Browsing by Subject "Gene Expression Regulation, Developmental"
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Item Apoptosis Determinants in Drosophila Melanogaster(2007-12-17) Chew, Su Kit; Abrams, John M.Apoptosis is a form of programmed cell death (PCD) that is governed by a core set of genes conserved across diverse metazoan phyla. Cells dying by apoptosis exhibit a characteristic series of morphological and biochemical changes that is also conserved. This form of PCD plays pivotal roles in homeostatic regulation of cell numbers, developmental sculpting of organs, damage and infection responses; conversely, its disregulation has profound implications in diseases such as cancers, immune disorders infertility and dystrophies. Common parallels in the regulation of the core apoptosis machinery have been elucidated in human and experimental model organisms, though many fundamental questions in our understanding of its regulation remain. A conserved node in the apoptosis pathway is the apoptosome, comprising the apical caspase and its adaptor protein. To understand the functions of this node, I generated a null allele of the apical caspase Dronc in the experimental model organism Drosophila melanogaster. Dronc is required for developmentally regulated apoptosis in multiple tissues during embryogenesis and larval development. Failure of apoptosis correlated with tissue hyperplasia. Notably, the removal of Dronc eliminated the cellular apopototic response to stresses in cells. In some of the stress contexts tested, Dronc depletion partially rescued cell viability to the same levels as pan-caspase inhibition by small peptide inhibitors, suggesting that Dronc functions map specifically to caspase activation and apoptosis. These and similar observations in its adaptor protein Dark point to the apoptosome as a key node for apoptosis in Drosophila. From these observations, I sought to use the induced apoptosis cellular response as a means to identify novel components and regulators in the apoptosis pathway. I optimized a cell culture system for high-throughput cell-based screening using RNA interference (RNAi) mediated gene silencing and a synthetic antagonist of inhibitors of apoptosis proteins (IAPs). From a genome-wide Drosophila RNAi library, I identified 42 potential genes required for apoptosis, of which I characterized 13 highly validated targets for their requirements in multiple stress contexts. One of these hits, Tango7, regulates pro-Dronc protein and represents an unprecedented point of apoptosis regulation. Collectively, my studies bolster the model for the crucial requirement of the apoptosome in apoptosis and identify new regulation entry-points into the apoptosis pathway.Item Compensation Between Foxp Transcription Factors Maintains Proper Striatal Function(August 2023) Ahmed, Newaz Ibrahim; Tsai, Peter; Chahrour, Maria; Roberts, Todd; Konopka, GenevieveSpiny projection neurons (SPNs) of the striatum are critical in integrating neurochemical information to coordinate motor and reward-based behavior. Mutations in the regulatory transcription factors expressed in SPNs can result in neurodevelopmental disorders (NDDs). Paralogous transcription factors Foxp1 and Foxp2, which are both expressed in the dopamine receptor 1 (D1) expressing SPNs, are known to have variants implicated in NDDs. Paralogous transcription factors are thought to have the ability to compensate for each other and previous work published by the lab supports the hypothesis that Foxp1 and Foxp2 have compensatory roles in D1 SPNs as well. For my dissertation work, I utilized mice with a D1-SPN specific loss of Foxp1, Foxp2, or both and a combination of behavior, electrophysiology, and cell type specific genomic analysis to address if there was compensation occurring. It is only upon the loss of both genes that motor behavior was impaired whereas Foxp1 mediated social behavior impairments were exacerbated upon the further loss of Foxp2 (Chapter Two). I also found that while loss of Foxp1 resulted in KLeak mediated hyperexcitability of D1-SPNs, this too was further impaired with the additional loss of Foxp2 (Chapter Three). Viral mediated re-expression of Foxp1 in the double knockouts was sufficient to restore both behavioral and electrophysiological impairments to baseline. I further studied the contribution of Foxp1 and Foxp2 to regulation of downstream targets genes using single-nuclei RNA-seq and found that in both juvenile and adult D1-SPNs, loss of both transcription factors resulted in differential expression of hundreds of genes (Chapter Four). I was able to use these experiments to also investigate how loss of these transcription factors from the D1-SPNs impacted gene expression in other cell-types (Chapter Five). I also utilized single-nuclei ATAC-Seq and again found that loss of both genes resulted in large scale dysregulation of chromatin state not seen in the single knockouts, including in regions enriched for Fox motifs (Chapter Six). I also began to address the open question of what the direct binding targets of Foxp1 and Foxp2 are using the newly developed CUT&RUN technique (Chapter Seven). The findings from my experiments point towards a form of compensation between Foxp1 and Foxp2 where one transcription factor maintains striatal function upon the loss of the other, which I discuss more in depth (Chapter Eight). I also discuss my involvement in a project where we further study the role of Foxp1 in D1- and D2-SPNs, which I am working on in collaboration with Dr. Nitin Khandelwal (Chapter Nine). I conclude by discussing the implications of my findings and suggest recommendations for further study (Chapter Ten).Item Defining Cardiac Conduction System Gene Regulatory Networks(2019-11-15) Bhattacharyya, Samadrita; Hon, Gary C.; Olson, Eric N.; Kittler, Ralf; Mammen, Pradeep P.; Munshi, NikhilThe cardiac conduction system initiates and propagates each heartbeat. Specialized conducting cells are a well-conserved phenomenon across vertebrate evolution, although