Apoptosis and cell death; Bioinformatics; Molecular Basis of Disease; Molecular and Cellular Biology; Molecular genetics; Neurobiology; Regulation of metabolism
My laboratory studies the cell-autonomous and non-cell-autonomous mechanisms of axon degeneration, a process akin to programmed cell death. In other words, we are attempting to elucidate what causes axon breakdown from within neurons and which external (glial) events trigger axon loss. Degeneration of axons is a hallmark in many neurodegenerative conditions, including those associated with abnormal glia. We have great hope that understanding why and how axons degenerate may lead to more efficient neuroprotective therapies tailored specifically to support axons and their surrounding glia. Axons are the longest cellular projections of neurons relaying electrical and biochemical signals in nerves and white-matter tracts of the nervous system. As such, they are critical for neuronal wiring and transport of neuronal maintenance signals. Because of their incredible length and energetic demand (human motor neurons can be one meter long), however, axons are very vulnerable and at continuous risk of damage. Axons do not exist in isolation but are inextricably and intimately associated with their enwrapping glia (Schwann cells and oligodendrocytes) to form a unique axon-glia unit. The most relevant neurological symptoms in a number of debilitating neurodegenerative conditions are due to compromised axon integrity. Thus, neuroprotective therapies promoting axon stability have great potential for more effective treatment. Recent studies indicate that axonal degeneration, at least in experimental settings, is an active and highly regulated process akin to programmed cell death (‘axonal auto-destruction’). Moreover, it is increasingly realized that axonal maintenance relies not only on neuron-derived provisions but also on trophic support from their enwrapping glia. The mechanism for this non-cell-autonomous support function remains unknown, but emerging evidence indicates that it is distinct from the glial role in insulating axons with myelin. We are pursuing the intriguing question of whether abolished support by aberrant delivery of metabolites and other trophic factors from glia into axons is mechanistically linked to the induction of axonal auto-destruction. This concept is supported by our recent finding that metabolic dysregulation exclusively in Schwann cells is sufficient to trigger axon breakdown.
Bioinformatics; Genomics and proteomics; Molecular and Cellular Biology; Molecular genetics; Gene Expression; Transcription and Translation
Our research group is interested in how regulatory proteins are targeted to the correct DNA binding sites at the correct time. Transcription factors are directed to their genomic targets by DNA sequence, local chromatin structure, and protein-protein interactions. These modulators of transcription factor binding are not independent but function both cooperatively and competitively to regulate where transcription factors bind. Understanding how these modulators affect transcription factor binding in vivo remains a major unsolved biological problem. We use the model organism Saccharomyces cerevisiae to address the disconnect between the presence of the correct DNA binding sequence and true regulatory protein binding, integrating both experimental and computational approaches to: i) investigate transcription factor binding in response to environmental stress, ii) identify and characterize the mechanisms directing transcription factor target selection, and iii) and develop bioinformatics tools to analyze and interpret ChIP-seq experiments and chromatin structural patterns.
Protein Function and Structure; Proteins and metalloenzymes; Vitamins and Trace Nutrient
Cytochrome P450 enzymes are ubiquitous catalysts that play integral roles in biochemical pathways throughout nature. In mammals, members of this class of enzyme serve a variety of functions that include drug metabolism, steroid biosynthesis and the activation and deactivation of vitamin D, to name a few. Cytochrome P450 enzymes are also heavily involved in bacterial and plant biochemistry. The overall goal of my lab is to use a combination of biochemical and biophysical tools to investigate structure and function in cytochrome P450 enzymes, thereby contributing toward an understanding of how this important class of enzymes work as well as informing the design of novel drugs. This goal is divided between two efforts. First, we are interested in characterizing the ligand binding interactions of the enzyme CYP24A1, the principle enzyme responsible for deactivating vitamin D. Describing the interaction between CYP24A1 and vitamin D has the potential to illuminate how the vitamin D structure becomes modified at a particular site. This insight could impact the design of vitamin D analogs with benefits for an array of human health conditions, including bone density disorders, diabetes and chronic kidney disease (CKD). A parallel effort in my lab is a structural study of the enzyme CYP121 of Mycobacterium tuberculosis, the disease-causing pathogen in tuberculosis (TB). The resurgence of standard TB and the rise of drug-resistant forms of TB are quickly becoming a global pandemic, with TB claiming more lives worldwide in 2014 than HIV. CYP121 is essential for survival of the bacterium and thus has emerged as one of the more promising antitubercular drug targets. Students and postdocs joining my lab will be exposed to a multidisciplinary set of research tools, including expression and purification of recombinant membrane protein, nuclear magnetic resonance, protein X-ray crystallography and P450 ligand binding assays.
