Browsing by Subject "Epigenetics"

In this dissertation, I reported several cellular reprogramming mechanisms in response to different factors, such as viral infection and oncogenic transformation, by utilizing molecular biology and high-throughput sequencing tools. In the first part of the dissertation, I investigated how hepatocytes contain HBV replication and promote their own survival by orchestrating a translational defense mechanism via the stress-sensitive SUMO-2/3-specific peptidase SENP3. We found that SENP3 expression level decreased in HBV-infected hepatocytes in various models including HepG2-NTCP cell lines and a humanized mouse model. Downregulation of SENP3 reduced HBV replication and boosted host protein translation. We also discovered that IQGAP2, a Ras GTPase-activating-like protein, is a key substrate for SENP3-mediated de-SUMOylation. Downregulation of SENP3 in HBV infected cells facilitated IQGAP2 SUMOylation and degradation, which leads to suppression of HBV gene expression and restoration of global translation of host genes via modulation of AKT phosphorylation. In the second part, I showed that, in Kras-mutant alveolar type II cells (AEC2), FOSL1-based AP-1 factor guides mSWI/SNF complex to increase chromatin accessibility at genomic loci controlling the expression of genes necessary for neoplastic transformation. I identified two orthogonal processes in Kras-mutant distal airway club cells. The first process was step-like in behavior and promoted their trans-differentiation into an AEC2-like state through NKX2.1. The second was linear and controlled oncogenic transformation through the AP-1 complex. Our results suggest that the chromatin state of the cell influences its response to oncogenic Kras. Other than the cell-type-specific effects, a cross-tissue conserved AP-1-dependent chromatin remodeling program regulates carcinogenesis.

The accurate and timely replication of eukaryotic DNA during S-phase is of critical importance for the cell and for the inheritance of genetic information. Missteps in the replication program can activate cell cycle checkpoints or, worse, trigger the genomic instability and aneuploidy associated with diseases such as cancer. Eukaryotic DNA replication initiates asynchronously from hundreds to tens of thousands of replication origins spread across the genome. The origins are acted upon independently, but patterns emerge in the form of large-scale replication timing domains. Each of these origins must be localized, and the activation time determined by a system of signals that, though they have yet to be fully understood, are not dependent on the primary DNA sequence. This regulation of DNA replication has been shown to be extremely plastic, changing to fit the needs of cells in development or effected by replication stress.

We have investigated the role of chromatin in specifying the eukaryotic DNA replication program. Chromatin elements, including histone variants, histone modifications and nucleosome positioning, are an attractive candidate for DNA replication control, as they are not specified fully by sequence, and they can be modified to fit the unique needs of a cell without altering the DNA template. The origin recognition complex (ORC) specifies replication origin location by binding the DNA of origins. The S. cerevisiae ORC recognizes the ARS (autonomously replicating sequence) consensus sequence (ACS), but only a subset of potential genomic sites are bound, suggesting other chromosomal features influence ORC binding. Using high-throughput sequencing to map ORC binding and nucleosome positioning, we show that yeast origins are characterized by an asymmetric pattern of positioned nucleosomes flanking the ACS. The origin sequences are sufficient to maintain a nucleosome-free origin; however, ORC is required for the precise positioning of nucleosomes flanking the origin. These findings identify local nucleosomes as an important determinant for origin selection and function. Next, we describe the D. melanogaster replication program in the context of the chromatin and transcription landscape for multiple cell lines using data generated by the modENCODE consortium. We find that while the cell lines exhibit similar replication programs, there are numerous cell line-specific differences that correlate with changes in the chromatin architecture. We identify chromatin features that are associated with replication timing, early origin usage, and ORC binding. Primary sequence, activating chromatin marks, and DNA-binding proteins (including chromatin remodelers) contribute in an additive manner to specify ORC-binding sites. We also generate accurate and predictive models from the chromatin data to describe origin usage and strength between cell lines. Multiple activating chromatin modifications contribute to the function and relative strength of replication origins, suggesting that the chromatin environment does not regulate origins of replication as a simple binary switch, but rather acts as a tunable rheostat to regulate replication initiation events.