mammalian and avian species harbor specific components unique to organisms with four-chamber hearts. Early histological studies in mammals provided evidence for a dominant pacemaker within the right atrium and clarified the existence of the specialized muscular axis responsible for atrioventricular conduction. Building upon these seminal observations, contemporary genetic techniques in a multitude of model organisms has characterized the developmental ontogeny, gene regulatory networks, and functional importance of individual anatomical compartments within the cardiac conduction system. Cis-regulatory DNA elements mediate transcriptional control (called enhancers in cases of transcriptional activation and silencers in cases of repression) by recruiting TFs. These collectively determine spatio-temporal regulation of each gene during development. Thus, determining the evolutionary history of participating enhancers provides key information on the origins of morphological trait diversity. My dissertation describes the development of novel genetic models, sensitive biochemical assays, and consistent genomic methodologies complemented with bioinformatics to highlight the gene regulatory networks that act during cardiac conduction system development and homeostasis with a particular emphasis on networks implicated in human electrical variation by large genome-wide association studies. As part of future directions of my doctoral work, I have also outlined some ongoing studies on determining the ontogeny of the cardiac pacemaker cells. I finally conclude with the translational impact of my research whereby we have made an impressive headway towards interrogating the development of the cardiac conduction system and onset of cardiomyopathies in human heart specimens.Item Epigenetic Regulation of Oligodendrocyte Development and Regeneration in the Central Nervous System(2016-10-26) He, Danyang; Kraus, W. Lee; Lu, Q. Richard; Johnson, Jane E.; Olson, Eric N.Oligodendrocytes (OLs) produce myelin sheaths that electrically insulate axons and promote rapid propagation of action potentials in the CNS. The onset and timing of CNS myelination and remyelination requires precise coordination between epigenetic programming and transcriptional regulation. In this thesis, I present my findings on two epigenetic regulatory complexes Chd7/Sox10 and lncOL1/Suz12 in CNS myelination and remyelination. First, we show that chromatin remodeler Chd7 is required for proper onset of CNS myelination and remyelination. Genome-occupancy analyses, coupled with transcriptome profiling, reveal that Chd7 interacts with Sox10 and targets the enhancers of key myelinogenic genes, and identify novel Chd7 targets including bone formation regulators Osterix/Sp7 and Creb3l2, which are also critical for oligodendrocyte maturation. Thus, Chd7 coordinates with Sox10 to regulate the initiation of myelinogenesis and acts as a molecular nexus of regulatory networks that account for the development of a seemingly diverse array of lineages including oligodendrocytes and osteoblasts, pointing to the hitherto previously uncharacterized Chd7 functions in white matter pathogenesis in CHARGE syndrome. To understand the role of lncRNAs in CNS myelination, we establish dynamic expression profiles of lncRNAs at different stages of oligodendrocyte development and uncover a cohort of stage-specific oligodendrocyte-restricted lncRNAs including a conserved chromatinassociated lncOL1. Genetic inactivation of lncOL1 causes defects in CNS myelination and remyelination following injury. Functional analyses illustrate that lncOL1 interacts with Suz12, a component of PRC2, to promote oligodendrocyte maturation in part through Suz12-mediated repression of a differentiation inhibitory network that maintains the precursor state. Collectively, these studies show that epigenetic circuitry between lncRNAs and transcription factors with chromatin-modifying complexes play roles in balancing inhibitory and activating gene program, allowing the timely CNS myelination and myelin repair.Item Evolution and Function of the Genomic Landscape in the Human Brain(2019-01-22) Fontenot, Miles Ray; Green, Carla B.; Konopka, Genevieve; Johnson, Jane E.; Monteggia, Lisa; Takahashi, JosephThe molecular mechanisms underlying human brain evolution are not fully understood; however, previous work suggested that expression of the transcription factor CLOCK in the human cortex might be relevant to evolution of the human brain and human cognition and disease. In this dissertation, we investigated this novel transcriptional role for CLOCK in human neurons by performing chromatin-immunoprecipitation sequencing for endogenous CLOCK in adult neocortex and RNA-sequencing following CLOCK knockdown in differentiated human neurons in vitro. These data suggested that CLOCK regulates expression of genes involved in neuronal migration, and a functional assay showed that CLOCK knockdown increased neuronal migratory distance. Furthermore, dysregulation of CLOCK disrupts co-expressed networks of genes implicated in neuropsychiatric disorders, and the expression of these networks are driven by hub genes with human-specific patterns of expression. Thus, these data support a role for CLOCK-regulated transcriptional cascades involved in human brain evolution and function. We further created a humanized mouse model with increased neocortical expression of CLOCK to mimic the human pattern of expression, providing a novel system for in vivo mechanistic studies of CLOCK function. Finally, we have conducted preliminary gene expression analysis of the human epileptic brain compared to control tissue from donors without neuropsychiatric disease. These data will be correlated with in-patient recordings of neuronal activity to identify potential new avenues for investigation into epilepsy. In total, we have contributed a rich dataset of genomics dataset related to CLOCK function in human neurons, generated a novel humanized mouse model, and initiated an exciting study