Cell growth, differentiation and development; Molecular Basis of Disease; Proteins and metalloenzymes; Gene Expression; Inherited Metabolic Disorders; Protein Function and Structure; Cell Cycle
Protein Methylation in Growth and Differentiation. Protein methylation was recently found by systems biology approaches to play a major role in regulating yeast cell growth. Consistent with this finding, we found that disruption of the gene encoding S-adenosylhomocysteine (SAH1) hydrolase markedly inhibited growth. S-adenosylmethionine (SAM) is the universal methyl donor,and SAH1 is the product of all methyltransferase(MTase) reactions.The SAH1 disruption leads to a 50% decrease in protein synthesis which,in turn leads to major decreases in the levels of Cln3p.Unexpectedly,when cells were transfected with a modified gene for Cln3 ,that desreased its rate of degradation,growth rates were normal.This result was unexpected because the basic defect of lacking SAH1 remained.We are currently testing the hypothesis that normal rates of growth are due to increased gene expression for multiple enzymes known to be involved in Met and SAM synthesis. We are also identifying substrates for specific MTases in yeast. Copper deficiency is known to affect brain development, and Menkes disease is fatal due to impaired brain development from low brain copper. A reduction in (SAH1) levels, as occurs in copper deficiency, may affect brain development by inhibiting protein methylation.We demonstrated that inhibiting SAH1 maredly inhibited development of two nerve cell models.
Neurology; Cytoskeleton and cell motility; Molecular and Cellular Biology; Molecular Basis of Disease; Molecular genetics; Neurobiology; Signal Transduction; Inherited Metabolic Disorders; Transgenic organisms
My laboratory seeks to understand the molecular basis of myelination and myelin diseases. Myelin is a multi-lamellar sheath that invests large axons and permits rapid conduction of nerve signals. Failure in myelin synthesis and myelin breakdown cause several important neurological diseases, including multiple sclerosis, leukodystrophies and peripheral dysmyelinating neuropathies. In some of these diseases, genetic mutations cause defects in cytoskeletal, adhesion and signaling molecules. I work with a team of undergraduate and graduate students, postdoctoral fellows, technicians, senior scientists and many international collaborators to discover how these molecules normally coordinate cell-cell and cell-extracellular matrix interactions to generate the cytoarchitecture of myelinated axons. We use a variety of approaches, including generation of mice carrying genetic abnormalities, cultures of myelinating glia and neurons, imaging, biochemistry and morphology to understand the role of these molecules in normal and pathological development. By comparing normal myelination to the abnormalities occurring in human diseases, we aim to identify molecular mechanisms that pharmacological intervention might correct. For example, we described how the protein dystroglycan associates with different proteins, some of which impact human neuropathies, depending on a proteolitic cleavage that can be regulated to improve the disease. Similarly, we found that molecules such as integrins and RhoGTPAses are required for glia to extend large processes that will become myelin around axons. In certain neuromuscular disorders, defective signaling pathways that converge on these molecules cause failure to produce or mantain an healthy myelin Finally, in collaborations with scientists and clinicians in the Hunter J. Kelly Research Institute, we are generating transgenic forms of GalC, an enzyme deficient in Krabbe leukodystrophy, to investigate which cells requires the enzyme. Investigating how GalC is handled may help find a cure for this devastating disease.
