At present, there are no means to counter functional loss in the aging brain underlying both normal cognitive decline and dementia-related neurodegenerative disease in the elderly. As such, defining key regulatory molecular signatures in the aging brain associated with age-related functional decline may help identify potential targets to restore cognition at old age. Given onset of cognitive impairments is not uniform across cognitive modalities, we sought to delineate the ages at which individual learning and memory processes exhibit dynamic deterioration. To define the temporal kinetics of age-related cognitive decline, we first assessed hippocampal-dependent learning and memory in the mouse species across the lifespan at young (3 months), mature (6 months), middle-aged (12 months), aged (18 months) and old (24 months) ages. We observed that the rate of cognitive decline was indeed different across distinct types of learning and memory, with recognition and associative memory impairments occurring at faster kinetics compared to spatial and working memory deficits. Young mice outperformed all age groups in recognition and associative memory, while mice that were middle-aged performed comparable to younger groups in spatial memory and working memory tasks. To define molecular changes associated with age-related cognitive decline, we performed single nucleus RNA sequencing (snRNA seq) and single nucleus chromatin accessibility sequencing (snATAC-seq) on hippocampi from young and aged mice and characterized differences in cell-type specific gene expression patterns and DNA regulatory elements during aging. DNA methylation has emerged as a critical modulator of neuronal plasticity and cognitive function. Notwithstanding, the role of enzymes that demethylate DNA remain to be fully explored. Here, we report that loss of ten-eleven translocation methylcytosine dioxygenase 2 (Tet2), which catalyzes oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), in adult neurons enhances cognitive function. In the adult mouse hippocampus, we detected an enrichment of Tet2 in neurons. Viral-mediated neuronal overexpression and RNA interference of Tet2 altered dendritic complexity and synaptic-plasticity-related gene expression in vitro. Overexpression of neuronal Tet2 in adult hippocampus, and loss of Tet2 in adult glutamatergic neurons, resulted in differential hydroxymethylation associated with genes involved in synaptic transmission. Functionally, overexpression of neuronal Tet2 impaired hippocampal-dependent memory, while loss of neuronal Tet2 enhanced memory. Ultimately, these data identify neuronal Tet2 as a molecular target to boost cognitive function.

Telomerase activation counteracts senescence and death-inducing telomere erosion caused by uncontrolled proliferation in cancer. Proliferation is highly correlated with telomerase activity across cancer types, suggesting there may be a mechanism linking these cancer hallmarks. In two of the major classes of adult diffuse glioma, high grade glioblastoma and low grade oligodendroglioma, telomerase reverse transcriptase promoter (TERTp) mutations are a predominant alteration underlying telomerase reactivation through recruitment of GA-binding protein (GABP). Very little is known about the mechanisms connecting drivers of proliferation to telomere maintenance and mutant TERTp regulation particularly in glioma. Epidermal growth factor receptor (EGFR) is commonly amplified in glioblastoma driving cell proliferation. EGFR amplification and TERTp mutations are frequent and significantly co-occur in glioblastoma. Oligodendroglioma lack EGFR amplification, but commonly rely on CIC mutations which modulate signaling downstream of receptor tyrosine kinase signaling. To determine if these two signaling pathways are functionally connected to mutant TERTp regulation in glioma, we combined analyses of copy number, mRNA, and protein data from primary tumor tissue with clinical pharmacologic treatments and genetic perturbations in cell lines and patient-derived cultures. From these data, we demonstrate that proliferation arrest lowers TERT expression in a GABP-dependent manner. We elucidate a proliferation-to-immortality pathway from EGFR to increased expression from the mutant TERTp through activation of AMP-mediated kinase (AMPK) and subsequent GABP upregulation. We demonstrate the phenotypic consequences of EGFR-AMPK on promoting telomerase activity and telomere length. These results implicate GABP as a direct link between proliferation signals and telomerase reactivation that is specific to TERTp mutant glioblastoma. In oligodendroglioma, we demonstrate that CIC inactivation upregulates TERT from the mutant TERTp through E24 transformation-specific (ETS) factors other than GABP, showing that mechanisms driving proliferation can simultaneously upregulate the mutant TERTp in a cancer-type specific manner.

To orchestrate the complex events of craniofacial morphogenesis requires a concerted effort between gene expression and tissue specific events. In the embryonic midface, facial prominences must execute a coordinated fusion between maxillary, medial and lateral nasal prominences at the lambdoidal junction, requiring breakdown and removal of epithelium to permit coalescence of underlying mesenchyme. When this process is disrupted by genetic mutations, cleft lip, the most common craniofacial birth defect in humans, is created. Key to understanding cleft lip pathogenesis is the reliance on mouse models. Models of cleft lip and studies of the role of epithelium are rare in comparison to cleft palate and research on underlying mesenchyme. The epithelium possesses discrete roles that facilitate fusion at the lambdoidal junction, including apoptosis and epithelial to mesenchymal transitions. To better understand its heterogeneity, we sought to enrich for the midface epithelium and isolate it from mesenchyme to gain fuller resolution of the lambdoidal junction transcriptome. This dissertation contains the product of that endeavor to focus on the role of the epithelium alone both in normal conditions and in cleft lip pathogenesis. In doing so, an atlas of lambdoidal junction epithelium at timepoints before, during and after fusion of the midface was created and validated. Through these methods, a deep analysis of each cluster resulted in the localization of a cluster undergoing cell cycle arrest and located at the fusion site of each prominence. A series of experiments were conducted which demonstrated that cell cycle arrest is characteristic of this population that is altered in cleft lip models. Additional research yielded ties to normal midface morphogenesis as well as novel orofacial cleft risk candidates, including Zfhx3 a gene with ties to cell cycle arrest. This thesis also expands upon the phenotypic characterization of a novel mouse cross which conditionally removes Bmpr1a from the ectoderm of the embryo, creating a model of cleft lip and palate. This work collectively produces knowledge of the midface epithelium at a scale of resolution not seen before as well as describes in detail how cell cycle arrest is a characteristic of primary palate fusion.