Taken together our data and analyses imply that the chromatin contains sufficient information to direct the DNA replication program.

Organisms are presented with a wide variety of environmental stimuli and must interpret and respond to these cues in to perform a wide variety of behaviors, such as foraging, mating, fleeing, and fighting. The ability of an organism to recognize various stimuli, such as pheromones, to identify mates or competitors through the activation of various circuits and molecular components in the brain is tightly regulated. In order to delineate how molecular changes occur in the brain during stimuli response we used Drosophila melanogaster as it has a well-defined nervous system. We focus in on the circuit which regulates sex-specific mating behaviors in male D. melanogaster. Sex-specific splicing regulates the expression of two genes known as fruitless (fruM) and doublesex (dsxM) in the courtship circuit. Here we demonstrate using in the fly olfactory system that Olfactory receptor 47b (Or47b) and Olfactory receptor 67d (Or67d) activity, through sensory experience, regulates the expression patterns of male-specific fruM through coincident activity of hormone binding transcription factors Gce and Met and histone acetyltransferase P300 activity. We also identify various genes which changes in various mutant and social contexts, including exon specific changes in fruitless transcripts as well as changes in the expression of hormone metabolism genes, and neuromodulators in antennae. Given these changes in neuromodulators and the known structure of the FruM and DsxM central circuits, we looked at changes in the chromatin state and expression levels and find changes in peripheral sensory neurons have downstream effects on higher order circuits. We identify that FruM regulates the chromatin structure of both itself and dsxM in whole brain lysates and that changes in chromatin structure depend on pheromone receptor and neurotransmitter activity across processing centers in the brain. Taken together, we identify potential candidates for future study, as well as lay the framework for understanding how sensory changes in the periphery have effects on various neuronal clusters in the brain.

Tumor recurrence following initial treatment is the leading cause of death among breast cancer patients. Epigenetic mechanisms are critical for regulation of gene expression and to facilitate appropriate responses to environmental cues. However, it is increasingly appreciated that epigenetic dysregulation directly promotes therapeutic resistance and tumor progression. While genetic alterations have been shown to promote tumor progression, the contribution of non-genetic drivers of recurrence remains unexplored. In the current work, we utilized genetically engineered mouse models of breast cancer recurrence to evaluate the contribution of epigenetic plasticity to tumor recurrence and chemoresistance. First, we found that recurrent tumors undergo dramatic epigenetic and transcriptional reprogramming, partially through acquisition of an epithelial-to-mesenchymal transition (EMT). EMT promoted epigenetic silencing of tumor suppressor Par-4 through a unique, bivalent histone configuration. This bivalent configuration conferred plasticity to Par-4, and Par-4 silencing was reversed with epigenetic inhibitors of EHZ2 and HDAC. Further, Par-4 re-expression sensitized recurrent tumors to commonly utilized microtubule-targeting chemotherapeutics through altered cytoskeletal regulation. Second, we found that recurrent tumor epigenetic and transcriptional rewiring conferred sensitivity to G9a inhibitors. G9a inhibition promoted recurrent tumor cell necroptosis through demethylation of genes involved in a pro-inflammatory cytokine program. Further, knockout of G9a protein delayed the time until mammary tumors recurred in vivo. Collectively, our studies demonstrate that epigenetic dysregulation is a key feature of breast cancer progression, and pharmacologic strategies designed to target epigenetic enzymes underlying these processes may be of clinical value in the treatment of recurrent breast cancer.