Bioinformatics; Genomics and proteomics; Immunology; Membrane Transport (Ion Transport); Molecular and Cellular Biology; Molecular Basis of Disease; Molecular genetics; Neurobiology; Gene Expression
The current focus of my lab is on iron metabolism in animals and humans. From the practical viewpoint, iron is an important nutrient, but its ability to act in the ferrous and ferric state also makes it toxic. Thus, iron deficiency is the most frequent disorder in the world and hereditary hemochromatosis (HH) is the most common Mendelian disorder in the United States. Our research is related to erythroid differentiation on the fundamental level and to genetic and acquired diseases on the applied level, with four long-term themes: 1.) analysis of the molecular basis of differential gene expression among tissues and during development, with hemoglobin synthesis and red blood cell (RBC) development as models; 2.) application of molecular and genetic advances to inherited diseases; 3.) iron metabolism; 4.) study of gene variation in populations and divergence of gene loci during evolution. New vistas have opened recently for the anemia of chronic diseases, leading us to re-exam how microbes and their human hosts fight for iron. We approach these issues by working on rodent models like the Belgrade rat, plus a series of genetically engineered mice. The rat has a hypochromic, microcytic anemia inherited as an autosomal recessive. The defect is in an iron transporter called DMT1 (or slc11a2, previously called Nramp2 or DCT1) that is responsible for iron uptake by enterocytes and is also responsible for iron exiting endosomes in the transferrin cycle. The rats appear to have a severe iron deficiency, and although dietary iron and iron injection increase the number of RBCs, they do not restore the RBCs nor the rat itself to a normal phenotype. Recent discoveries show that DMT1 is ubiquitous and responsible for transport of other metals such as Mn and Ni. It occurs in the kidney, brain and lung at even higher levels than in the GI tract or in erythroid cells. It also has multiple isoforms, and we have cloned them and developed cell lines that express high levels of particular isoforms. We have specific antibodies to the isoforms and assays for each of the mRNAs too. Future projects in my lab will continue to address whether DMT1 is dysregulated in HH. We will also tackle how DMT1 functions in neurons, pneumocytes and other tissues, look at isoforms of DMT1 under circumstances where we suspect that they must have different functions from one another, and examine DMT1’s relevance to iron metabolism and human disease. Because we cloned the gene and identified the mutation, a number of molecular and cellular approaches can now be used. As evidence indicates that metal ion homeostasis fails in Parkinson’s disease, Alzheimer’s disease and Huntington’s disease, research on DMT1 has opened new vistas for these disorders.
Bioinformatics; Cell growth, differentiation and development; Genomics and proteomics; Molecular and Cellular Biology; Molecular Basis of Disease; Molecular genetics; Neurobiology; Gene Expression; Stem Cells; Transgenic organisms
My research goal is to gain a better understanding of how proteins that interact with DNA regulate RNA transcription, DNA replication and metazoan development. I mentor undergraduate and graduate students in my lab; we focus on the structure and function of the Nuclear Factor I (NFI) family of site-specific DNA binding proteins, and we are investigating their roles in development. Our work has been made possible by our development of loss-of-function mutations of the NFI genes in the mouse and C. elegans. We are addressing four major questions in my laboratory and in collaboration with a number of talented collaborators: What is the structure of the NFI DNA-binding domain? How does NFI recognize and interact with DNA? Does NFI change the structure of DNA when it binds? What proteins interact with NFI to stimulate RNA transcription and/or DNA replication? These research questions are explored in my lab through two major projects focused on the role of NFIB in lung development and the role of NFIX in brain development. When NFIB is deleted from the germline of mice the animals die at birth because their lungs fail to mature normally. This provides a good model for the problems that occur with premature infants, whose lungs also fail to mature normally. We are using this model to determine how NFIB promotes lung maturation with the goal of being able to stimulate this process in premature infants. In our NFIX knockout animals, the brains of the animals are actually larger than normal and contain large numbers of cells in an area known to be the site of postnatal neurogenesis. We have evidence that NFIX may regulate the proliferation and differentiation of neural stem cells, which produce new neurons throughout adult life. Our aim is to understand the specific target genes that NFIX regulates in the adult brain to control this process of neurogenesis.
Bioinformatics; Cell growth, differentiation and development; Gene Expression; Genomics and proteomics; Molecular genetics; Signal Transduction
Research in my laboratory investigates the genetic regulatory circuitry that controls how cell fates are determined during development. We focus on two key aspects, intercellular signaling and transcriptional regulation, using primarily the fruit fly Drosophila melanogaster due to its extremely well-annotated genome and amenability to experimental manipulation. All conclusions, however, are expected to relate directly to mammalian (including human) gene regulation. Recently, we have also started investigating the regulatory genomics of other insect species of both medical and agricultural importance, beginning with the development of methods for regulatory element discovery in species with fully sequenced genomes but little functional, experimental data. A defining feature of my laboratory is that it takes both wet-lab and computational/bioinformatics approaches to studying the same set of problems about development and transcriptional regulation; hypotheses and ideas generated using one set of methods are tested and explored using the other. Current research in the laboratory falls into two main areas: 1) discovery and characterization of transcriptional cis-regulatory modules (CRMs), and 2) mechanisms of specificity for receptor tyrosine kinase (RTK) signaling. The combined results of these studies will provide insight into gene regulation, genome structure, intercellular signaling, and the regulatory networks that govern embryonic development. My group is also heavily involved in biocuration through our development and maintenance of REDfly, an internationally-recognized curated database of known Drosophila transcriptional cis-regulatory modules (CRMs) and transcription factor binding sites (TFBSs). Despite more than 25 years of experimental determination of these elements, the data have never been collected into a single searchable database. REDfly seeks to include all experimentally verified fly regulatory elements along with their DNA sequence, their associated genes, and the expression patterns they direct. REDfly is by far the most comprehensive database of regulatory elements for the higher eukaryotes and serves as an important resource for the fly and bioinformatics communities.