into the gene expression and function of the epileptic brain.Item Examining the Role of PRDM13 in Dorsal Interneuron Specification(2016-07-29) Uruena, Ana Cristina; Wu, Jiang I.; Johnson, Jane E.; MacDonald, Raymond J.; Morrison, Sean J.PTF1A is a transcription factor transiently expressed as neural progenitor cells become post-mitotic and begin to express neuronal specific genes. PTF1A specifies these cells to become GABAergic (inhibitory) neurons while suppressing the glutamatergic (excitatory) program. A fundamental principle in bipotential fate decisions is the necessity to repress gene programs in the alternative fate. Our lab identified PRDM13, a zinc finger transcription factor and direct downstream target of PTF1A that may serve this function in the inhibitory/excitatory neuron fate choice. Overexpression of PRDM13 in chick neural tube shows it represses markers of the excitatory neuronal lineage. To explore PRDM13 function in vivo and expand these findings to regions outside the neural tube, a Prdm13GFP_KI and Prdm13ΔZF mutant mouse strains was generated and are null for PRDM13 expression. These mice die neonatally and at E10.5 show an increase in the dorsal neural tube excitatory neuron population at the expense of the inhibitory neurons. These phenotypes recapitulate that seen in Ptf1a null mice. These models have revealed additional insights into the function of PRDM13 in the developing spinal cord. First, PRDM13 negatively regulates Ptf1a providing a mechanism for downregulating PTF1A as development progresses. Second, in contrast to the phenotype seen with Ptf1a mutants, late stage mutant embryos show only a partial loss of the inhibitory interneuron population, possibly due to the higher levels of PTF1A in these mutants. Finally, ChIP-Seq and RNA-Seq analysis of heterozygote vs homozygote Prdm13 mutants revealed a novel function of PRDM13 to keep neuronal subtype specification genes for the ventral neural tube suppressed in the dorsal region. These mouse models has placed PRDM13 in a pivotal role in the specification of neuronal subtypes in the spinal cord, a function that will likely extend to the retina and cerebellum where PRDM13 is also present.Item GATA Like Protein-1: A Somatic Cell Factor Required for Normal Ovarian Development and Function(2010-09-20) Strauss, Tamara Joy; Hammes, Stephen R.Oogenesis and follicular maturation are processes that require organized and precisely timed communication through paracrine and endocrine signals of neighboring tissues. Deviations in the cross talk between ovarian cells, or aberrant gene expression within one of the cell populations, can lead to germ cell loss and infertility in the adult female. Expression of Glp-1 in the somatic cells of the ovary is required for normal fertility in female mice, as a deficiency for Glp-1 leads to the absence of oocytes at birth and ovarian tubular formation in the adult. However, the nature of germ cell loss and tubular adenoma formation, in the setting of a somatic cell protein deficiency, is not well understood. In this report, I characterize the embryonic germ cell loss phenotype in Glp-1LacZ null mice. Immunohistochemical analyses of Glp-1LacZ null mouse ovaries show that germ cells are appropriately specified and migrate to the nascent gonad similarly to wild type. After their arrival at the gonad, precocious loss of the germ cells begins at or around E13.5. This loss is completed by birth and is accompanied by defects in the expression of oocyte-specific genes associated with meiotic entry. Interestingly, somatic pregranulosa cells retain their ability to secrete paracrine signaling molecules to the oocyte and are still able to form the basement membrane surrounding the germline cysts. In the adult, the structure of the germline cyst persists, albeit without germ cells, and there is loss of HPG axis communication. The loss in HPG communication in Glp-1LacZ null mice can be accounted for by loss of regulated steroidogenesis through the GATA4-dependent transcriptional activation of StAR. These data imply that the somatic cell protein Glp-1 regulates 1) germ cell survival early in embryogenesis and 2) steroidogenesis through StAR promoter activation.Item Gene Regulatory Networks in Striated Muscle Pathologies(2023-05-01T05:00:00.000Z) Shah, Akansha Mahavir; Munshi, Nikhil; Olson, Eric N.; Mendell, Joshua T.; Kliewer, Steven A.The striated musculature, comprised of skeletal muscle and cardiac muscle tissues, is essential for vertebrate life. Skeletal muscles are composed of bundles of long and parallel multinucleated myofibers that constitute approximately 40% of the human body mass. The cardiac muscle is much smaller and consists of a branched network of short, mononucleated or binucleated cardiomyocytes that are connected by intercalated discs. Both these tissues partake in force generation through contractile units called sarcomeres to support movement, respiration (skeletal muscle), and to pump oxygenated blood throughout the body (cardiac muscle). Gene regulatory networks, the interactions between lineage or stage-determining transcription factors and the mRNAs they govern, tightly control cell function and striated tissue development and homeostasis. A disruption or change in these complex networks underlies most skeletal and cardiac muscle-related diseases. In this dissertation, we used transcriptomic and epigenomic approaches at single cell or bulk tissue resolution to provide a molecular framework for the function of TWIST2 in rhabdomyosarcoma pathogenesis as well as the mechanisms of heart remodeling and repair following myocardial infarction. In this process, we elucidated critical nodes in complex pathways that can be manipulated to derail the pathological process of FN-RMS or stimulate regeneration and repair in postnatal hearts.Item Genome-Wide Analysis of Transcription Factors Ascl1 and Ptf1a in Development and Cancer(2013-11-26) Borromeo, Mark Dominic; Yu, Gang; Lu, Q. Richard; Kim, Tae-Kyung; Johnson, Jane E.Cell fate specification