely to respond in a positive way. Additionally, biomarkers that can be frequently tested and monitored are very helpful in determining drug response more effectively. Our collaboration has sought to identify multiple strong FXS biomarkers in human white blood cells (WBCs) using a blood test. We find that FXS individuals (ages 12-38 years) display about 1600 statistically significant changes in RNA levels and alternative pre-mRNA processing (splicing) events. One of our goals is to determine whether similar RNA alterations are also detected in FXS children. Another goal is to assess whether WBC RNA levels and/or splicing can be predictive of cognitive ability. WBC RNA from FXS patients that have been stratified based on IQ scores will be deep sequenced and analyzed for differential expression and splicing. If we are able to successfully co-stratify IQ with RNA mis-regulation in WBCs, we will explore several new avenues of investigation. For example, we will ascertain whether drugs used to treat FXS patients alter the WBC RNA population and correlate with success of the drug and whether a specific RNA signature might be predictive of a drug therapy outcome.

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    Deciphering the role of Gasdermin C in intestinal inflammation and colorectal cancer

    Cell death is a normal part of cellular function, and this important behavior can occur through several different mechanisms. One form of cell death—pyroptosis—is characterized by the dying cells spilling their inner contents after they bursts, and can result in inflammation that attracts the attention of the immune system. A key step in pyroptosis is the *cleavage* of a protein called Gasdermin D, but little is known about the cell death-related roles played by other members of the Gasdermin family. To improve our understanding of the mechanisms of cell death, Dr. Ketelut-Carneiro is focusing on a novel Gasdermin protein that is highly expressed in normal colon cells, but not when there is inflammation. Specifically, she is seeking to define the pathways leading to the activation of this protein, characterize its role in cell death, and test how it impacts intestinal inflammation and colorectal cancer. Understanding its role in the intestine could then be reveal new potential targets for cancer drugs as well as open up new avenues for future research in cancer therapy.

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    The Role of Monoclonal Gammopathy of Undetermined Significance Diagnosis in Healthcare Utilization

    Monoclonal Gammopathy of Undetermined Significance (MGUS) is an understudied precursor of multiple myeloma (MM), the second most commonly diagnosed hematologic malignancy in the United States. Patients with MGUS progress to MM at a rate of 1% per year throughout their lifetime, resulting in continuous clinical surveillance and associated anxiety. MM patients with a prior MGUS diagnosis may have better prognosis than MM patients without a diagnosis, although the mechanisms are unknown. Gaining a better understanding of overall healthcare utilization by patients with MGUS may provide insight into preventative healthcare measures that may improve their overall health. Therefore, this proposal focuses on investigating the role of an MGUS diagnosis on healthcare utilization practices and gaining insight into the processes involved in managing care for patients with MGUS. The Specific Aims are to: (1) Describe the sociodemographic, clinical characteristics, follow-up patterns, and laboratory value trajectories of patients with MGUS; (2) Determine if an MGUS diagnosis is associated with changes in healthcare utilization that differ according to patients’ sociodemographic and clinical characteristics; and (3) Understand the patient- and provider-level drivers of healthcare utilization in patients with MGUS and the predisposing, enabling, and need factors associated with care-seeking practices. To address these objectives, this proposal will use two data sources. Aims 1 and 2 will analyze a cohort of patients with MGUS (n=429) identified by a novel case-finding algorithm using health claims and electronic health record data, identified from a community-based population of patients seeking care in central Massachusetts. For Aim 3, we will conduct qualitative semi-structured interviews with patients with MGUS diagnosed in central Massachusetts and providers who treat patients with MGUS. The knowledge generated by the successful completion of these Aims will inform stakeholders on the role of an MGUS diagnosis in healthcare utilization and will assess the factors contributing to healthcare utilization in this population. In addition, the results of this study will elucidate potential clinical and sociodemographic characteristics that may lead to improvement in the long-term overall health of patients with MGUS and to the identification of targets for future interventions. This dissertation proposal also includes a multi-faceted, comprehensive training plan that will support Maira A. Castaneda-Avila’s development as an independent investigator, including advanced level training in quantitative and qualitative methods, cancer prevention and control, hematological malignancies, further training in the ethics of conducting research and grant writing, and exposure to national and international cancer epidemiologists through presentations and attendance at national research conferences. The skills gained through the proposed training plan will greatly augment the proposed research plan and ensure the successful completion of the project’s Specific Aims.