The long term goal of the research conducted in my lab is to learn about the general principles that organisms use to acquire and metabolize the essential nutrient iron. Since in eukaryotes, iron metabolism depends on the activity of copper-containing enzymes called ferroxidases, we examine the trafficking copper in cells as well. The first challenge for a cell is to scavenge these two metals from the environment. This is true for a yeast cell in culture, or for an epithelial cell in your intestine. The second challenge is to efficiently and correctly partition these metals in the cell for subsequent utilization and storage. Ultimately the cell or organism will have to regulate the accumulation of these metals and to ensure that they are not allowed to roam "free" since both are toxic. Iron and copper are essential micronutrients. They are required in fundamental cellular processes such as cellular respiration in all organisms, and for vital physiologic functions such as oxygen transport in blood and muscle. The brain has a strong requirement for iron to support the elevated energy metabolism needed to support neuronal function. However, both iron and copper are also intrinsically toxic. This toxicity results from their strong tendency to generate oxygen radicals which in turn destroy key cellular components. For example, iron uptake into the brain must be tightly regulated, a process we focus in our research. Failure of this regulation can result in a variety of brain pathologies particularly those that result in degeneration of neuronal function.
Research in my laboratory is focused on stem cell biology, engineering, and therapeutic applications with an emphasis on cardiovascular repair. We have explored the immunomodulatory property of bone marrow mesenchymal stem cells (MSCs) in our cell transplantation studies, and found that large quantities of human and porcine MSCs can be implanted in immunocompetent pigs, mice, and hamsters without inducing inflammatory immune responses in the host. Our research shows that MSCs improve cardiac function in the porcine myocardial ischemia and hamster heart failure models. Implanted MSCs promote tissue regeneration by recruiting bone marrow progenitor cells and activating local host stem cell niches. These processes are mediated by inter-tissue cross-talk mechanisms involving signaling molecules such as JAK/STAT3, integrins, VEGF receptors, and Wnt/b-catenin. Our long-term goal is to generate clinically relevant stem cell information that may be used to achieve robust therapeutic effects for a broad spectrum of human diseases and lower the cost of future stem cell therapy.
Bioinformatics; Gene Expression; Genomics and proteomics
My research is focused on developing bioinformatics algorithms especially through sequencing analysis and data integration, to understand better transcriptional and epigenetic regulation. Transcription factor often binds to DNA and interferes with transcription machinery to enhance or repress gene expression. Epigenetic features such as histone modification, chromatin remodeling factor binding, DNA methylation, and chromatin 3D organization add yet another layer of information, making it more complex to understand the regulation dynamics within the nucleus. With advancing sequencing technology, however, such information now can be measured and quantified in genome scale, though the growing number of big genomic datasets creates challenges as well as opportunities for bioinformatics methodologies. The focus of our lab is to build algorithms, analysis platforms and databases to integrate big datasets from the public domain into various biological questions and disease models. The MACS (Genome Biology 2008) algorithm, on which I worked to develop, is one of the most widely-used algorithms for predicting cis-regulatory elements from Chromatin Immunoprecipitation with high-throughput sequencing (ChIP-seq). The algorithm has been evolving over years to accommodate various factor types from punctuate transcription factor binding to long-range histone modifications. It has been used to process hundreds of publicly-available datasets in the mod/ENCODE project, and it continues as a focus of my lab. I also worked to build an integrative platform for ChIP analysis based on Galaxy framework, named Cistrome (Genome Biology 2011). This platform provides both a user-friendly interface and rich functionality for biologists to manage and process their high-throughput genomic data and to publish the results conveniently over the Internet. The Cistrome platform will continue as a collaborative project between my UB lab and research partners at Harvard University. I have also been involved in many collaborative research projects, such as circadian binding of histone deacetylase and nuclear receptor Rev-Erba in mouse liver (Science 2011), and the modENCODE consortium project to elucidate chromatin factor functions of C. elegans (Genome Research 2011 and Science 2010).