in the developing embryo relies on combinations of transcription factors to regulate tissue specific gene programs. Many of the same transcription factors can be found in multiple tissue types and are crucial for their development, and at other times these same factors can be misused in disease states. The basic helix-loop-helix (bHLH) factors Ascl1 and Ptf1a are examples of factors that give rise to and function in multiple tissues. Ascl1 and Ptf1a are essential for generating the correct number and sub-type of neurons in multiple regions of the nervous system. In addition, Ptf1a is required in the developing pancreas for both its formation and maturation, while Ascl1 is crucial for tumor growth in malignant small cell lung carcinoma (SCLC). It is unknown if Ascl1 and Ptf1a directly regulate different genes programs in these disparate tissues. Furthermore, Ptf1a and Ascl1 are members of same transcription factor family, which recognize and bind a similar DNA sequence. How these two factors achieve specificity of DNA binding and gene target selection in vivo is unknown. These questions have long been unanswered due in part to the lack of known direct transcriptional targets. Thus, to understand how Ascl1 and Ptf1a function in these processes, the direct transcriptional targets were identified genome-wide in the multiple tissues using ChIP-Seq and RNA-Seq. Overwhelmingly, Ascl1 and Ptf1a directly regulate different gene programs important for each tissue. Within a given tissue, the specificity of Ascl1 and Ptf1a function is partly explained by their differences in E-box sequence preferences. Ptf1a and Ascl1 are co-expressed in a subset of cells in the dorsal neural tube, and comparative analysis of their binding sites show that they bind a common E-box. However, Ptf1a can also bind a distinct E-box, which is found enriched in binding sites unique to Ptf1a. However, analysis of Ascl1 binding in SCLC and Ptf1a binding in the developing pancreas, shows that their E-box preferences change and does not reflect the same type of E-box they bind in the neural tube. Mechanisms in addition to E-box specificity are likely in use because tissue specific binding coincides with tissue-specific chromatin accessibility and enrichment of lineage-specific transcription factor binding motifs. Thus, Ascl1 and Ptf1a make use of different tissue-specific co-factors to regulate tissue-specific genes. This study provides insights into how a single factor can regulate the transcription of different genes in different tissue types, and how two related E-box binding proteins regulate distinct genes.Item Investigating the Transcriptional Regulation of PTF1A and the Function of PRDM13 to Specify Dorsal Spinal Interneurons(2019-03-28) Mona, Bishakha; Zhang, Chun-Li; Johnson, Jane E.; Wu, Jiang I.; Meeks, Julian P.Transcriptional regulation is a fundamental process required for cell fate specification. During neural development, complex interactions of diverse transcription activators and repressors regulate lineage specific programs. The multitudes of neuronal subtypes generated during development is dependent on spatio-temporally controlled expression of these transcription factors such as bHLH factors, required for cell fate specification. PTF1A is one such factor whose expression is required for specification of the inhibitory fate while silencing the excitatory fate in the neural tube. Previous studies from our lab determined that PTF1A directly activates the inhibitory neuron lineage specific genes to direct neural progenitors towards an inhibitory neuronal fate and initiates a repressive program to suppress the excitatory neuron gene expression program through PRDM13. My thesis work uncovered additional functions of PRDM13 including suppression of ventral neural tube specification genes in the dorsal neural tube through repressing the activity of multiple bHLH transcriptional activators. Additionally, PRDM13 feedback inhibits PTF1A through a Ptf1a auto-regulatory enhancer. Given the indispensable requirement of PTF1A in neural specification, I continued to explore the regulatory network in place for Ptf1a expression. An enhancer with activity specifically in the dorsal neural tube specific was identified for Ptf1a using reporter says in mice and chick. The function the auto-regulatory enhancer and the dorsal neural tube specific enhancer was tested in situ. Mutations in the auto-regulatory enhancer, but not the dorsal neural tube enhancer, resulted in reduced levels of PTF1A, decreased the number of inhibitory neurons generated during dorsal spinal cord development, and exhibit a spontaneous scratching phenotype. The increased sensitivity to itch can be attributed at least in part to loss of BHLHB5;PAX2+ inhibitory neurons, known to play a role in itch. The absence of altered sensitivities in other somatosensory modalities whose information is known to be modulated in dorsal spinal cord circuits suggests itch circuitry is more dependent on PTF1 levels than the other sensory pathways. Together, these findings examine the role of two specific factors PTF1A and PRDM13 in neuronal specification during development.Item Mash1 Defines Lineage Restricted Neuronal and Oligodendrocytic Precursor Cells in Spinal Cord Development(2007-05-21) Battiste, James Douglas; Johnson, Jane E.Recent advances have defined distinct neural progenitor and early interneuron pools in the developing spinal cord and the molecular events that influence progenitor cell fate. However, these early neurons have not been traced to adult neuron types. The transcription factor Mash1 is transiently expressed in a subset of neural progenitors and possesses a pro-neural function. The transient nature of its expression limits the ability to trace Mash1+ progenitors. To study the developing neural tube from progenitor to adult neuron, transgenic mouse strains were generated that express GFP, Cre recombinase, and tamoxifen-inducible Cre recombinase. The M1-GIC mouse line, showed faithful Mash1 expression