While there are many FDA-approved therapies to treat relapsing-remitting multiple Sclerosis (MS), there are far less options for treating neurodegeneration in progressive disease. Intriguingly, similar to other neurodegenerative diseases (e.g. Alzheimer’s disease and frontotemporal dementia), a hallmark feature of progressive disease in MS is the loss of synapses and gray matter atrophy. Our lab recently discovered a striking loss of synapses in the visual thalamus of MS patient tissue and MS-relevant mouse and marmoset models concomitant with visual impairment. This was particularly intriguing since prolonged visual impairment is historically attributed to demyelination of the optic nerve and is a frequent occurrence in MS patients. As complement proteins were previously shown to mediate synapse elimination by phagocytic microglia in neurodegenerative disease and genetic variants in complement proteins have recently been correlated with visual impairment in MS patients, Dr. Schafer’s lab has been exploring this pathway in synapse loss in the visual thalamus in MS. First, Dr. Schafer’s lab showed that complement proteins C1q and C3 were both increased in a mouse and non-human primate model of MS (experimental autoimmune encephalomyelitis, EAE). However, unlike in development, C1q did not localize to synapses in this context. Instead, C3 was highly synaptic in EAE to induce microglia-mediated phagocytosis and elimination of synaptic material. In contrast to C3 at synapses, Dr. Reich and Dr. Schafer identified that C1q was particularly high in microglia surrounding chronic active MS lesions and loss of C1q in microglia in the mouse EAE model attenuated the inflammatory response of microglia. Still, it is unclear how C1q is modulating inflammation, and whether C1q is working upstream of C3 to regulate synapse loss or if synapse loss is occurring through the alternative pathway, independent of C1q. Also, there are many other molecules that regulate complement proteins and it is unclear how many of these complement-related proteins contribute to MS-related disease. Therefore, the overall goal of this proposal is to gain a more comprehensive understanding of how complement proteins are regulated in demyelinating disease.

B cells are essential immune cells that protect the host from infections via antibody production. This requires B cells to acquire e9ector functions and di9erentiate from naïve into germinal centers, and eventually into antibody-secreting plasma cells. Cell- cell interactions underpinning e9ective B cell response have been extensively studied, yet, less focus has been placed on soluble factors involved in this process, notably, mechanistic insights into lipid production and sensing on B cell immunity is still lacking. To this end, I will characterize the role of the liver X-receptors (LXR), nuclear hormone receptors regulating cholesterol homeostasis, during a B cell response. I aim to understand, with the highest granularity, how B cells integrate intrinsic and extrinsic lipid metabolic cues. Using cutting edge approaches, including conditional and inducible murine knock-out models, dietary interventions, targeted epigenetic profiling, single-cell RNA sequencing (ScRNAseq), and spatial transcriptomic to 1) Investigate LXR requirements for germinal center B cell and plasma cell di9erentiation, proliferation and maintenance in both homeostasis, vaccination and infection, in di9erent tissues; 2) Elucidate the molecular mechanisms of LXR transcriptional activity and regulation in germinal centers and antibody-secreting plasma cells; and 3) Identify the natural LXR ligand(s) that specifically shape B cell responses. My research will help resolve with high granularity how lipid metabolite sensing tunes humoral immunity in steady state and inflammation. Furthermore, it will help better understand B cell immuno-metabolic circuits that are tuned by lipid metabolites and that might be leveraged to design harmacological interventions enhancing antibody-mediated immunity.