Microbial Pathogenesis; Molecular and Cellular Biology; Gene Expression; Regulation of metabolism
The adaptive success of bacteria depends, in part, on the ability to sense and respond to their environment. Metals such as iron and manganese are important nutrients that can often be limiting, and therefore cellular metabolism must be modified to either scavenge the nutrients or use alternative processes that do not require the metal. Bradyrhizobium japonicum belongs to a group of related organisms that form close or intracellular and related bacteria that form an intracellular relationship with eukaryotes in a pathogenic or symbiotic context. This bacterium serves as a model to study related pathogens that are refractive to genetic and biochemical study. One project involves understanding the mechanisms by which cells maintain iron homeostasis at the level of gene expression. We discovered the global transcriptional regulator Irr that controls iron-dependent processes. Irr is stable only under iron limitation, where it positively and negatively controls target genes. We are interested in understanding the mechanism of this conditional stability, how Irr regulates genes, and the functions of numerous genes under its control. We initiated a new project to understand the requirement for manganese in cellular processes, how it is acquired from the environment, and how manganese controls gene expression. Also, we identified cross-talk between regulators that control iron and manganese homeostasis and are pursuing this unique mechanism.
Bioinformatics; Cell growth, differentiation and development; Neurobiology
My laboratory seeks to understand the transcriptional regulatory network governing the differentiation of oligodendrocytes and central nervous system (CNS) myelination, with the long-term goal of translating this knowledge into the treatment of demyelinating diseases. CNS myelination by oligodendrocytes is important not only for saltatory conduction of action potentials but also for trophic support of nerve axons. An improved understanding of how the differentiation of oligodendrocytes is regulated for CNS myelination should provide a firm basis on which to develop more effective therapeutics for demyelinating diseases. Toward this goal, we are currently pursuing two different research directions. The first is to elucidate the functional mechanism of Myrf, a key transcription factor for CNS myelination. Conditional knockout mice in which Myrf is knocked out in the oligodendrocyte lineage cells completely fail to develop CNS myelin and exhibit severe neurological symptoms, eventually prematurely dying. Recently, we and the Emery laboratory have independently made the surprising discovery that Myrf is generated as an integral membrane protein that is auto-cleaved by its ICA domain into two fragments. This discovery invokes a number of fundamental questions about how Myrf drives the differentiation of oligodendrocytes for CNS myelination. We employ both computational and experimental laboratory methodologies to elucidate the functional mechanism of Myrf. The second direction is to identify new transcription factors for CNS myelination. By taking advantage of our computational expertise, we have performed integrated computational analysis of functional genomics data that are publicly available to predict a number of new transcription factors for oligodendrocyte differentiation. We are currently characterizing them using primary oligodendrocyte cultures. Promising hits will be further analyzed by generating knockout mice to test for in vivo relevance.
One research goal is to investigate the structure-function relationships and regulation of the human pyruvate dehydrogenase complex (PDC). We investigate the catalytic mechanism of the pyruvate dehydrogenase (PDH) component and its interactions with the dihydrolipoamide acetyltransferase (E2) component of PDC. We also determine the loci of interactions between PDH kinases (four PDK isoenzymes) and the lipoyl domains of E2. Using a PDC-knockout mouse line we investigate the importance of glucose metabolism as a source of energy for fetal development as well as the role of PDC in glucose-stimulated insulin secretion by pancreatic beta cells. Another research goal is to investigate diet-induced metabolic programming during early life. We investigate (i) the effects of an altered nutrition during the immediate postnatal life on development of adult-onset obesity and (ii) the effects of maternal obesity on fetal programming. Current research focuses on the role of the hypothalamic signaling pathways in rodents with diet-induced obesity and also in the progeny of obese mothers.
