Our laboratory studies transcriptional processes that are disrupted in disease. We identified a novel transcriptional signaling pathway in the heart that mediates the heart’s ability to regulate whole body metabolism. Through a combination of pharmacological and genetic gain- and loss-of-function studies in mice, we found the heart is capable of regulating whole body metabolism through a mechanism that is governed by MED13 and miR-208a. MED13 is a particularly interesting component of the Mediator complex because it functions as a molecular bridge between the core complex and kinase submodule, providing a mechanism for spatial and temporal control of Mediator-dependent regulation of transcription. In addition, we are studying the function of multiple components of Mediator including CDK8, CDK19, MED12 and CycC. We primarily utilize mutant mouse models to study the proteomic, molecular, bioinformatic and biochemical methods to study the molecular signaling events controlling cardiac response to stress.
My primary research interest is in the genetic determinants of intellectual disability (ID), formerly referred to as mental retardation (MR). I specifically study the roles of copy number variation and somatic structural variation in the context of a “genomic mutational burden” hypothesis of ID. This hypothesis is investigated using a combination of conventional cytogenetics methods (chromosome analysis and fluorescence in situ hybridization) and new molecular, high throughput, and high data volume, genomic technologies including single nucleotide polymorphism (SNP) arrays, gene expression microarrays, comparative genomic hybridization (CGH) arrays as well as custom targeted, whole exome, and whole genome massively parallel DNA sequencing. We perform all our own bioinformatics and are actively engaged in the development of new analysis tools to better meet our needs and those of the scientific community. Drosophila melanogaster and the well-established GAL4/UAS/RNAi system are used to evaluate candidate ID genes with the use of a validated olfaction learning technique (T-Maze). Candidate genes are derived from our extensive clinical database of patients with ID that have already undergone diagnostic chromosomal microarray testing.
Cellular protein misfolding leads to the occurrence of amyloid oligomers and protein aggregates inside the cell. Protein misfolding is the cause of cellular dysfunction associated with numerous diseases such as cardiomyopthy, Huntington’s disease, Alzheimer’s disease, adult onset diabetes, cancer and others. To protect themselves against toxic stressors and concomitant protein aggregation, all cells possess highly conserved surveillance and repair mechanisms. One of the most central of these is the stress responsive transcriptional program called the ‘heat shock response’, controlled by the heat shock transcription factor 1 (HSF1). HSF1 upregulates a set of cytoprotective proteins, the so-called heat shock proteins (HSPs) which help refold and/or degrade damaged proteins to restore homeostasis. Previously, we have shown that in the animal model Caenorhabditis elegans the upregulation of HSPs upon protein misfolding is not regulated cell autonomously by the amounts of protein misfolding, but instead, is under the control of the organism’s nervous system. Our lab works on understanding how the nervous system of C. elegans controls the cellular response to stress and protein misfolding. Specifically, we use the powerful genetic techniques afforded to us by the use of C. elegans to ask:
a) What genes and signaling pathways are involved in how the nervous system detects suboptimal environmental conditions, or stressb) What signaling pathways are responsible for transmitting this information to non-neuronal cellsc) How is the accumulation of protein damage in non-neuronal cells communicated to the nervous systemd) How do these signaling mechanisms ultimately result in an adaptive, organimal response to macromolecular damage and stress.
Our research focuses on the evolutionarily conserved process of meiosis, using C. elegans as a model system. Meiosis enables sexual reproduction by the production of haploid gametes. A successful meiotic division relies on the formation of crossover events between each pair of homologous chromosomes. These crossover events are formed in the context of the synaptonemal complex (SC), a protein complex that bridges paired homologous chromosomes during meiotic prophase I. Hence, in the absence of a functional SC, meiosis is abrogated. Perturbation of meiosis can lead to chromosome nondisjunction, which is the leading cause for miscarriages and birth defects in humans. These observations, combined with evidence from studies of infertile patients, suggest a connection between SC dysfunction and chromosomal nondisjunction in human reproduction. In our lab we aim to discover and characterize novel genes essential for various aspects of meiosis, including genes essential for chromosome pairing, recombination, and the regulation of SC assembly and disassembly. To accomplish this goal, we are engaged in genetic screens targeted to isolate genes in these processes. This approach has already resulted in the identification of a novel class of mutants affecting SC disassembly. We combine our genetic approaches with high-resolution microscopy to investigate the role of these proteins in meiosis. These studies will result in a better understanding of the various fundamental processes unfolding in C. elegans meiosis and will lead to insights into this basic process in other organisms as well. In-depth investigation of meiosis is of central importance for progress in developing methods to prevent and treat infertility and birth defects stemming from meiotic chromosome nondisjunction in humans.