recapitulation and traces Mash1+ progenitors mainly to dI3 and dI5 interneurons. This supports data from the Mash1 null mutant where these populations are decreased or absent. Using M1-GIC;R26R-lacZ mice, I was able to trace Mash1 expressing cells to neurons and oligodendrocytes in the adult mouse, but tracing to astrocytes was never observed. These data refute the conventional understanding that Mash1 is purely pro-neuronal, and is consistent with recent findings of Mash1 descendents in the early postnatal subventricular zone. Using M1-CRE-ER™;R26RlacZ and M1-CRE-ER™;R26R-YFP, Mash1+ cells trace into adulthood in a temporally-dependant manner. Cells expressing Mash1 at E10.5 become neurons of the dorsal horn in lamina I-IV while cells expressing Mash1 at E15.5 become oligodendrocytes spread over both gray and white matter. As a control, Nestin- CreERT2;R26R-lacZ and Nestin-CreERT2;R26R-YFP mice were used to confirm that common progenitors of all neural cell types can be traced from E10.5 to P21. This data provides evidence that Mash1 defines lineage restricted precursors that exit the cell cycle rapidly, and Mash1 is necessary for efficient loss of common progenitor characteristics as seen in the Mash1 null mutant. This data refines our understanding of progenitor characteristics and Mash1 function in the developing spinal cord.Item Modulation of Transcription Factor Chromatin Association and Gene Transcription Program in Embryonic Stem Cell and Triple Negative Breast Cancer by Poly (ADP-Ribose) Polymerase 1(2016-03-30) Liu, Ziying; Zhang, Chun-Li; Kraus, W. Lee; Kim, Tae-Kyung; Morrison, Sean J.; Li, BingPoly(ADP-ribose) polymerase-1 (PARP-1), also referred to as ADP-ribosyltransferase Diphtheria toxin-like 1 (ARTD1), is an abundant nuclear protein that plays key roles in a variety of nuclear processes, including the regulation of transcription. PARP-1 possesses an intrinsic enzymatic activity that catalyzes the transfer of ADP-ribose (ADPR) units from nicotinamide adenine dinucleotide (NAD+) onto target gene regulatory proteins, thereby modulating their activities. Although great strides have been made in the past decade in deciphering the seemingly opposing and varied roles of PARP-1 in gene regulation, many puzzles remain in this field. Using a combination of cell biology, molecular biology, genomics and biochemistry methods, I investigated the functions of PARP-1 in regulating gene transcription program in mouse embryonic stem cells and human triple negative breast cancer cells. I found that in mouse embryonic stem cells, PARP-1 functions as a pre-pioneering factor, stabilizing transcription factor Sox2 interaction with nucleosomes. This function is required for maintaining gene transcription program in embryonic stem cells. Depletion of PARP-1 causes disrupted embryonic stem cell gene expression profile, including decreased expression of Nanog, as well as increased expression of differentiation genes. Furthermore, using human triple negative breast cancer cells, I showed that this gene transcriptional regulation mechanism through PARP-1-Sox2 interplay is conserved in different physiological models. Interestingly, inhibiting PARylation activity causes gain of Sox2 binding to a set of genomic locations in TNBC cells, indicating that PARylation activity plays an antagonizing role in PARP-1-regulated Sox2 chromatin interaction. In summary, our results illustrate how PARP-1 can act at the level of the nucleosome to produce global effects on transcription factor binding and biologically important gene expression outcomes.Item Molecular Dissection of Hand2 During the Formation of Pacemaker-Like Myocytes During Direct Reprogramming(2019-03-06) Fernandez-Perez, Antonio; Zhang, Chun-Li; Cleaver, Ondine; Olson, Eric N.; Munshi, NikhilDirect reprogramming of one cell type into another has great promise for regenerative medicine, disease modeling, and lineage specification. Currently, the conversion of fibroblasts into induced cardiomyocytes (iCM) by Gata4, Mef2c, and Tbx5 (GMT) represents an important avenue for generating de novo cardiac myocytes. Recent evidence has shown that iCM formation and diversity can be enhanced by the addition of Hand2 to GMT (GHMT). These four transcription factors give rise to a heterogenous CM population, consisting of atrial (iAM), ventricular (iVM), and pacemaker myocytes (iPM). However, the molecular mechanisms that drive this plastic fate conversion remain poorly understood. Although chromatin and single-cell studies in GMT-iCM have shown the existence of a set of temporal steps that orchestrate iCM formation, little is known about how Hand2 enhances this process. In the present study, we seek to characterize these Hand2-dependent mechanisms. We hypothesize that Hand2 regulates a discrete pacemaker regulatory network that becomes active during GHMT-iCM reprogramming. To test this, we compared the transcriptional and genomic profiles of fibroblasts, GMT, GHMT, and endogenous mouse Pacemaker cells. We observe similar chromatin landscape and gene expression profiles between Hand2-iPM and endogenous sinoatrial node (SAN), however several known key PM pathways are not active. Activation of these networks further enhances iCM-iPM fo Moreover, we show that Hand2 enhances chromatin accessibility in regions related to sarcomere function and electrical coupling, as well as promoting the closing of regions related to alternative fates. Utilizing integrative genomics between ATAC-seq and RNA-seq datasets, we identify the desmosome machinery as an important feature of iPM formation. In parallel, we define a novel Hand2 domain region that regulates cardiac subtype diversity. Taken together, our results showcase Hand2-dependent mechanisms for iPM formation and gives insight into the improvement of future iPM engineering.Item Multifunctional Regulators of Cardiac Disease and Development(2008-09-12) Kim, Yuri; Olson, Eric N.Embryogenesis requires delicate regulatory mechanisms. A single cell embryo divides into millions of daughter