Long noncoding RNAs (lncRNAs) have been described in cell lines and various whole tissues, but lncRNA analysis of development in vivo is limited. Here, we comprehensively analyze lncRNA expression of the adult mouse subventricular zone neural stem cell lineage. We utilize complementary genome-wide techniques including RNA-seq, RNA CaptureSeq, and ChIP-seq to associate specific lncRNAs with neural cell types, developmental processes, and human disease states. By integrating data from chromatin state maps, custom microarrays, and FACS purification of the subventricular zone lineage, we stringently identify lncRNAs with potential roles in adult neurogenesis. shRNA-mediated knockdown of two such lncRNAs, Six3os and Dlx1as, indicate roles for lncRNAs in the glial-neuronal lineage specification of multipotent adult stem cells. Using our lncRNA pipeline, we identify Pinky (Pnky) as a long non-coding RNA (lncRNA) that regulates the transition between NSCs and neurogenic progenitors. Pnky is expressed in NSC populations and becomes downregulated during neurogenesis. shRNA-mediated knockdown of Pnky expands the pool of neurogenic progenitors, increasing neurogenesis. Pinky is conserved and expressed in NSCs of the developing mouse and human cortex. In embryonic mouse brain, Pnky knockdown accelerates neurogenesis and depletes this embryonic NSC population. Pnky physically interacts with PTBP1, a regulator of mRNA splicing, and Pnky depletion leads to the differential splicing of PTBP1 targets. These data thus identify Pinky as a conserved lncRNA that regulates a critical stage of neurogenesis from NSCs in the adult and embryonic brain. Taken together, our data forms the foundation of a resource for the study of lncRNAs in development and disease. In depth study of the novel lncRNA Pnky highlights the utility of this resource for the identification of lncRNAs that play key roles in neural lineage progression.

The acquisition of cell fate is dependent on gene regulatory networks that are regulated spatiotemporally by cell type specific transcription factors (TFs). In binding to a class of cis-regulatory elements known as enhancers, TFs stimulate transcription at target genes. Studies on enhancers have revealed that enhancers exist in multiple different states and interconvert between them. The establishment of an active, transcriptionally promoting enhancer state from an inactive state is a stepwise process initiated by TFs and then facilitated by chromatin regulators. Here, we investigate the molecular processes required to establish an active, transcriptionally promoting enhancer state. To study enhancer activation, we utilize an in vitro embryonic stem cell differentiation system known as the naive to formative transition that recapitulates gene regulatory events that occur in vivo during mouse early embryogenesis. We first investigate the generalizability of current molecular models of enhancer activation in Chapter 2. Using the naive to formative transition and genetic deletions we find that the homologous enzymes MLL3/4 (KMT2C/D) are required for activation of some but not all enhancers as previously thought. Moreover, surprisingly, there is an underwhelming impact on gene expression changes during the transition despite loss of MLL3/4 and the loss of “active” molecular signatures at many enhancers. This work demonstrates the existence of multiple modes of enhancer activation and suggests a more cell-context specific role for the key chromatin regulators MLL3/4 than known before. In ongoing work in Chapter 3, we are investigating the current prevailing molecular model of enhancer activation more deeply. We built a new synthetic system using the TF Grhl2 for rapid and conditional induction of enhancer activation. We are currently employing this system to identify the mechanistic relationships between Grhl2, MLL3/4, other key chromatin regulators, and transcription. Our discoveries on the molecular fundamentals of enhancer state dynamics serve as a foundation on which to better understand drivers of human development and disease.

In this study, I present and validate the first version of MAPES, an agent-based model (ABM) that facilitates the study of international migration (IM) by providing a framework within which theories from different fields can be easily modeled and reconciled. To validate this model, I examine three scenarios that test the capabilities of the model and provide opportunities to make substantive contributions to the field of international migration studies. I use the first of these scenarios, the bilateral economic gradient scenario, to show how rational decisionmaking by individuals gives rise to anti-immigrant sentiment and the flow patterns predicted by macro-economic migration theory. The second scenario, which I call the trilateral equal scenario, exhibits the theoretical phenomenon of path dependence, in which small random changes lead to large differences in migration system outcomes. Finally, I use a scenario that models the real-world border between Mexico and the US to predict the impacts of raising the cost of border crossings from south to north. Through these efforts, I show that MAPES is a research tool which can used to make valuable contributions to the field of IM studies.