Prostaglandins are transiently acting hormones that are synthesized at their sites of action by cyclooxygenase (COX) enzymes, the targets of Aspirin and Advil, to mediate a variety of biological activities, including inflammation, sleep, reproduction, and cancer development. How do prostaglandins regulate these diverse, cellular events? To address this question we have developed Drosophila oogenesis as a new a new and powerful model for studying prostaglandin signaling. Using both pharmacology and genetics, we discovered that prostaglandins mediate Drosophila follicle development, identified the Drosophila COX1 enzyme, Pxt, and revealed that genetic perturbation of prostaglandin signaling can be used to exam the function of prostaglandins. This research on prostaglandin signaling implicates it in modulating actin/membrane dynamics, cell migration, stem cell activity, and the timing of gene expression during Drosophila follicle development. The lab is currently pursuing how prostaglandin signaling regulates actin dynamics and invasive cell migrations during Drosophila follicle development. By using a multifaceted experimental approach that combines Drosophila genetics, cell biology, live imaging, and biochemistry to we can begin to work out the mechanisms by which prostaglandins regulate these processes, and provide general insight into how prostaglandins regulate the cytoskeleton and migration at a cellular level. Such mechanisms of prostaglandin action are likely to be reutilized throughout development, including mediating the changes that occur during cancer progression and metastasis.
The protozoan parasite, Leishmania chagasi, causes the fatal human disease visceral leishmaniasis. L. chagasi express an abundant surface protease GP63, which is important for parasite survival. GP63 is encoded by >18 tandem MSP genes, falling into 3 homologous classes whose expression varies throughout the parasite life cycle. MSPL genes are expressed in logarithmic, whereas MSPS genes are expressed in stationary phase when parasites achieve maximal virulence and express high levels of GP63 protein. Our studies focus on the post-transcriptional mechanisms regulating expression of different MSP gene classes. These include mRNA T½, the efficiency of trans-splicing, and protein T½. Using reporter gene constructs and transfection techniques we are localizing unique sequences in MSP 3'UTRs that interact with regulatory proteins. Additionally, using MALDI-TOF mass spec we are examining products of specific MSP class genes that are expressed in different parasite stages. An ongoing epidemic of visceral leishmaniasis in northeast Brazil has led to our studies genetic loci associated with different outcomes of human L. chagasi infection (asymptomatic versus fatal). Using molecular genotyping methods (microsatellites, SSCP, RFLP, sequencing) we are examining polymorphic alleles of candidate genes for their contributions to disease susceptibility, in collaboration with Dr. Selma Jeronimo of Natal, Brazil. These studies will extend to a genome-wide scan and fine mapping of loci linked to different disease outcomes.
Our research focuses on cell-cell signaling events that lead to intracellular calcium release. We integrate in vivo image analysis coupled with molecular-genetic tools to elucidate the role of calcium-dependent signaling networks critical in developmental processes such as body plan formation and organogenesis in the zebrafish. The zebrafish model system for vertebrate developmental biology has many attributes including genetics, rapid development and translucent embryos. We defined a class of Wnt signaling ligands that modulate intracellular calcium release and are investigating the mechanisms by which this Wnt/calcium class mediates its biological effect on the developing embryo. As inappropriate Wnt signaling has been associated with a high frequency of tumors, we are also investigating spontaneous tumor formation in genetic backgrounds that disrupt the function of Wnt/calcium class ligands. Additionally, we have determined that the calcium release requires G-protein signaling. To identify potential intracellular regulators of calcium release dynamics, we are characterizing members of the regulators of G-protein signaling protein family. We have cloned and characterized a few members of the RGS family and find they have essential roles in sensory neuron and somite patterning. Due to the remarkable conservation of developmental processes and mechanisms among vertebrates, we also use zebrafish as a model for human disease and test candidate genes. Of note are studies involving retinal degeneration and Bardet-Biedl Syndrome.