cells to form an organism comprised of various organs with different shapes and function. Organogenesis is mainly controlled by genes that are expressed in a tissue-specific manner. Thus, regulation of gene expression is a critical step in development. In this thesis, I present my findings on two cardiac transcription factors MEF2 and Yap that play multiple roles in development. First, I show a novel function of myocyte enhancer factor 2 (MEF2) transcription factors in development of endochondral bone. MEF2 proteins are widely known as essential regulators of development of various tissues such as striated muscle and brain. Based on expression patterns of Mef2 genes and skeletal defects present in Mef2c +/-; Mef2d +/- mice, I hypothesized that MEF2is an important regulator of skeletogenesis and generated mice lacking MEF2C and MEF2D in chondrocytes using Mef2c and Mef2d conditional mutant alleles. From this study, I demonstrated that MEF2 proteins are also critical regulators of chondrocyte hypertrophy at least partly through their regulation of procollagen, type X, alpha 1 (Col10a1). I also explored another function of MEF2 protein, which is to mediate stress-dependent cardiac remodeling. Mef2d null mice show impaired response to cardiac remodeling stresses such as pressure overload and chronic Β- adrenergic stimulation; hypertrophy, chamber dilation, fibrosis, and fetal gene activation were blunted in the absence of MEF2D. Conversely, overexpression of MEF2D is sufficient to drive pathological remodeling of the heart. These findings reveal an important role of MEF2D in stress-dependent cardiac growth and reprogramming of gene expression in the adult heart. Finally, I demonstrate that yes-associated protein (Yap) serves as a critical regulator of cardiac function and angiogenesis by generating a Yap conditional mutant allele. Deletion of Yap in cardiomyoctes leads to lethal cardiomyopathy resulting from compromised cardiac angiogenesis and ischemia. I also identify Yap as a coactivator of GATA4, a trascription factor that functions as a regulator of angiogenesis in the heart. Moreover, my studies on deletion of Yap in other tissues suggest the possible role of Yap as a global angiogenic factor. Collectively, these studies show that key transcriptional regulators of cardiogenesis play a significant role not only in heart development, but also in development of other organs. These findings imply that combinatorial actions of transcriptional regulators in a tissue-specific manner are critical in embryogenesis.Item Nuclear Receptors in Lung Cancer(2007-05-22) Jeong, Yangsik; Mangelsdorf, David J.; Minna, John D.Lung Cancer is a fatal disease with new diagnoses of more than 150,000 Americans every year. Although it has a relatively well-known etiology (e.g. smoking) and has been widely researched, clinical tools and markers for early diagnosis, prognostic prediction, and therapeutic interventions remain limited. Here, for the first time, I propose a novel translational approach for providing diagnostic, prognostic, mechanistic, and therapeutic information by studying of the expression of the nuclear receptor (NR) superfamily in lung cancer. Using quantitative real-time PCR, mRNA expression levels for the 48 members of the NR superfamily were profiled in 56 lung cell lines. Based on the resulting dataset, further analysis was performed to show the diagnostic and therapeutic potential of the NR profile using both an in vitro cell response assay and an in vivo mouse xenograft model with cognate ligand treatment for selected nuclear receptors. In addition, the NR profiles of 30 microdissected and pair-matched patient tissue samples provided a subset of NRs showing dramatic differences in expression and subgroupings that demonstrate individual variations between the normal and corresponding tumor. Furthermore, I identified several individual NRs as well as a subgroup of NRs with prognostic power. The relevance of NRs to disease pathogenesis was then studied in genetically manipulated human bronchial epithelial cells (HBEC3) and in transgenic K-rasV12 mice, a well-known genetic model for lung adenocarcinoma. In the HBEC3 panel, the induced expression of peroxisome proliferator activating receptor gamma (PPARγ) in the parental HBEC3 introduced by oncogenic K-rasV12 is decreased in a subset of tumorigenic clones derived from the parental cells. It appears to be strongly correlated to the expression of cylooxygenase 2 (COX2), which is shown to be decreased with PPARγ ligand treatment. In the transgenic model, I demonstrated that expression of a subgroup of NRs in wild type mice becomes altered in histologically normal tissues that harbor the K-ras mutation, and become further altered in tumor tissues of the mutant. This observation suggests that NR profiling also provides a valuable tool for understanding disease pathogenesis in lung cancer.Item Post-Transcriptional Regulation of Maternal mRNA Shapes Early C. Elegans Embryogenesis(2014-03-17) Burleson, Marieke Oldenbroek; Buszczak, Michael; Abrams, John M.; Cleaver, Ondine; Hobbs, Helen H.Much of early embryogenesis is controlled through complex networks comprised of maternally provided factors. Oocytes are packed with protein and RNA that are ready to spring into action after fertilization to guide early embryonic development. The regulation of maternally provided factors is therefore critical and is a fundamental goal of developmental biology. During my studies, I investigated how two maternally provided mRNAs, zif-1 and mom-2, are regulated post-transcriptionally through their 3’ untranslated region (3’ UTR) to ensure proper spatio-temporal protein expression. I discovered that seven RNA binding proteins bind directly to the zif-1 3’ UTR in a combinatorial fashion thereby ensuring that zif-1 is only translated in somatic blastomeres, beginning at the four cell stage embryo. Interestingly a similar set of RNA binding proteins (nine total) regulate the spatio-temporal expression of mom-2 in a similar fashion