The eukaryotic epigenome has an instrumental role in determining and maintaining cell identity and function. Epigenetic components such as DNA methylation, histone tail modifications, chromatin accessibility, and DNA architecture are tightly correlated to central cellular processes, while their dysregulation manifests in aberrant gene expression and disease. The ability to specifically edit the epigenome holds the promise of enhancing understanding how epigenetic modifications function and enabling manipulation of cell phenotype for scientific or therapeutic purposes. Genome targeting technologies, such as the CRISPR/Cas9 system, have successfully been harnessed to create epigenome editing tools to alter gene expression. Prominently, two leading CRISPR-based technologies, CRISPRa and CRISPRi, were shown to be highly specific and effective in controlling gene transcription levels. These tools, however, often lead to formation of complexes that affect a multitude of endogenous factors, thus mitigating our ability to elucidate the role of individual epigenetic marks. Moreover, changes in epigenetic marks are associated with numerous health conditions, therefore the development of tools that can modify specific marks may help in creating disease models, or the restoration of a “healthy” epigenome. We first created a suite of CRISPR-based epigenome modifiers (CRISPR-GEMs) that were aimed to catalyze the removal or addition of specific histone tail marks. Next, we tested a few promising CRISPR-GEMs on multiple target genes to characterize their effect on gene expression and chromatin marks. Furthermore, we utilized these tools to deepen our insights into the relationship of individual histone marks and gene expression in different contexts and to better our understanding of the kinetics and dynamics of several of these novel tools alongside existing ones. Additionally, we decided to use the CRISPRa platform to explore senescence, a cellular process that is at the epicenter of aging and has been shown to play a key role in various age-related diseases. Using the CRISPRa platform in an inducible-senescence cell model, we found and validated multiple transcription factors (TFs) that regulate senescence-associated growth arrest (SAGA). Lastly, we characterized genetic pathways that are pivotal to successful inhibition of SAGA, thereby demonstrating a new application of epigenome editing in a senescence model that enhanced our understanding of the pathways that govern SAGA.

The ability to regenerate after injury is quite astonishing, yet not all organisms share this ability. Mammalian genomes likely encode all gene products required to regenerate an amputated limb, yet they lack the correct instructions for strategically modulating those gene products to accomplish limb regeneration. While the catalogue of defined cell dynamics and molecular factors in tissue regeneration is expanding, we know comparatively little of how genes involved in regenerative events are engaged upon injury, despite decades of research. Certain non-mammalian vertebrates like salamanders and zebrafish possess these instructions, which exist as cis-regulatory elements that can direct expression of their target genes during regeneration. To identify candidate tissue regeneration enhancer elements (TREEs) important for zebrafish fin regeneration, we performed ATAC-seq from bulk tissue or purified fibroblasts of uninjured and regenerating caudal fins. We identified tens of thousands of DNA regions from each sample type with dynamic accessibility during regeneration, and assigned these regions to proximal genes with corresponding changes in expression by RNA-seq. To determine the extent to which these profiles reveal bona fide TREEs, we tested the sufficiency and requirements of several sequences in stable transgenic lines and mutant lines with homozygous deletions. Our study generates a new resource for dissecting the regulatory mechanisms of appendage generation and reveals a range of requirements for individual TREEs in control of regeneration programs.

*Designated as an Exemplary Master's Project for 2014-15*

Epigenetics is the study of inheritable mechanisms and factors that regulate gene expression. Although the underlying genetic sequence is the same in every cell, it is the epigenome that controls the expression of these genes and accounts for differences in phenotype. Epigenetic controls have clinical ramifications from cancer to autoimmune disorders to psychiatric pathologies. The main tool to study epigenetics is chromatin immunoprecipitation (ChIP), which probes the relationship between the underlying DNA and its structural histone proteins. Standard benchtop ChIP has five major drawbacks: (1) it requires a large input volume of cells, (2) it is very time consuming, work intensive, and low throughput, (3) it suffers from poor chromatin yield and sensitivity, (4) ChIP antibodies can be non-specific, vary by batch, and have low sensitivity, (5) and ChIP performs bulk tissue analysis which loses the granularity necessary to detect cell-to-cell variations. Digital microfluidic biochips (DMFBs) have proven successful at utilizing small volumes of reagents and samples to perform high throughput analyses using a variety of assaying techniques, making them an ideal platform for ChIP adaptation. Droplet manipulation using electrowetting-on-dielectric, in conjunction with magnetic bead control using magnetic field gradients generated by a current running through a wire on the device, provide all the necessary functionality to successfully run ChIP more efficiently on a DMFB. Translation of the benchtop ChIP protocol onto a DMFB addresses the issues facing epigenetic study workflow. The smaller volumes reduce reaction time, decrease reagent and sample use, and increase sensitivity and granularity towards single-cell resolution. Automation makes ChIP less labor consuming. DMFB platforms can be expanded for parallel operation and multiplexing thus increasing throughput. Finally, streamlining all the steps of ChIP onto one device greatly reduces sample loss, thereby expanding the type of studies possible. Herein, specifically modified nucleosomes and human chromatin were detected in a new semi-quantitative fluorescent immunoassay on a DMFB. Furthermore, chromatin was immunoprecipitated using a new targeted biotinylated technique. Successful chromatin capture and detection is a powerful tool for ChIP protocol development. This approach provides a rapid method to screen for antibody specificity and sensitivity as well as a confirmatory check point in the overall ChIP protocol to ensure that the target analyte has been isolated prior to any downstream analyses. Finally, a new modified ‘pull-through’ DMFB design was introduced to enhance the capture and detection of analyte-bound magnetic beads. The contributions from the studies described in this dissertation have provided the first steps towards ChIP implementation on a DMFB: 1) Developed new fluorescent confirmatory chromatin and nucleosome immunoprecipitation assays.