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    Understanding the role of aggregates in Huntington's disease

    Huntington’s disease (HD) is caused by a defect in the Huntingtin (HTT) gene involving the expansion of a DNA segment, called “CAG,” that repeats multiple times in a row. Over time, CAG repeat length can undergo further expansion, which produces a short protein (HTT1a) that may form aggregates. Whether eliminating HTT1a can delay HD is unknown. Utilizing novel RNA drugs that can “silence” HTT1a or machinery involved in CAG repeat expansion, Dr. O’Reilly will study the contributions of CAG expansion and HTT1a to aggregate formation and disease progression in mouse models of HD. His findings may identify effective HD treatments.

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    Spatial organization of the mTOR Complex 2 regulates lipid metabolism

    The Hallmarks of Cancer are specific abilities that many cancer cells acquire to grow uncontrollably. One of these is the ability to produce fats, or lipids, from sugar taken up from outside the cell. This is called lipogenesis. Only liver and fat cells normally undergo lipogenesis, but many types of cancer are more aggressive after acquiring the ability to produce their own fats. These fats are used to make new cell parts, activate genes, and signal to other cells to grow and divide. The mTORC2, an energy sensing signaling protein complex, is required for lipogenesis in normal cells and highly activated in cancer cells. It is plausible that mTORC2 is also required for lipid formation in cancer cells, but this hypothesis has not been tested. Our lab’s data indicates that two enzymes involved in this pathway are activated by a signaling molecule, called Akt, downstream of mTORC2. Preliminary data in cancer cells demonstrates that signaling to these enzymes requires both mTORC2 and Akt. I hypothesize that Akt requires mTORC2 activation to signal to its lipogenic substrates, because these substrates are located together at a signaling hub within the cell. Therefore, I take a two-step approach to examine the interactions of these proteins within the cell. First, I will identify the entire set of proteins that interact with mTORC2 during its activation using a state-of-the-art technology called proximity labeling. This technique will likely yield new information about the mTORC2 activation pathway which is currently poorly understood despite its upregulation in many types of cancer. Next, I will use classic cell biology and biochemistry to examine where in the cell Akt interacts with these enzymes and if this localization is necessary and sufficient for their activation. This proposal has the potential to identify new modes of mTORC2 activation and new interactors between the proteins in this lipogenic pathway. Currently, there are mTOR inhibitors which target mTORC2 and mTORC1, but these have many off target effects and are poorly tolerated by patients. This mechanistic project will identify new protein interactors for mTORC2 to place us one step closer to creating a novel mTORC2 specific drug.

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    The role of immunity in shaping Mycobacterium tuberculosis metabolism

    Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, and the leading cause of death by infectious disease. Mtb is an intracellular pathogen which is dependent on host nutrients in order to survive. Amongst these are the four primary carbon sources that Mtb uses for energy production and the production of metabolic intermediates. However, since Mtb resides in the macrophage phagolysosome, the bacteria are exposed to a variety of cellular stresses, including reactive nitrogen species (RNS) stress. Previous work from Rhee, et al. evaluating the effect of RNS stress on Mtb identified multiple essential proteins within glycolysis and metabolic intermediate production which were targeted by this type of intracellular stress. Additionally, unpublished work from our own lab showed that genes involved in glycolysis and glycerol metabolism are less essential than genes involved in fatty acid and cholesterol metabolism. Based on these data, we hypothesized that RNS stress specifically targets glycolysis which leads to the bacterium becoming more dependent on fatty acids and cholesterol for energy production. This project focuses on defining the effect of cellular reactive nitrogen species stress on carbon utilization for Mycobacterium tuberculosis and how Mtb regulates these changes in response to immune pressures.

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    Characterization of topological machines that control chromosome conformation