Loss of the breast cancer susceptibility (BRCA1 or BRCA2) genes in hereditary breast and ovarian cancer (HBOC) is characterized by defects DNA repair by homologous recombination (HR) and in the protection of replication forks (known as fork protection (FP)). It is thought that HR and FP deficiencies produce points of vulnerability in cancer cells because they cannot fix or prevent DNA double stranded breaks (DSBs) and therefore cells are sensitive to DNA damaging agents such as to cisplatin and Poly (ADP-ribose) polymerase (PARP) inhibitors (PARPi). Our recent findings provide a counter model in which these therapies induce single stranded DNA (ssDNA) gaps that sensitize BRCA deficient cells due to a defect in gap suppression (GS). Several BRCA mutant cell models support gaps in mediating response, however, each model of resistance maintains at least two functions. Thus, it is not certain which function underlies the resistance, leaving a knowledge gap that limits clinical insight. The development of effective therapies requires identifying whether HR, FP, and/or GS is the fundamental mediator of response. This goal of this study is to systematically disrupt and retain each function (HR, FP, GS) within BRCA2 to define what function is critical for therapy resistance, elucidate a unified mechanism of resistance, and provide insight into inhibiting pathways of resistance to inform therapeutic choices. To do this we aim to determine the molecular mechanism of GS through mapping the GS domain(s) in BRCA2 (Specific Aim 1). In BRCA2 deficient cells complemented with wild-type vs a series of BRCA mutants that either delete or selectively target well-characterized domains (i.e., HR or FP), protein interacting regions, or DNA binding sites, we will analyze gap induction in our routine DNA fiber and immunofluorescence assays. If not already well characterized, we will assess mutants for HR proficiency in standard assays and FP via examination of nascent strand degradation in DNA fiber assays. We will use CRISPR/CAS9 to make additional mutants in the identified GS domain(s) to further characterize the critical residues mediating GS. We will also test PARPi sensitivity of these mutant expressing cells in order to assess the link of HR, FP, or GS to response. We also aim to determine if apoptosis underlies loss of cell viability in BRCA2 deficient cells following genotoxins (Specific Aim 2). Apoptosis will be measured using standard assays in BRCA2 mutants following treatment with cisplatin or PARPi. In addition, we will treat cells with apoptosis inhibitors and determine if sensitivity to PARPi or cisplatin is suppressed. We will verify the time and dose in which DSBs are induced compared to apoptosis and assess if inhibition of apoptosis reduces DSB formation. The rationale for the proposed research is that BRCA2 deficiency will be most effectively treated by therapies that form gaps, gap formation will be a biomarker of tumor response, and to maximize therapy response, pathways limiting gap formation should be targeted. The insight gained from the experiments proposed will have implications for cancer and provide new opportunities for therapeutic intervention.

The overarching goal of this proposal is to gain a deeper understanding of oligodendrocyte progenitor cell (OPC) phagocytosis and determine how it compares to the phagocytic ability of microglia and its relevance for neurological disease. Oligodendrocyte progenitor cells (OPCs) are a pool of progenitors found in the adult brain that give rise to mature, myelinating oligodendrocytes throughout life. Recent work has established that OPCs function as phagocytes and engulf synapses within both the developing and adult brain. This challenges the long-held notion that the sole function of OPCs is to generate new oligodendrocytes and establishes a role for OPCs in synapse pruning, similar to microglia. However, there is currently no understanding of how OPC phagocytosis impacts their progenitor function or how their phagocytic function compares to microglia. It is also unknown if OPCs function as phagocytes in the context of neurodegeneration. I will now use a combination of in vitro and in vivo phagocytosis assays and live cell imaging to compare OPC and microglial phagocytosis under steady-state conditions and during neurodegeneration and determine how phagocytosis impacts OPC function as progenitor cells. In Aim 1, I will use in vitro phagocytosis assays coupled with live cell imaging, OPC differentiation analyses, and RNA-sequencing to compare microglia and OPC phagocytosis of synaptic substrates and determine how phagocytosis impacts OPC differentiation. In Aim 2, I will utilize an animal model relevant to multiple sclerosis (MS) where the Schafer lab has shown early synapse loss and AAV-driven inhibition of complement C3 deposition to determine if OPCs function as phagocytes in the context of neurodegeneration, and if this phagocytosis is complement C3-dependent. The proposed studies will build on emerging work that challenges the current thinking that microglia are the primary phagocytes of the CNS. It will reveal the role of OPC phagocytosis in modulating the differentiation capacity of OPCs. It will also determine if OPC phagocytosis is dependent on the deposition of the complement protein C3. These results will have implications for neurodegenerative diseases, as lack of OPC differentiation is observed in multiple neurodegenerative states. Additionally, complement is now a target for therapeutic intervention in a variety of neurological diseases, so understanding how this pathway regulates other cells of the brain is essential for the effective use of these therapeutics. Finally, the experiments outlined here combine my strength in OPC biology from graduate school with the expertise in microglia and mechanisms of phagocytosis in my postdoctoral lab. Together, these studies will give me ideal training to achieve my goal of becoming an independent researcher studying glia-glia interactions in neurodegeneration.