2) Demonstrated that the immunoprecipitation assays were detectible on-chip without any complex downstream analyses nor specialized fluoroscopy instrumentation.

3) Demonstrated that the immunoprecipitation assays performed at higher sensitivity than traditional benchtop ChIP.

4) Developed a single-channel pixel intensity measurement system for semi-quantitative analysis of chromatin and post-translationally modified nucleosomes directly on-chip.

5) Designed a new DMFB for improved capture of magnetic beads with twice the measured signal intensity using a new pull-through droplet scan method with on-chip embedded magnetic controls.

Primary glioblastoma (GBM) is the most common and lethal primary malignant brain tumor, with a median patient survival of only 15 months from the time of diagnosis. GBM is particularly challenging to treat due to its aggressive and invasive nature, and has proven resistant to therapeutic advances, with no significant improvement in outcomes over the past several decades. Understanding of the molecular characteristics of GBM, however, has improved dramatically, with genetic, epigenetic, and transcriptomic classifications now able to divide GBM into subtypes that provide prognostic information and guide the organization of clinical trials. One of the most frequent genetic alterations that has been identified in GBM is homozygous deletion of the methylthioadenosine phosphorylase (MTAP) gene, which occurs in 50% of all GBM cases. Despite its common occurrence, it is unclear what contribution MTAP loss makes in the pathogenesis of GBM or whether this genetic alteration can be used as a therapeutic target.

MTAP is a metabolic enzyme in the salvage pathway of adenine and methionine and its absence results in the accumulation of its metabolic substrate, methylthioadenosine (MTA), within and around tumor cells. MTA is known to inhibit activity of methyltransferases, raising the possibility that MTA accumulation is interfering with regulatory processes within the cell.

We utilized patient-derived GBM cell lines in vitro and GBM xenografts in vivo, to characterize consequence of MTAP deletion in GBM through analysis of DNA methylation, gene expression, and response to therapeutic agents. We show that MTAP loss promotes the formation of glioma stem-like cells through epigenomic dysregulation. We show these epigenetic changes influence gene expression patterns and alter the sensitivity to epigenome-modifying drugs. We also demonstrate that MTAP-null GBM cells are more tumorigenic in experimental models and that patients with MTAP deletion have poor disease outcomes. Finally, we show that targeting metabolic liabilities of MTAP-null cells through inhibition of de novo purine synthesis specifically depletes the therapy-resistant, stem-like cell subpopulation of GBM.

As the final component of this work, we explore the impact of MTA accumulation in the tumor microenvironment. We found that MTA alters the function of immune cells through adenosine receptor signaling, suggesting that modulation of adenosine receptor signaling in GBM may improve the native immune response and the efficacy of immunotherapeutics in the treatment of this disease.