    The goal of this research is to investigate the molecular mechanisms responsible for chromosome conformation. Chromosome structure is important for controlling genomic processes such as transcription, replication, and chromosome segregation, and disruptions to this structure are found in genetic diseases and cancer cells. There are three main levels of chromosome organization: chromosome territories, chromosome compartments, and topologically associating domains (TADs). The current model is that TAD formation occurs due to dynamic loop extrusion of chromatin fibers, which is blocked in a directional manner by CTCF bound to TAD boundaries. However, the components and molecular mechanism of this proposed topological machine which forms these chromatin loops are currently unknown. Previous studies suggest that topoisomerases and histone variants may have a role in regulating chromosome conformation and topological machine activity. In addition, recent molecular modeling research has implicated loop extrusion e.g. dynamically extruded DNA loops, as an important characteristic of chromosome structure, however this has not yet been tested experimentally. This study will use genomic methods such as Hi-C, ChIP-seq, and TMP-seq in combination with cell biological and functional genetic approaches to study the molecular components and dynamics of the topological machine. Three complementary aims will be performed to address this question: 1) Assess the role of topoisomerases that have recently been identified as a part of the CTCF complex at TAD boundaries. 2) Investigate the role of histone variants that decorate key elements involved in TAD biology. 3) Develop new methods to determine chromatin dynamics inside TADs to test recently proposed models of TAD formation by dynamic loop formation. Together, completion of these aims will lead to new insights about the function and regulation of chromosome conformation.

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    Understanding how dicer partner proteins establish microRNA target specificity

    MicroRNAs (miRNAs) influence every physiological process, and dysregulated microRNAs have been linked to human diseases, including cancer. MicroRNAs are processed from the double-stranded stem of stem-loop precursor RNAs (pre-miRNAs) by the ribonuclease Dicer. Human Dicer and fly Dicer-1 (Dcr-1) can also process long double-stranded RNA (dsRNA) into small interfering RNAs in vitro, but exclusively make miRNAs in vivo. Moreover, Dicer can process some pre-miRNAs into multiple miRNA isoforms—i.e., isomiRs—whose cellular profiles change dynamically during differentiation and development. Here, I propose single-molecule biochemical and structural studies to answer the following questions: What restricts Dicer to miRNA processing? How do Dicer partner proteins affect kinetics of miRNA processing? How does Dicer make isomiRs?

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    Mechanism of epigenetic silencing by the HUSH complex

    This project has led to my interest in studying the co evolution of how newly integrated proviruses are incorporated into cellular programing and in turn how these provirus shape the cellular response. The HUSH complex bridges exogenous retroviruses and endogenous retroelements and may contribute to driving evolutionary adaptation of genetic networks. By gaining a better understanding of how exogenous reteroviruses are able to escape transcriptional control evolved to repress and limit the damage caused by parasitic genetic elements we can gain deep knowledge about the evolutionary pressures driving our evolution. Retroelements make up over 50% of our genome and the rules of how they are regulated by the genome and how they antagonize each other in a arms race will unravel many new discoveries about how we evolved and came to be. The major differences between us and our nearest relatives are not the coding sequences of proteins but the gene regulation of those proteins which may in part be driven by the need to regulate species specific transposable elements. By gaining a better understanding of how the HUSH complex recognizes and makes decisions as to what elements to repress it will unravel new interesting biology.

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    Dynamic role of Prmt5 in chromatin organization during adipogenesis

    The prevalence of obesity is astounding, and obesity-related health care costs amount to more than $147 billion a year in the US. Adipocytes in adipose tissue play a major role in obesity, as adipocytes are responsible for storing excess fat. Our lab previously found that the arginine methyltransferase Prmt5 is required for adipocyte differentiation, in part due to its ability to mediate enhancer-promoter loops, cis- interactions between transcribed genes on the same chromosome, and trans-interactions between adipocyte- specific regulatory sequences on different chromosomes. A major goal of this study is to identify how Prmt5 mediates higher order chromatin interactions genome-wide and whether this corresponds to transcriptional activation prior to and during adipogenesis. To test this, I will investigate Prmt5’s chromatin binding sites genome-wide prior to and during adipogenesis and relate it to chromatin conformation changes with and without Prmt5 knockdown. I will utilize high-level computational approaches to draw conclusions by integrating my chromatin landscape and genome organization studies with publicly available datasets in the adipogenic model, the 3T3-L1 cell line. These studies will provide critical insight into how Prmt5 and other factors regulate the chromatin landscape, higher order chromatin structure and transcriptional activation during adipogenesis. My studies will shed light on this relatively understudied protein arginine methyl transferase and may lead to identification of novel therapeutic targets for modulating adipogenesis and targeting obesity and obesity-related diseases.