Negative affect and stress experienced during alcohol abstinence can be a major factor contributing to relapse in alcohol use disorder, yet underlying neurobiological mechanisms remain ill-defined. Corticotropin-releasing factor (CRF)-expressing neurons in the bed nuclei of the stria terminalis (BNST) are involved in anxiety and stress responses, and they play a major role in the withdrawal. A subcommissural population of CRF neurons in the BNST (vBNSTCRF) is heavily innervated by hindbrain noradrenergic neurons that co-express prolactin releasing peptide (PrRP). Because these PrRP neurons are sensitive to both stress and interoceptive state, they are likely involved in the development of stress hypersensitivity following withdrawal. However, the role of PrRP neurons in alcohol-related behaviors has not been studied. I hypothesize that signaling of PrRP neurons to vBNST during acute ethanol withdrawal contributes to the development of negative affective behaviors, and that ethanol withdrawal potentiates vBNSTCRF responses to stress and PrRP neuronal activation. In the proposed project, mice will undergo chemogenetic silencing of PrRP+ neurons projecting to vBNST during acute ethanol withdrawal to examine the necessity of the circuit in the development of negative affective behaviors (Aim 1). Then, the influence of ethanol withdrawal on in vivo calcium responses of vBNSTCRF neurons to stress and chemogenetic activation of PrRP+ neurons will be explored using fiber photometry (Aim 2). Finally, monosynaptic tracing and whole brain imaging will be used to define additional brain regions innervating vBNSTCRF neurons that may be modulated concomitantly during ethanol withdrawal (Aim 3). The results of these studies will provide an improved understanding of neurobiological mechanisms impacting affective behavioral changes during ethanol abstinence. This project also provides a strong platform to expand and strengthen the trainee’s expertise in modern behavioral neuroscience approaches, including intersectional viral strategies for chemogenetic manipulation of neural circuits, observation of cell-type specific in vivo calcium signaling, and characterization of monosynaptic inputs to specific cell populations.

"Small noncoding RNAs <70 nucleotides long (sncRNAs) regulate diverse biological processes, including development and epigenetic inheritance. For example, in the germ cells of male placental mammals PIWI-interacting RNAs (piRNAs) and microRNAs (miRNAs) guide Argonaute proteins to silence transposable elements and mRNAs, thereby ensuring the development of functional sperm. As sperm mature, piRNAs and miRNAs are replaced by tRNA fragments, which are believed to mediate epigenetic inheritance. Methods to clone and identify sncRNAs have played a key role in understanding their biogenesis and function. Notably, the strategies typically used to sequence Argonaute protein-associated small RNAs only capture RNAs with 5′-monophosphate (P) and 3′ hydroxyl (OH) termini. Consequently, they fail to detect tRNA fragments or other sncRNAs with different terminal groups. Methods that convert all termini to 5′-P and 3′-OH can capture diverse sncRNA types but fail to retain knowledge of the original terminal groups, information critical to deducing sncRNA biogenesis and function. To fill this gap, we have developed Terminus-Specific Ligation and sequencing of sncRNAs (TSL-Seq), a high-throughput sequencing framework that captures diverse sncRNAs while identifying the terminal groups of each RNA. Here, I propose to: 1. Maximize the efficiency of the TSL-Seq cloning procedure; 2. Use TSL-Seq to capture the diversity of mouse sncRNAs in developing and mature male germ cells; 3. Use TSL-Seq to capture the diversity of mouse sncRNAs in female germ cells and zygotes. Optimizing TSL-seq will decrease the amount of input RNA required, allowing characterization of sncRNAs from rare cell types or mutant tissues. This project will produce the first comprehensive catalog of sncRNAs in developing mouse sperm and oocytes, uncover paternally and maternally deposited sncRNAs, and reveal the fates of sncRNAs after fertilization before the activation of zygotic transcription"

Vascular dysfunction and cerebral blood flow (CBF) reduction have been increasingly implicated in the pathogenesis of Alzheimer’s disease (AD). Interestingly, a subset of microglia, are known to reside in a vascular niche. Microglia have now been implicated in regulating blood flow and vasodilation and, in AD, microglial depletion produces a change in Aβ deposits in blood vessels. It is unclear whether these effects are solely attributed to juxtavascular microglia (JVM) and whether JVM represent a unique population of microglia that are preferentially susceptible to the aging and neurodegenerative process in AD. Thus, I will test if the crosstalk between the microglia and the vasculature is altered by aging and if this alteration is restricted to areas more vulnerable to neurodegeneration. To do so, I will implement an innovative approach called MERFISH to look at 100’s of genes directly in tissue. Ultimately, this study could result in identifying new therapeutic targets.