This work thus establishes MTAP deletion as a pathogenic genetic alteration in the process of gliomagenesis by illustrating it’s contribution to the formation of the cancer cell epigenomic landscape, stemness characteristics, growth, and response to therapeutic agents.

Neural tube defects (NTDs) are a common class of human birth defects with a complex, multifactorial etiology. Although many contributing factors have been identified, an estimated 60% of human population risk remains unexplained. A portion of that risk is likely attributable to gene-gene and gene-environment interactions which have yet to be fully elucidated. In one project, we used whole-exome sequencing to identify candidate genetic factors in a multiplex anencephaly family, revealing an aggregation of rare and common variants in planar cell polarity genes among the affecteds. In the second project, we profiled the methylomes of a pair of monozygotic twins discordant for anencephaly and identified several differentially methylated sites which could contribute to NTD risk, particularly the mir-886 locus. Finally, we performed whole-exome and whole-methylome sequencing of mouse strains with differential susceptibility to fumonisin-induced NTDs, in combination with a human SNP association study. We identified epigenetic changes and variant associations which implicate Wnt and Hippo signaling genes as modifiers of the metabolic impacts of fumonisin exposure. These findings underscore the complexity of NTD pathogenesis and highlight the need to elucidate gene-gene and gene-environment interactions contributing to NTD etiology.

Fluctuations in nutrient availability occur for nearly all species, and adaptation to endure starvation conditions is essential. Genetic pathways involved in regulating starvation resistance are implicated in aging and complex diseases such as cancer, diabetes, and obesity in humans. Consequences of experiencing starvation persist later in life and subsequent generations, suggesting epigenetic regulation. However, much is still unknown about how starvation resistance is regulated and the contributions of different types of regulation. The roundworm Caenorhabditis elegans reversibly arrests development in the absence of food and can endure starvation for several weeks. Here, we investigate how transcriptional, epigenetic, and genetic regulation impact starvation resistance during developmental arrest in C. elegans. Gene expression dynamics change quickly during the first few hours of starvation, and broadly conserved transcription factors are required for starvation survival. However, temporal- and tissue-specific requirements of transcription for supporting starvation survival and recovery are largely unknown. In chapter 2, we used mRNA-seq combined with temporal degradation of RNA Polymerase II in the soma and germline to better understand gene regulation throughout arrest. We find that transcription is required in the soma for survival early in starvation but is dispensable thereafter, and known transcriptional regulators primarily act early in arrest. In contrast, the germline is transcriptionally quiescent throughout starvation, but germline transcripts are relatively stable compared to somatic transcripts. This reveals alternative gene-regulatory strategies in the soma and germline during starvation-induced developmental arrest, with the soma relying on a robust early transcriptional response while the germline relies on mRNA stability to maintain integrity. Phenotypic plasticity is facilitated by epigenetic regulation, and remnants of such regulation may persist after plasticity-inducing cues are gone, even affecting germ cells to impact subsequent generations. However, the relationship between plasticity and transgenerational epigenetic memory is not understood. Dauer diapause provides an opportunity to determine how a plastic response to the early-life environment affects traits later in life and in subsequent generations. In chapter 3, we find that, after extended diapause, postdauer worms initially exhibit reduced reproductive success and greater interindividual variation. In contrast, F3 progeny of postdauers display increased starvation resistance and lifespan, revealing potentially adaptive transgenerational effects. Transgenerational effects are dependent on the duration of diapause, indicating an effect of extended starvation. In agreement, RNA-seq demonstrates a transgenerational effect on nutrient-responsive genes. This work reveals complex effects of nutrient stress over different time scales in an animal that evolved to thrive in feast and famine. Many conserved genes and pathways regulate starvation resistance, but most genetic analysis in C. elegans has been restricted to a single genetic background, potentially restricting identification of additional genes. Hundreds of genetically distinct wild strains of C. elegans have been whole-genome sequenced and can be used for GWAS. In chapters 4 and 5, we implemented two high-throughput sequencing approaches, RAD-seq and MIP-seq, to determine relative starvation resistance of over 100 wild strains over time. We used GWAS to identify QTL associated with starvation resistance, near-isogenic lines to validate QTL, and CRISPR gene editing to modify specific genes within QTL. We focused on genes in the insulin receptor-like domain (irld) family, as this family has been virtually uncharacterized, but the genes share homology with the sole known insulin-like receptor in C. elegans, DAF-2, which is a major regulator of starvation resistance. We found that specific variants in two members of the irld family confers increased starvation resistance in multiple genetic backgrounds, and this is dependent on the transcription factor downstream of insulin signaling, DAF-16/FOXO. Thus, this work shows that natural genetic variation in novel modifiers of insulin-signaling regulates starvation resistance.