Peroxisome proliferator activated receptors (PPAR's) are ligand activated transcription factors which have a pleiotropic role in many physiological processes. PPARγ is the molecular target of the thiazolidinediones class of drugs which are used to treat patients with non-insulin dependent diabetes mellitus (NIDDM). Endothelial dysfunction, which develops in patients that are diabetic or chronically hypertensive, is thought to contribute to the progression of carotid artery disease, cerebral vascular dysfunction and stroke. PPARγ is expressed in vascular endothelium and smooth muscle and therefore is a potentially important factor in the regulation of vascular function and blood pressure. PPARγ has been reported to inhibit responses to vasoconstrictors such as endothelin, stimulate the release of vasodilators, and increase expression of CuZn-SOD in vascular muscle and endothelium. Importantly, patients carrying dominant negative mutations in PPARγ exhibit early onset type II diabetes and hypertension. Current data suggests that PPARγ exerts a protective effect in the vessel wall and we hypothesize that PPARγ plays an important role in the regulation of vascular function and blood pressure. We are currently testing this hypothesis using a variety of genetic, computational and Bioinformatic tools. We are: 1) using adenoviruses over-expressing wildtype and dominant negative mutations of PPARγ in blood vessels from normotensive and hypertensive mice to test whether they can alter endothelial function, 2) developing novel transgenic mice with expression of the wild-type and dominant negative mutants of PPARγ targeted specifically to vascular muscle and endothelial cells using cell-specific promoters, 3) using microarrays to determine the transcriptional targets of PPARγ, and 4) using computational and Bioinformatic tools to scan genomic sequences for PPARγ response elements (PPRE).
My research interest is the control of neuronal gene expression. The major focus of the lab is on the neuropeptide CGRP and its role in migraine. The role of CGRP in migraine is supported by the ability of CGRP to cause headache and the recent efficacy of a CGRP antagonist as an antimigraine drug. We have found that the CGRP gene is up-regulated by cytokine-induced MAP kinases and repressed by antimigraine drugs that appear to act via an unusually prolonged calcium signal. We are currently investigating these mechanisms using adenoviral-mediated gene transfer to cultured trigeminal neurons and intact ganglia in vivo. We are also using gene transfer and transgenic mice to regulate CGRP receptor activity in the vasculature and nervous system by overexpressing the RAMP1 subunit of the CGRP receptor. The RAMP1 transgenic mice are sensitized to CGRP-induced neurogenic inflammation. The RAMP1 transgenic mice display a unique phenotype has raised the possibility that these mice may provide a model for some aspects of migraine, which is currently being explored. In collaborative projects, we are studying the beneficial effects of CGRP against hypertension and following myocardial infarction in the RAMP1 mice. Other collaborative projects include the regulation of serotonin biosynthesis, which may be important in migraine and behavioral disorders, and use of the CGRP promoter to target a dominant negative oncogene to specific neuroendocrine cells. The overall goal of these projects is to develop effective diagnostic and therapeutic strategies.
Our laboratory is interested in how proteins are degraded in lysosomes. This is a fundamental process of all eukaryotic cells necessary for regulating a variety of cell surface proteins. This process is often termed “downregulation”, and is a central feature of virtually all physiological processes that rely on cell surface membrane proteins. Failure to properly downregulate particular proteins can lead to or exacerbate a variety of pathophysiological conditions such as cancer, hypertension, and cardiac disease.. Our overall goal is to understand the protein machinery common in all cell types that controls the delivery of membrane proteins to the lysosome. There are two processes that we examine: the first is how proteins are designated and recognized for delivery to the lysosome. The second is how transport through the endocytic pathway via endosome membrane fusion events is controlled.
Our primary focus is one finding out how individual “cargo” proteins are selected and designated for transport and degradation in the lysosome. Membrane proteins can initiate their journey to lysosomes from a number of cellular compartments. At the cell surface, proteins can enter the endocytic pathway via internalization. At the Golgi apparatus, proteins can be sorted into transport vesicles that are targeted to endosomes. Perhaps the most critical sorting step controlling the degradation of membrane proteins in lysosomes, is the incorporation of protein cargo into vesicles that bud into the lumen of the endosome. This sorting step occurs within endosomes and leads to the formation of multivesiculated bodies (MVBs) that accumulate lumenal membranes. These lumenal membranes are then subject to degradation by lysosomal lipases and proteases, thereby ensuring the complete destruction of integral membrane proteins.. A variety of studies in both yeast and mammalian cells have established that this sorting step is conferred by the post-translational attachment of ubiquitin, a 76 amino acid peptide that is covalently linked to lysine residues via an isopeptide bond.. Currently we are determining how ubiquitinated cargo is recognized and incorporated into transport vesicles destined for the lysosome.
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