RNAi-based drugs are emerging as a new class of therapeutic modalities that are transforming pharmaceutical development. The National Institute of Arthritis and Musculoskeletal and Skin Diseases has granted a K99/R00 Pathway to Independence Award to Dr. Qi Tang to support his career transition to an independent principal investigator position at a U.S. academic institution and to fund his research on developing a programmable siRNA-based therapeutic platform for gene silencing in the skin. Dr. Tang will receive integrated scientific and career training during his K99 phase at UMass Chan, and in R00 phase, he will work to establish his independent laboratory with a focus on designing and expanding the utility of siRNA therapeutics for treating inflammatory skin diseases that currently lack sufficient treatments or are undruggable by conventional modalities.

Aging is the major risk factor for Alzheimer’s Disease (AD). Senescence, a hallmark of aging, is a process by which a cell no longer can proliferate. Since microglia, the primary immune cell of the central nervous system, become senescent in AD, I will investigate if they initiate the inflammatory process in the aging brain and study if senescent cells are present in specific locations within the brain in AD. To do so, I will implement an innovative approach called MERFISH to look at 100’s of genes directly in tissue. Ultimately, this study could result in identifying new therapeutic targets.

Smoking tobacco is one of the leading causes of preventable mortality in adults worldwide. However, there is currently a lack of pharmacological smoking cessation aids and their efficacy is limited. Therefore, there is an urgent need to better understand the neural mechanisms that underlie nicotine addiction for the development of novel smoking cessation treatments. Much of our understanding of the reinforcing effects of nicotine are derived from studies focused on canonical mesolimbic dopamine reward circuitry in the brain. Research suggests that the habenulo-interpeduncular pathway consisting of the medial habenula (MHb) and interpeduncular nucleus (IPN) contributes to the aversive motivational properties of high nicotine doses. It is speculated that activation of nicotinic cholinergic receptors (nAChRs) stimulates excitatory acetylcholine/glutamate (ACh/Glu) co-releasing projection neurons which activate ɣ-aminobutyric acid (GABA) neurons in the IPN. However, silencing IPN neurons in rodent models has also been shown to reduce nicotine consumption and reward. Thus, it is possible that modulation of MHb→IPN circuit activity may contribute to the balance of nicotine reward and aversion, a critical component of nicotine addiction liability. However, it is unknown how MHb→IPN neurons respond to nicotine within the context of reward related behavior. It is also unclear whether nicotine-induced activation of this circuit is necessary to elicit nicotine’s aversive properties. In the proposed project, I aim to test the hypothesis that modulation of IPN GABAergic neuron activity by nicotine is critical for balancing nicotine reward/aversion. I also aim to test the hypothesis that nicotine-induced control of ACh/Glu projection neuron activity from the MHb to IPN controls nicotine reward/aversion balance. Thus, I will assess the influence of rewarding or aversive nicotine doses on MHb or IPN neural activity during the performance of conditioned place preference/conditioned place aversion (CPP/CPA) procedures using in vivo fiber photometry combined with genetically encoded calcium indicators (GCaMPs). Finally, I will use in vivo optogenetics to selectively activate or silence MHb or IPN neurons to investigate whether specific components of this circuit are sufficient to drive nicotine reward/aversion balance. The goal of this project is to provide a better understanding of circuit-specific mechanisms that may contribute to nicotine reward/aversion balance, which will aid in the development of precise pharmacological treatments for smoking cessation.

Down syndrome (DS) is caused by trisomy for human chromosome 21 and affects more than five million individuals worldwide. Autism spectrum disorder (ASD) is a common co-occurring condition in persons with DS. Because individuals with DS-ASD often present with deleterious behaviours, such as self-injurious behaviour, aggression, sleep disturbances, and feeding difficulties, identifying effective treatments for the behavioural sequelae of DS-ASD has the potential to improve quality of life for individuals with DS and their caregivers. However, recent research has focused on diagnosing DS-ASD and describing its behavioural profile. The underlying molecular mechanisms that contribute to DS-ASD risk remain unexplored. Other DS-associated phenotypes likely result from dysregulation of the Sonic hedgehog (Shh) signalling pathway, which is a critical developmental pathway. Because Shh signalling is required for the differentiation of serotonergic neurons and because the serotonergic system is implicated in the pathogenesis of ASD, we hypothesized that disruption of Shh could result in behavioural phenotypes relevant to ASD. To address this hypothesis, we will perturb Shh signalling in developing zebrafish and assess serotonergic neuron morphology, overall brain structure, brain activity, and behaviour. How trisomy 21 contributes to misregulation of Shh also merits further study. Based on a previous screen of chromosome 21 genes, we selected HMGN1 as a likely regulator of Shh. We will overexpress HMGN1 and explore its effects on Shh signalling, serotonergic neurons, behaviour, gene expression, and epigenetic modifications. Successful completion of this project will lay the foundation for drug screens relevant to DS-ASD and cognition in larval zebrafish models.