despite the fact that mom-2 has a reciprocal expression pattern when compared to zif-1. My studies on zif-1 and mom-2 regulation indicate that a “code” is embedded within the 3’ UTR of mRNAs to mediate translational regulation. The precise combination of RNA binding proteins present in a particular cell at a particular time, each with the intrinsic capability of binding to regulatory sequences contained in this “code”, determines when and where mRNAs get translated. I also investigated mechanisms by which maternal mRNAs get degraded. Zygotic transcription activation is often linked to maternal mRNA degradation, which I showed to be the case in C. elegans embryos. Specifically, I discovered a gene termed vet-5 that is first transcribed in the somatic blastomeres of the four-cell embryo and is sufficient to degrade at least several maternal mRNAs when provided exogenously as dsRNA. vet-5 maps to a highly repetitive locus and has been shown to be a target of siRNA production. Consistent with vet-5 derived siRNA production I found that the siRNA pathway is, at least partly, required for the degradation of maternal mRNAs and that removing components of the siRNA pathway affects vet-5 expression. Therefore, I hypothesize that siRNAs could be produced from the vet-5 locus that target maternally provided mRNAs for degradation.Item Transcriptional and Translational Regulation of Cardiac Progenitors in the Mouse and Zebrafish(2009-01-09) Cordes, Kimberly Rene; Srivastava, DeepakIn vertebrates, the heart is the first organ to function and cardiac progenitors are among the first cell lineages to be established. Transcriptional networks control the specification of cardiac progenitors, however, it is not fully understood how some transcription factors function in particular cardiac progenitor populations. The basic helix-loop-helix, bHLH, transcription factor, Hand2 has been discovered over a decade ago, and has a severe loss-of-function cardiac phenotype in vivo, yet its function is still not completely known. It is expressed in the early cardiac progenitors of the neural crest cells and second heart field lineages. The first part of my thesis touches on the beginnings to understand the role of Hand2 in the cardiac neural crest progenitors. Generally, expression levels in vertebrates reflect the combined transcription of both alleles of the gene being transcribed. Although there are notable exceptions (i.e., X chromosome genes), the presence of only one functional copy or more than two copies of a gene can have detrimental effects on the development of the organism. Many of the genetic examples of congenital heart disease, which affects 1% of live births, are a result of a haploinsufficient gene dose. Like Hand2, which acts in a dosage-sensitive manner to regulate ventricular formation, the precise dose of proteins can be very important in regulating cardiac development. One way to fine-tune the activity of genes is through the newly identified class of small RNAs, microRNAs (miRNAs), which translationally repress the production of proteins by binding to target sites on messenger RNA (mRNA). MiRNAs provide a sophisticated way to adjust protein levels in a spatiotemporal manner. One miRNA may control several mRNAs, including transcription factors, which are the 'master switches' that regulate gene expression. And cooperatively, cell type-specific transcription factors can regulate the tissue-specificity of miRNA expression. Together with transcription factors, miRNAs function in cell fate determination, cell differentiation, proliferation and disease progression. Similar to transcription factors, which activate or repress a set of genes in a particular cell type, miRNAs create an environment, tailored for each cell type, allowing translation of some genes to occur, while repressing others. To date, less than a handful of miRNAs have been identified that function during heart development. The latter half of my thesis represents efforts to identify cardiac progenitor miRNAs and understand their function during development. I found that miRNA function is important in the cardiac mesodermal progenitors. In addition, I present a family of miRNAs, miR-143 and miR-145, that is specific to cardiac and smooth muscle progenitors, and I discuss their function in regulating their respective environments during cardiovascular development and disease.Item Transcriptional Regulation of Neonatal Heart Regeneration and Direct Cardiac Reprogramming(2021-05-01T05:00:00.000Z) Wang, Zhaoning; Sadek, Hesham A.; Olson, Eric N.; Xu, Jian; Kliewer, Steven A.The adult mammalian heart has limited capacity for regeneration following injury, whereas the neonatal heart can readily regenerate within a short period after birth. Deciphering the molecular underpinnings of neonatal heart regeneration and the blockade to regeneration in later life may provide novel insights for heart repair. To elucidate the transcriptional responses of the different cellular components of the mouse heart following injury, we performed single cell RNA-sequencing on neonatal hearts at various timepoints following myocardial infarction, and coupled the results with bulk tissue RNA-sequencing and H3K27ac ChIP-sequencing data collected at the same timepoints. This approach provides detailed transcriptional dynamics of heterogeneous cardiac cell types during neonatal heart regeneration. Concomitant single cell ATAC-sequencing exposes underlying dynamics of open chromatin landscapes and regenerative gene regulatory networks of diverse cardiac cell types, and reveals previously unknown extracellular mediators of cardiomyocyte proliferation, angiogenesis, and fibroblast activation. Furthermore, using single-nucleus RNA sequencing, we mapped the dynamic transcriptional landscape of five distinct cardiomyocyte populations in healthy, injured and regenerating mouse hearts. We identified immature cardiomyocytes that enter cell-cycle following injury and disappear as the heart loses the ability to regenerate. These