All DNA-templated events, including replication and gene transcription, occur in the context of the local chromatin environment. The passage of the replication machinery results in disassembly of chromatin, which must be re-assembled behind the replication fork to re-establish the epigenetic state of the cell. Many of the factors and mechanisms regulating DNA replication and chromatin assembly have been identified from elegant in vitro biochemical experiments, work in model systems like Saccharomyces cerevisiae, or novel proteomic approaches. In spite of current advances in the field, it is still not clear how the chromatin landscape is organized and re-assembled during this process.

Current methods, while informative, lack the genome-wide base-pair resolution required to assess the dynamics of chromatin assembly and maturation in a spatial-temporal manner. To overcome the limitations of these studies, I have taken advantage of an epigenome mapping technique based on micrococcal nuclease (MNase) digestion followed by paired-end sequencing. This approach facilitates the analysis of chromatin structure by capturing not only nucleosomes, but also smaller DNA binding protein footprints in a factor-agnostic manner. I have developed a technique based on this approach that generates Nascent Chromatin Occupancy Profiles (NCOPs) to study the dynamics of chromatin assembly following passage of the DNA replication fork at a genome-wide level and at single base-pair resolution in S. cerevisiae. It employs a nucleoside analog to specifically enrich for nascent chromatin, which can be captured following a chase over different periods of time. Thus, NCOPs resolve the structure of nascent and mature chromatin, facilitating the analysis of chromatin maturation across the entire genome.

Using NCOPs, I provide a comprehensive description of the maturation process across different genomic regions and the dynamics of small DNA binding factor association with nascent and mature chromatin states. Our results support previous work characterizing the structure of nascent chromatin as being more disorganized and having poorly positioned nucleosomes. Importantly, using positioning and occupancy scores, I provide new details on the structure of nascent and mature chromatin at intergenic regions, including replication origins, and at highly transcribed and poorly transcribed genes. I uncovered that local epigenetic footprints have the potential to shape the dynamics of chromatin assembly, generating a chromatin maturation landscape that is dependent on the parental chromatin. Finally, I resolved patterns of transcription factor occupancy with nascent and mature chromatin, and observed transient factor association in the nascent state.

In all, this work provides insight into the dynamics of chromatin assembly, and allows for genome-wide and base-pair resolution investigation of chromatin maturation. The genomic and bioinformatic approaches developed here open the door for further investigation of the dynamics of epigenetic inheritance and the role of known and unknown players in re-establishing the eukaryotic epigenome following passage of the DNA replication fork.

During the immune response, helper T cells must proliferate and upregulate key metabolic programs including glucose and glutamine uptake. Metabolic reprogramming is imperative for appropriate T cell responses, as inhibition of glucose or glutamine uptake hinders T cell effector responses. Glutamine and glutaminolysis use in cancer cells has partially been explored. However, the role of glutamine and its downstream metabolites is incomplete and unclear in T cells. The first step of glutamine metabolism is conversion to glutamate via the hydrolase enzyme glutaminase (GLS). To target glutaminolysis, two different methods were employed: 1) genetic knockout of GLS using a CRE-recombinase system specific for CD4/CD8 T cells, and 2) pharmacological inhibition of GLS via the potent and specific small molecular CB839. These two models of glutaminase insufficiency were used as a tool to target glutamine metabolism during T cell activation and differentiation both in vitro and in vivo.