Physical frailty, characterized by decreased physiologic reserve and increased vulnerability to stressors, and cognitive impairment, ranging from mild impairment to dementia, often co-occur in older adults. Both are associated with considerable adverse health outcomes, high healthcare costs, and substantial caregiver burden, and highly prevalent in U.S. community-dwelling older adults. However, for older adults receiving long-term care in nursing homes, data is scarce on the prevalence of the two conditions over their stay. Community-based studies suggest heterogeneous clinical presentation of physical frailty, which may have implications for its management. It is unknown if such heterogeneity is similar in older nursing home residents and if it is influenced by cognitive impairment. Further, physical frailty and cognitive impairment share risk factors and predict future onset of one another but the mechanism of this complex interplay remains unclear. Lastly, depression is strongly correlated with both conditions, yet findings regarding the impact of antidepressants on the progression of physical frailty and cognitive impairment are inconsistent. This proposed F99/K00 project seeks to address these gaps by two specific aims with population longitudinal data and advanced statistical methods. Aim 1 (dissertation research) focuses on older nursing home residents and will describe the prevalence of physical frailty and cognitive impairment; identify subgroups of physical frailty and examine the variation of subgroups by cognitive impairment levels; and delineate the developmental trajectories of physical frailty and cognitive impairment and examine the correlations between trajectories. Aim 2 (post-doctoral research) expands to older adults in the community and will assess the reciprocal association between physical frailty and cognitive impairment; quantify the impact of cumulative exposure to antidepressants on trajectories of physical frailty, cognitive impairment and depressive symptoms; and examine the effect of depressive symptoms as a mediator of physical frailty on cognitive impairment with causal mediation analysis. Methodological innovations include the use of latent class analysis, group-based trajectory models, structural equation models (autoregressive cross-lagged panel analysis; autoregressive latent trajectory model), and causal mediation. This proposal is directly relevant to the growing aging population in the U.S., including those residing in the nursing homes and those living in the community, since it uses the national nursing home database Minimum Data Set 3.0 (Aim 1) and the nationally-representative Health and Retirement Study linked to Medicare Part D Drug Event Files and the Harmonized Cognitive Assessment Protocol (Aim 2). This project will shed light on the concurrent progression of age-related physical and cognitive conditions. Results will inform future work to develop diagnostic tools and prediction models to facilitate timely identification of older adults at risk for accelerated functional decline, and implement care tailored to older adults’ needs to effectively delay the onset of negative health outcomes, enhance quality of life, and foster a healthy longevity.

Dr. Ceglia will advance our understanding of glomerulonephritis development by investigating the molecular mechanisms underpinning oxysterol production and function in the kidneys. This work may also lead to the identification of oxysterol regulation as a new target for prevention and treatment of IgA nephropathy.

Dr. Afshari will seek to define the underlying mechanisms of photosensitivity and disease pathogenesis in patients with cutaneous Dermatomyositis (DM), an autoimmune disease characterized by skin rash and muscle weakness. Additionally, studies will aim to dissect how skin epithelial and immune cells in DM respond to ultraviolet B exposure and explore novel specific disease markers with potency to be used as therapeutic targets.