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    Deep Molecular Profiling of Fragile X Mouse and Human Cells

    In 1991, the gene responsible for the Fragile X syndrome, FMR1, was first identified. Since then, important advances have been made in understanding the genetic inheritance of this gene, its regulation and the potential roles of its protein product, FMRP. Fragile X research has greatly benefited from animal models that have a deletion of the Fmr1 gene: Fmr1 knockout (KO) animals. Mouse models of Fragile X have been extremely useful in guiding research efforts, but they may not recapitulate the heterogeneity of symptoms and severity that are manifest in humans. But mice may not recapitulate the range of symptoms and severity seen in humans. Several therapies based on mouse models have been developed, which have not been as effective as hoped when treatment is applied to human patients. Additional approaches are needed. The advent of human cell cultures of FXS patients is promising for basic research, drug discovery and pre-clinical validation. Human pluripotent stem cells (iPS cells) derived from Fragile X patients contain the CGG triplet repeat expansion that cause Fragile X syndrome in humans. Thus they can be used to identify features of human FXS that can be recapitulated in-vitro. These cells have been used to study many aspects of the syndrome, such as epigenetic regulation of FMR1 gene silencing, defects in gene expression, neuronal differentiation and synaptic plasticity. In our studies, we aim to harness the power of patient-derived stem cells to generate excitatory neurons that would mimic the molecular profile of neurons in FXS patients. This provides us with an excellent system that can give us a meaningful snapshot of changes that would occur in humans.

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    Identifying regulators of PD-L1 to restore antitumor immunity

    The immune system should recognize cancer as foreign in the same way is does viruses and bacteria. One way in which it goes wrong is the expression of a protein PD-L1 on the surface of tumor cells that acts as a stop sign to immune cells when it binds to another protein, PD-1, on the surface of immune cells. Recently, the Food and Drug Association has approved several drugs that inhibit PD-L1 and PD-1 that allows for immune cells to attack tumors for the treatment of several cancers. These drugs have been widely successful in fighting cancers such as triple-negative breast cancer, melanoma and small cell lung cancer that don’t respond to traditional chemo-therapeutics. However, the drugs have one major unwanted side effect: autoimmunity. The drugs are not intended for long-term use because they ramp up the immune system too well. When this happens, patients develop autoimmunity and the immune system attacks normal, healthy tissue. We are working to identify the genes that cause PD-L1 to be expressed on cancer. If successful, they could potentially find drugs to inhibit these genes, and thus allow the immune system to attack tumors but spare normal tissue.

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    A genome-wide, biochemical approach to study the biology of chromosome folding

    Eukaryotic genomes are organized into a highly-compacted nucleoprotein complex known as chromatin. Hi-C and high-resolution microscopy studies reveal organization of chromosomes at multiple levels, from chromosome territories, to MB-scale functional domains that are spatially separated from each other, to shorter contact domains often called topological associated domains (TADs). While genetic studies in cell culture coupled with Hi-C or high-resolution microscopy reveal key roles for a variety of factors in higher-order chromatin folding, our understanding of chromatin fiber folding largely derives from biochemical studies in vitro. Mostly, these in vitro studies rely on artificial, homogenous chromatin templates that do not reflect the heterogeneous local folding properties of in vivo chromatin. The aim of this project is to approach higher-order chromatin folding and chromosome organization biochemically. Micro-C detects internucleosomal interactions, which refer to local chromosome folding, by identifying nucleosomal DNA sequences and is therefore equally suitable for in vivo and in vitro studies. I will use chromatin prepared from cells (ex vivo) and in vivo-like in vitro reconstituted chromatin for chromatin compaction studies. With Micro-C I will be able to measure autonomous salt-dependent chromatin folding driven by all endogenous factors (ex vivo chromatin) or solely by histone-DNA interactions. Biochemical manipulation will allow to remove or test individual candidate factors. This biochemical approach will help to decipher the regulatory role of higher-order chromatin organization on DNA templated processes, such as gene regulation.