GLS-deficient T cells had decreased activation at early time points compared to control. Over several days, these GLS-deficient T cells differentiated preferentially to Th1-like effector cells. This was reliant on increased glucose carbons incorporating into Tri-Carboxylic Acid (TCA) metabolites. This increased effector response in vitro occurred in both CD4+ T helper cells and CD8+ cells (Cytotoxic lymphocytes, or CTLs). Differentiation of CD4+ T cells to Th1 or Th17 subsets showed decreased Th17 differentiation and cytokine production, while Th1 effector responses were increased. This increased Th1 function was dependent on IL-2 signaling and mTORC1, as reducing IL-2 or inhibiting mTORC1 with rapamycin prevented GLS inhibition-induced Th1 effector function. Th17 cells, meanwhile, were inhibited by changes in reactive oxygen species, and recovery of Th17 function was achieved with n-acetylcysteine treatment.

T cells lacking GLS were unable to induce inflammation in a mouse model of Graft vs Host disease, an inflammatory bowel disease model, or in an airway inflammatory model. Importantly, Chimeric Antigen Receptor (CAR) T cells made from GLS knockout cells were unable to maintain B cell aplasia in recipient mice. Contrary to this, temporary inhibition of GLS via small-molecule inhibition increased B cell killing in vitro and enhanced T cell persistence in both the B cell aplasia and in a vaccinia virus recall response. These results indicate a balance, where permanent deficiency of GLS is detrimental to T cell responses, but acute inhibition can actually promote T effector responses and survival. Overall, this work aims to understand how perturbations in glutamine metabolism in T cells affects differentiation and function and the role of glutaminolysis and improve therapies for inflammatory disease and cancer.

Genomic imprinting is the parent-of-origin dependent monoallelic expression of select developmentally important genes that are regulated by epigenetic mechanisms. It is believed to have co-evolved with placentation in the Therian lineage, but it is unclear whether this phenomenon arose in a convergent or divergent manner in the Metatherians (those with a rudimentary placenta) and Eutherians (true placental mammals). Moreover, the precise epigenetic mechanisms involved in establishing genomic imprinting (DNA methylation or histone modifications) are still poorly defined. Thus, I studied Metatherian orthologues of Eutherian imprinted loci using Monodelphis domestica as a model organism. L3MBTL and HTR2A were monoallelically expressed; PEG1/MEST had one imprinted and one non-imprinted transcript, while IMPACT, COPG2 and PLAGL1 were not imprinted, thus revealing that this phenomenon is conserved at some, but not all loci between the two groups of Therians. Moreover, differential methylation patterns and the associated regulatory non-coding RNA are also not conserved amongst them, exemplified by the novel DMR identified within IGF2R which had no associated anti-sense transcript. However, histone modifications, specifically the activating H3 Lysine 4 dimethylation mark at the active allele's promoter seems to be important in both lineages and probably serves as the primordial imprint mark. Although the evidence does not resolve the issue of convergence or divergence, it raises the intriguing possibility that both forms of evolution occurred during establishment of imprinting in these mammals.

The imposition of functional haploidy in the genome by such epigenetic mechanisms necessarily makes imprinted genes more susceptible to deleterious mutations and regulatory perturbations. Thus, imprinting is implicated in a number of developmental disorders, but its role in the etiology of complex human diseases and neurological disorders, like autism and schizophrenia, remains to be determined. I chose to investigate the imprint status of the duplicated locus DGCR6/DGCR6L lying within the 22q11.2 microdeletion causative of DiGeorge Syndrome (DGS), because our lab previously predicted genes at this genomic location to be imprinted. My studies revealed that both genes DGCR6 and DGCR6L are monoallelically expressed in the primate lineage, but not in a parent-of-origin dependent manner. Interestingly, DGCR6L is not present in the mouse, and Dgcr6 is expressed from both parental alleles.