An understanding of how epigenetic changes rewire the regulatory network of cancer cells would facilitate the development of new therapeutic strategies. However, tumor heterogeneity hinders the study of epigenetic landscapes in cancer. I propose to leverage a new single-cell genome profiling technology I recently developed to map epigenetic changes in single cells, allowing me to characterize different cell populations within ovarian tumors, including cancer stem cells (CSCs) and more differentiated cell types. These studies will uncover how the epigenome is re-wired upon inhibition of epigenetic enzymes implicated in multiple cancers. EZH2 is a histone methyltransferase that is often overexpressed in different types of cancer, including ovarian cancer, and inhibition of EZH2 has been shown to strongly reduce the aggressiveness of tumors by impairing metastasis, reducing invasiveness, and promoting differentiation. Yet, how EZH2 overexpression alters the repressive histone mark, H3K27me3 in each tumor cell type remains unknown. I propose three Aims, the first of which will use a combination of single cell Multi-CUT&Tag—a single cell profiling technology I recently developed—and scRNA-seq to characterize the epigenomic landscapes in each ovarian cancer cell type. In Aim 2, I will examine how perturbation of EZH2 alters the epigenetic architecture of tumor sub-populations, including CSCs, and identify how EZH2 promotes CSC self-renewal and tumor progression. Finally, in Aim 3, I will uncover the roles of genes and pathways subjected to epigenomic remodeling, in order to identify potential weaknesses that can be exploited therapeutically. Successful completion of these studies will provide considerable insight into the epigenetic regulation of CSCs in ovarian cancers and candidates for new therapies for ovarian cancer treatment. In addition, these studies will provide extensive training in tumor biology and single cell bioinformatics, which will be essential for my goal of running an independent group focused on tumor epigenetics.

Abstract Pneumonia is the leading cause of infection-related deaths worldwide, a fact that is set to rise exponentially with the SARS-CoV2 pandemic. Recovery from pneumonia requires both clearance of the pathogen and resolution of infection, the latter of which is critical to resume normal lung function. While both processes are important to host health, there is vastly less known about the mechanisms that regulate resistance to and resolution of tissue injury during pneumonia, representing a large knowledge gap in our understanding of the biology of the lung and its repair processes. Here, we propose that lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) modulates acute pulmonary inflammation in way that promotes resolution through reprogramming of leukocyte response. LOX-1 is a class E scavenger receptor, primarily known for its role in promoting vascular inflammation during atherosclerosis. In direct contrast, our data suggests that LOX-1 has a unique function in the lung, where it prevents edematous lung injury and inflammation, independent of bacterial clearance in murine models of Escherichia coli and Streptococcus pneumoniae pneumonia. Moreover, LOX-1 and its major ligand oxidized low-density lipoprotein (oxLDL) are elevated in patients with ARDS as a result of a confirmed diagnosis of pneumonia. Analysis of the cellular expression of LOX-1 in the lung revealed that alveolar macrophages and recruited (airspace) neutrophils are uniquely enriched for LOX-1 expression. Hematopoietic cells are also likely sources of LOX-1-dependent protection, as LOX-1-/- (WT recipient) chimeras are significantly more protected from injury than WT (LOX-1-/- recipient) chimeras during pneumonia. Assessment of the specific effects of LOX-1 inhibition on alveolar macrophages demonstrated that with inhibition macrophages are skewed towards inflammation and exhibit metabolic changes associated with increased glycolysis and lower fatty acid oxidation consistent with inflammatory macrophages. Moreover, we discovered that recruited neutrophils differ in their expression of LOX-1, where about half of neutrophils are positive during infection. Curiously, we also found phenotypic differences associated with LOX-1+ neutrophils that suggest increased cholesterol metabolism, which may uniquely promote tissue resolution. Taken together, leukocytes are an important source of LOX-1 and are likely responsible for LOX-1-dependent protection during pneumonia. However, whether and how LOX-1 elicits its protective effects on leukocytes is not known. Thus, we propose a central hypothesis that LOX-1 signaling evokes tissue-protective mechanisms in leukocytes (K99), that are associated with metabolic changes consistent with reduced inflammation and increased tissue recovery (R00). Results from our investigations will be the first to elucidate how LOX-1 is regulated at the transcriptional and metabolic level in the unique microenvironment of the lung, where it likely facilitates recovery from pneumonia and lung homeostasis.

Small interfering RNAs (siRNAs) are an emerging class of drugs that target disease-causing RNA for degradation in a sequence-specific manner. Such targeting is promising for hereditary diseases, where we want to deactivate (or "silence") the disease-causing protein encoded by a specific gene. Unfortunately, siRNAs face challenges when injected into the body, as they are unstable and not delivered to their target organ/cells. However, recent advances have led to the first approvals of siRNA drugs, in this case drugs that target hereditary diseases in the liver. This success is due to the use of highly specific targeting ligands and nanoparticles capable of protecting and delivering the drug to the liver. Dr Fakih, with the Khvorova lab, plans to develop and optimize new nanoparticles capable of delivering siRNA drugs beyond the liver, specifically to the brain.