Gene Expression; Molecular Basis of Disease; Molecular and Cellular Biology; Molecular genetics; Protein Function and Structure; Transcription and Translation
Our laboratory utilizes combined genetic, biochemical and molecular biological approaches to investigate the molecular mechanisms involved in the initiation and regulation of eukaryotic transcription. Previous work in our laboratory utilizing both the budding yeast Saccharomyces cerevisiae and human cells resulted in the identification and biochemical characterization of mutants of nuclear RNA polymerase II (RNAPII) and the general transcription factors TFIIB and TFIIF that coordinately affect transcription start site utilization and transcript elongation. These studies supported a model where yeast and human TFIIF induce global conformational changes in RNAPII that result in structural and functional changes in the polymerase active center. Our current studies are focused on elucidating the mechanisms of kinetoplast transcription by the mitochondrial RNA polymerase of Trypanosoma brucei. T. brucei is a protozoan parasite that is the causative agent of African sleeping sickness (trypanosomiasis) in humans and nagana in animals. Procyclic trypanosomes growing in the midgut of the tsetse fly have a fully functional mitochondrion whereas trypanosomes in the mammalian bloodstream exhibit repressed mitochondrial function. The mitochondrial DNA in trypanosomes is unusual in its structure, comprising a highly catenated network of maxicircles and minicircles termed kinetoplast DNA (kDNA). Surprisingly, very little is known about the cis-acting elements and the trans-acting factors governing the transcription of maxicircles and minicircles. Our objective is to elucidate the mechanisms and regulation of T. brucei kDNA transcription with the ultimate goal of developing therapeutic agents.
Neurodegenerative disorders; Ion channel kinetics and structure; Membrane Transport (Ion Transport); Molecular Basis of Disease; Signal Transduction; Protein Function and Structure; Neuropharmacology
I focus my research on the activation mechanisms of fast neurotransmitter receptors. We seek to define the activation pathway, modulatory mechanisms and structure-function relationships of the N-methyl-D-aspartate (NMDA) receptor to better understand the roles played by this protein in the brain. NMDA receptors are the most abundant glutamate-stimulated, Ca2+-conducting ion channels in brain and spinal cord. They are the predominant molecular devices for controlling synaptic development and plasticity and govern memory and learning processes. Understanding the mechanisms that control their activity may lead to more effective strategies to treat neuropathies including stroke, neurodegenerative conditions, chronic pain and addiction as well as mental disorders such as schizophrenia and epilepsy.
Cell growth, differentiation and development; DNA Replication, Recombination and Repair; Gene Expression; Molecular and Cellular Biology; Proteins and metalloenzymes; Signal Transduction; Transcription and Translation
The main goal of my research group is to understand the role of N-terminal methylation on human development and disease. I identified the first eukaryotic N-terminal methyltransferases, NRMT1 and NRMT2, and am now working to identify how these enzymes and this new type of methylation affect cancer development and ageing. Our laboratory has shown that NRMT1 functions as a tumor suppressor in mammary glands, and its loss sensitizes breast cancer cells to DNA damaging chemotherapeutics. We have also created the first NRMT1 knockout mouse and shown it to have developmental defects, as well as, exhibit phenotypes of premature ageing. Currently, we are working to understand the exact biochemical pathways that lead from loss of N-terminal methylation to these phenotypes. We are also studying how post-translational modifications on the N-terminus of proteins may interact and dictate protein function, similar to the post-translational modifications found on histone tails.
Genomics and proteomics; Molecular and Cellular Biology; Gene Expression
My laboratory is interested in understanding the transcriptional control mechanisms that dictate epithelial cell development and differentiation. Specifically, we seek to understand the functional role of a p53-family member, p63 and Ets family of proteins in epithelial cells such as those of the skin and mammary glands. Towards this end, we have developed and characterized transgenic mice in which the normal expression pattern of these crucial factors is altered by both gain-of-function (Tet-inducible transgenic system) and loss-of-function (knockout) experiments. Our broad objectives are to elucidate the molecular mechanism by which transcription factors such as p63 and Ets proteins regulate their target genes and how such regulation of specific pathways dictate cell fate, development and differentiation. We utilize broad biochemical and genetic approaches, cell culture systems and state of the art genome-wide interrogation techniques to answer questions about differentiation of progenitor/stem populations and to examine molecular consequences of altered expression of transcription factors. These studies will not only help better understand the normal physiological processes but also lead to novel mechanistic insights into the pathophysiology of wide range of disease including cancer.