proliferative neonatal cardiomyocytes display a unique transcriptional program dependent on NFYa and NFE2L1 transcription factors, which exert proliferative and protective functions, respectively. Cardiac overexpression of these two factors conferred protection against ischemic injury in mature mouse hearts that were otherwise non-regenerative. Together, these findings provide mechanistic insights into the molecular basis of neonatal heart regeneration, and offer various pathways that can be manipulated to facilitate cardiac repair after injury. Direct reprogramming of fibroblasts into induced cardiac-like myocytes using cardiac transcription factors offers another possible therapeutic approach for cardiac repair. To elucidate the gene regulatory network during direct cardiac reprogramming, we performed a genome-wide analysis of cardiac transcription factors binding sites and active enhancers during reprogramming. We found reprogramming factors cooperatively activate enhancers involved in cardiac development and maturation, and further delineated the regulatory relationships between reprogramming factors and cardiac gene expression. These findings reveal synergistic activation of the cardiac epigenetic landscape by cardiac transcription factors and key signaling pathways that govern direct cardiac reprogramming.Item Translational Repression of G3BP in Cancer and Germ Cells Suppresses Stress Granules and Enhances Stress Tolerance(2020-08-01T05:00:00.000Z) Lee, Anna Kunyoung; Chook, Yuh Min; Potts, Patrick Ryan; Conrad, Nicholas; Rice, Luke; Thomas, Philip J.Melanoma antigen (MAGE) genes are conserved in all eukaryotes and encode for proteins sharing a common MAGE homology domain. Although only a single MAGE gene exists in lower eukaryotes, the MAGE family rapidly expanded in eutherians and consists of more than 50 highly conserved genes in humans. A subset of MAGEs initially garnered interest as cancer biomarkers and immunotherapeutic targets due to their antigenic properties and unique expression pattern that is primary restricted to germ cells and aberrantly re-activated in various cancers. However, further investigation revealed that MAGEs not only drive tumorigenesis, but also regulate pathways essential for diverse cellular and developmental processes. Therefore, MAGEs are implicated in a broad range of diseases including neurodevelopmental, renal, and lung disorders, as well as cancer. Recent biochemical and biophysical studies indicate that MAGEs assemble with E3 RING ubiquitin ligases to form MAGE-RING ligases (MRLs) and act as regulators of ubiquitination by modulating ligase activity, substrate specification, and subcellular localization. Here, we present a comprehensive guide to MAGEs highlighting the molecular mechanisms of MRLs, their physiological roles in germ cell and neural development, oncogenic functions in cancer, and potential as therapeutic targets in disease. Stress granules (SG) are membrane-less ribonucleoprotein condensates that form in response to various stress stimuli via phase separation. SG act as a protective mechanism to cope with acute stress, but persistent SG have cytotoxic effects that are associated with several age-related diseases. Here, we demonstrate that the testis-specific protein, MAGE-B2, increases cellular stress tolerance by suppressing SG formation through translational inhibition of the key SG nucleator G3BP. MAGE-B2 reduces G3BP protein levels below the critical concentration for phase separation and suppresses SG initiation. Importantly, knockout of the MAGE-B2 mouse ortholog or overexpression of G3BP1 confers hypersensitivity of the male germline to heat stress in vivo. Thus, MAGE-B2 provides cytoprotection to maintain mammalian spermatogenesis, a highly thermo-sensitive process that must be preserved throughout reproductive life. These results demonstrate a mechanism that allows for tissue-specific resistance against stress and could aid in the development of male fertility therapies.Item Understanding the Role of SCL in Early Mammalian Development Using Mouse Embryonic Stem Cell Differentiation as a Model(2010-05-14) Ismailoglu, Ismail; Kyba, MichaelHow a complete organism develops from a single cell is among the most complicated questions in life sciences. Early experimental studies on the development of animals were performed on amphibians and birds due to the size and accessibility of their embryos, while studies in placental mammals have been limited by the difficulty posed by in utero development. In vitro differentiation of ES cells provides a convenient model for the study of the mammalian development. Since ES cells can be grown and maintained in a pluripotent state virtually forever, ample amount of research material for molecularbiological studies can be produced; differentiating ES cells are easily accessible and they can also be manipulated genetically. I have used the ES cell differentiation model to study the bHLH factor SCL, a critical regulator of the formation of the hematopoietic lineage in the early embryo and the maturation of erythrocytes and megakaryocytes later on. The latter function of the protein has been studied extensively, but a complete molecular analysis of the former function has been lacking. My work shows that SCL can skew the patterning of the mesoderm towards the hematopoietic lineage. This function required the interaction of SCL with LMO2. Transcriptional profiling revealed organizer genes FoxA2 and Chordin as novel downregulated targets of SCL during this time. Differentiation of human pluripotent cells to be used in cellular therapy or to generate replacement tissues; is considered to be one of the most promising branches of medical research. Considering the importance of SCL in hematopoiesis, we hypothesized that SCL can direct differentiation of pluripotent cells to this lineage in a simple culture system. Ectopic expression of SCL induced hematopoiesis at low levels. Co-expression of LMO2 and GATA2 increased efficiency of the programming significantly.