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.

Successful specification of the two mouse blastocyst inner cell mass (ICM) lineages (the primitive endoderm (PrE) and epiblast) is a prerequisite for continued development and requires active fibroblast growth factor 4 (FGF4) signaling. Previously, we identified a role for p38 mitogen-activated protein kinases (p38-MAPKs) during PrE differentiation, but the underlying mechanisms have remained unresolved. Here, we report an early blastocyst window of p38-MAPK activity that is required to regulate ribosome-related gene expression, rRNA precursor processing, polysome formation and protein translation. We show that p38-MAPK inhibition-induced PrE phenotypes can be partially rescued by activating the translational regulator mTOR. However, similar PrE phenotypes associated with extracellular signal-regulated kinase (ERK) pathway inhibition targeting active FGF4 signaling are not affected by mTOR activation. These data indicate a specific role for p38-MAPKs in providing a permissive translational environment during mouse blastocyst PrE differentiation that is distinct from classically reported FGF4-based mechanisms.

Male infertility impacts >10% of couples trying to conceive, yet the molecular causes of this disease remain mostly indeterminate. The expression of microRNAs (miRNAs), a class of small regulatory RNAs that post-transcriptionally regulate gene expression, is often aberrant in infertile men, and disruption of miRNA biogenesis blocks germ cell development in mice. One family of miRNAs in particular, the miR-34/449-5p family, accounts for >40% of miRNAs in male mouse meiotic germ cells. Reduction of miR-34/449-5p family members in human seminal plasma was linked with oligoasthenoteratozoospermia and sertoli cell only syndrome. In mice, constitutive depletion of all miR-34/449-5p family members leads to oligoasthenoteratozoospermia, infertility, defective neurodevelopment, and perinatal lethality due to aberrant somatic ciliogenesis. Given the essential role of the miR-34/449-5p family in ubiquitous somatic ciliogenesis, I employed mouse germ cell-specific mutants of these miRNAs. These mutants are infertile bu otherwise physically indistinguishable from control mice. The cell-type-specific comparisons of mutant and control mice have enabled identifying molecular targets of these miRNAs in male germ cells. In the coming year I propose to confirm the importance of miR-34/449-5p-dependent repression of these targets for spermatogenesis, though genetic and biochemical approaches. The findings from this study promise to inform on potential treatments of male infertility.

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.

Neuroendocrine prostate cancer (NEPC) is an aggressive form of PCa that is associated with rapid progression, drug resistance and a very poor prognosis. It most commonly evolves from pre-existing adenocarcinoma by upregulation of regulators that drive stemness and lineage plasticity in response to pressure from therapies. Our lab discovered that vascular endothelial growth factor (VEGF) signaling in tumor cells is mediated primarily by neuropilins-2 (NRP2). VEGF/NRP2 signaling contributes to stemness associated with aggressive cancer cells. Interestingly, the genes for NRP2 and VEGF-A are significantly amplified in NEPC. Further, VEGF/NRP2 signaling regulates programmed cell death 1 ligand (PD-L1), which contributes to tumor progression cell-autonomously by promoting stemness and inhibiting anti-tumor immunity non-autonomously. Currently, there are no established therapies for treating NEPC. Importantly, treatments that can reverse stemness could sensitize NEPC cells to therapies. NRP2 can be an effective therapeutic target using function-blocking Abs, making it a promising target to reduce the aggressive behavior of NEPC and sensitize it to other therapies. I hypothesize that VEGF/NRP2 signaling contributes to the aggressive behavior of NEPC by sustaining the expression of PD-L1, which contributes to both stemness and immune evasion. Consequently, targeted inhibition of NRP2 is a promising therapeutic strategy for NEPC to inhibit stemness, activate anti-tumor immunity and synergize with PD pathway blockade. The objective of this proposal is to understand the role of VEGF/NRP2 signaling in regulating stemness and aggressive behavior of NEPC cells and to develop new therapeutic strategies for NEPC.

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.

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.

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.

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.

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.

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.

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.

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.

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?

This project has led to my interest in studying the co evolution of how newly integrated proviruses are incorporated into cellular programming 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 retroviruses 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.

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.

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.

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.

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.

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.

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.

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.