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    Elucidation of exocyst tethering function by single molecule assays and EM

    The packaging, transport, recognition, and fusion of vesicle-enclosed cargo is an essential hallmark of eukaryotes, and must be tightly regulated for basic cellular organization. A key step in vesicular trafficking is a tethering event where vesicles are attached to their pre-determined target membrane prior to SNARE mediated fusion by tethering complexes. Multi-subunit tethering complexes (MTCs) are one such categorization of conserved tethering proteins, and are required for a majority a membrane trafficking steps within the cell. However, despite their name, there is a dearth of biochemical evidence showing the capacity of these MTCs to recruit and hold vesicles to a target membrane prior to fusion. Thus, I propose a series of experiments to reconstitute vesicle tethering in vitro using post-Golgi secretory vesicles and the MTC exocyst to definitively observe the proposed tethering activity of the complex and to gain structural and mechanistic insights into tethering events. To accurately and sensitively observe vesicle tethering, we seek to employ a single molecule assay TIRF microscopy based assay to observed individual tethering events and changes to exocyst conformations in real-time. Furthermore, we seek to gain structural insights into the overall architecture of the exocyst complex and the binding sites for various proteins that are thought to participate in the tethering event using negative stain electron microscopy techniques. The information gained from these experiments will not only determine the proposed tethering activity of exocyst and serve as a platform for other MTCs to be tested, but reveal mechanism details of the poorly understood, but essential tethering set in membrane trafficking.

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    Regulation of Immune Gene expression and inflammatory diseases by the Cellular nucleic acid binding protein (CNBP)

    An inducible program of inflammatory gene expression is a hallmark of antimicrobial defenses. Germline-encoded receptors recognize microbial products and activate signaling pathways that lead to the expression of hundreds of inflammatory response genes. This proposal expands on these studies by defining a new regulator of immune gene expression; the CCHC-type zinc finger protein cytosolic nucleic acid binding protein (CNBP). We have generated mice lacking CNBP and found that CNBP-deficient macrophages fail to induce transcription of the IL-12/IL23 family. Cnbp resides in the cytosol of macrophages and translocates to the nucleus in response to multiple microbial ligands and pathogens. Cnbp regulates IL12 via c-Rel, an NFkB/Rel family member known to control IL12b gene transcription. c-Rel nuclear translocation and DNA binding activity require Cnbp. Furthermore, Cnbp itself a DNA binding protein bound the IL12b promoter. CNBP-deficient mice were more susceptible to acute toxoplasmosis associated with reduced production of IL12b, as well as a reduced Th1 cell IFNg response essential to control parasite replication. Collectively, these findings identify Cnbp as a new signaling molecule downstream of multiple Pattern Recognition Receptors, that acts as a key regulator of IL12b gene transcription and Th1 immunity. This proposal will test the hypothesis that CNBP represents a novel signaling molecule that acts as a transcriptional coactivator to bind the genome and coordinate expression of IL12p40 to regulate IL12 and IL23 in innate cells and direct TH1/TH17 dependent adaptive immunity and inflammation. We will explore these hypotheses using the following specific aims: (1) defining detailed molecular mechanisms of CNBP-dependent control of the IL12/IL23 gene family; (2) defining the cell type specific contributions of CNBP in control of Th1 immunity to Toxoplasma gondii and TH17 responses to Candida Albicans; and (3) defining the role of CNBP in controlling Inflammatory Bowel Diseases using DSS colitis and related models.

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    Identification and functional characterization of chromatin-associated long non-coding RNAs (ca-lncRNAs) involved in innate immune responses

    Long non-coding RNAs (lncRNAs) are important regulators of gene expression in diverse biological contexts. Their role in immune regulation is less understood. Recently, work from the Fitzgerald lab has revealed important functional roles of lncRNAs in innate-immunity. One of these, lincRNA-EPS controls the basal expression of immune genes through regulation of chromatin accessibility. How lincRNA-EPS restrains immune gene expression in macrophages and its in vivo functions remains to be better understood. Combining both the Fitzgerald lab’s expertise in lncRNAs and innate immunity with my own experience in immunity we will unveil lincRNA-EPS mechanism of action and in vivo functions. In addition, we will expand these studies to characterize new lncRNAs that control the inflammatory response. To this end we will define proteins involved in the lincRNA-EPS complex and study these RNA-protein complexes in vivo. lncRNAs are often co-expressed with protein-coding genes. However, not all lncRNAs that are co-expressed are functional. To date, function of only a handful of lncRNAs have been described. We hypothesize that functional lncRNAs regulate transcription through their association with chromatin in immune cells similar to lincRNA-EPS. We propose to characterize chromatin-associated lncRNAs in immune cells and identify their genomic targets. The conceptual innovation of this proposal lies in bridging our understanding of gene regulation in innate-immunity and inflammatory response with the expanding field of lncRNAs. These studies will provide critical insights into the inflammatory response and have the potential for the discovery of new biomarkers or targets for infectious and inflammatory diseases.

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