Although DGS primarily manifests as facial, limb and heart abnormalities in children, a number of these patients also ultimately present with variable neurocognitive defects. Thus, I focused my studies on determining the effect of the microdeletion at this chromosomal region on DGCR6 and DGCR6L expression because of their potential role in neural crest cell migration. This revealed that DGS subjects have a highly dysregulated pattern of DGCR6 and DGCR6L expression as compared to that in controls. Moreover, increased expression of these genes correlated significantly with decreased performance in sustained-attention tests. This provides the first evidence that disruption of the normal monoallelic expression pattern of DGCR6 and DGCR6L by hemizygous deletion is involved in the variability in neurocognitive symptoms associated with DiGeorge Syndrome. The results of my studies highlight the importance of searching for novel imprinted domains to better understand not only their evolution, but also the potential role of such epigenetically labile regions in modulating complex human diseases and neurological disorders.

The goal of this dissertation is two-fold: 1) to identify novel biological pathways implicating individual differences in reward and threat processing in the emergence of risk and resilience for psychopathology, 2) to identify novel genetic and epigenetic predictors of the inter-individual variability in these biological pathways. Four specific studies are reported wherein blood oxygen-level dependent functional magnetic resonance imaging (BOLD fMRI) was used to measure individual differences in threat-related amygdala reactivity and reward-related ventral striatum (VS) reactivity; self-report was used to measure of mood and psychopathology as well as the experience of stressful life events. In addition, DNA was derived from peripheral tissues to identify specific genetic and epigenetic markers.

Results from Study 1 demonstrate that individuals with relatively low reward-related VS reactivity show stress-related reductions in positive affect, while those with high VS reactivity remain resilient to these potentially depressogenic effects. Heightened VS reactivity was, however, associated with stress-related increases in problem drinking in Study 2. Importantly, this effect only occurred in individuals showing concomitantly reduced threat-related amygdala reactivity. Study 3 demonstrates that using a multilocus genetic profile capturing the cumulative impact of five functional polymorphic loci on dopamine signaling increases power to explain variability in reward-related VS reactivity relative to an approach considering each locus independently. Finally, Study 4 provides evidence that methylation in the proximal promoter of the serotonin transporter gene is negatively correlated with gene expression and positively correlated with threat-related amygdala reactivity above and beyond the effects of commonly studied functional DNA-sequence based variation in the same genomic vicinity.

The results from these studies implicate novel biological pathways, namely reward-related VS reactivity and threat-related amygdala reactivity, as predictors of relative risk or resilience for psychopathology particularly in response to stressful life events. Moreover, the results suggest that genetic and epigenetic markers may serve as easily accessible peripheral tissue proxies for these neural phenotypes and, ultimately, risk and resilience. Such markers may eventually be harnessed to identify vulnerable individuals and facilitate targeted early intervention or prevention efforts.

Background

Results

Conclusions

As a major component of the adaptive immune system, CD4+ T cells play a vital role in host defense and immune tolerance. The potency and accuracy of CD4+ T cell-mediated protection lie in their ability to differentiate into distinct subsets that could carry out unique duties. In this dissertation, we dissected the roles and interplays between two emerging mechanisms, miRNAs and epigenetic processes, in regulating CD4+ T cell-mediated responses. Using both gain- and loss-of-function genetic tools, we demonstrated that a miRNA cluster, miR-17-92, is critical to promote Th1 responses and suppress inducible Treg differentiation. Mechanistically, we found that through targeting Pten, miR-17-92 promotes PI3K activation. Strong TCR-PI3K activation leads to the accumulation of DNMT1, elevated CpG methylation in the foxp3 promoter, and suppression of foxp3 transcription. Furthermore, we demonstrated that an epigenetic regulator, methyl CpG binding protein 2 (MeCP2), is critical to sustain Foxp3 expression in Tregs, and to support Th1 and Th17 differentiation in conventional CD4+ T cells (Tcons). In Tregs, MeCP2 directly binds to the CNS2 region of foxp3 locus to promote its local histone H3 acetylation; while in Tcons, MeCP2 enhances the locus accessibility and transcription of miR-124, which negatively controls SOCS5 translation to support STAT1, STAT3 activation and Th1, Th17 differentiation. Overall, miRNAs and epigenetic processes may crosstalk to control CD4+ T cell differentiation and function.