DNA Replication, Recombination and Repair; Genome Integrity; Molecular and Cellular Biology; Molecular genetics; Protein Function and Structure
In my laboratory, we are interested in the general problem of maintaining genome stability. To this end, we focus on two distinct aspects of genome stability: 1) the roles of mismatch (MMR) proteins in multiple pathways for DNA repair and 2) the manner in which regulation of dNTP pools, through the regulation of ribonucleotide reductase (RNR) activity, impacts genome integrity. 1) MMR proteins recognize many different types of DNA lesions and then target the lesion for the appropriate repair pathway. We are interested in the mechanism(s) by which recognition of a lesion is translated into the appropriate DNA repair pathway, using the yeast Saccharomyces cerevisiae as a model system. Is it through differential protein-nucleic acid or protein-protein interactions? To address these questions as well as the regulation of DNA repair pathway selection, we use a combination of genetic, biochemical and biophysical approaches. 2) RNR activity modulates the level of dNTPs that are available in a cell at a given time. Higher levels of dNTPs lead to higher mutation rates. We are interested in the various ways in which misregulated dNTP pools might affect cellular metabolism and affect the stability of the genome.
DNA Replication, Recombination and Repair; Gene Expression; Genome Integrity; Microbiology; Molecular and Cellular Biology; Protein Function and Structure; Signal Transduction
We are interested in developing an integrated mechanistic view of how organisms coordinate the actions of their DNA replication machinery with those of other cellular factors involved in DNA repair and damage tolerance. Failure to properly coordinate these functions leads to mutations, genome instability, and in extreme cases, cell death. We utilize a combination of biochemical, biophysical, and genetic approaches to investigate the molecular mechanisms of DNA replication, DNA repair, and error-prone DNA damage tolerance functions in Escherichia coli. The primary mechanism for damage tolerance involves direct bypass of damaged bases in the DNA. This process is inherently error-prone, and is the basis for most mutations. Current efforts are focused on understanding the mechanisms by which the actions of high fidelity and error-prone lesion bypass DNA polymerases are coordinated with each other, as well as other proteins involved in DNA metabolism. Our goal in this work is to develop methods that enable us to control the fidelity of DNA repair for therapeutic gain. We are also interested in understanding the mechanisms that contribute to DNA mutagenesis in the opportunistic human pathogen, P. aeruginosa. P. aeruginosa is a particular problem for individuals afflicted with cystic fibrosis. Persistent colonization of cystic fibrosis airways with P. aeruginosa serves as a major source of morbidity and mortality for these patients. The ability of P. aeruginosa to persist in the airways relies in part on its ability to adapt to the continuously changing environment within the diseased airways. We are particularly interested in determining the contribution of mutagenesis and DNA repair to adaptive mutations that contribute to clonal expansion and pathoadaptation of P. aeruginosa during colonization of cystic fibrosis airways.
Cell growth, differentiation and development; Membrane Transport (Ion Transport); Molecular and Cellular Biology; Signal Transduction; Inherited Metabolic Disorders; RNA
Regulation of Kidney Epithelial Cell Growth, Transport and Differentiation Our laboratory is investigating the molecular mechanisms by which hormones, growth factors and extracellular matrix proteins regulate kidney tubule epithelial cell growth and functional differentiation in vitro. An established canine kidney epithelial cell line, MDCK, and isolated "mutants" are currently being utilized to examine the actions of growth regulatory on the expression of several proteins including the Na+, K+-ATPase and laminin, a glycoprotein in the extracellular matrix. The effects of novel growth regulatory factors on the expression of proteins involved in gluconeogenesis, membrane transport, renal disease and growth control in primary renal cell cultures are being examined. Primary kidney epithelial cells differentiate into nephrons in a reconstituted extracellular matrix proteins is the subject of study.
Cell growth, differentiation and development; Microbiology; Molecular Basis of Disease; Molecular and Cellular Biology; Regulation of metabolism; Signal Transduction; Toxicology and Xenobiotics; Vitamins and Trace Nutrient
Dr. Willsky’s research focuses on the role of oxovanadium compounds in cellular metabolism. V is a trace metal believed to be required for growth. Oral administration of oxovanadium compounds alleviates the symptoms of Diabetes in animal models and humans. The techniques of genetics, microbiology, molecular biology, biochemistry, pharmacology, magnetic resonance spectroscopy, and cell physiology are used. The diabetes-altered gene expression of genes involved in lipid metabolism, oxidative stress and signal transduction is returned to normal by V treatment of rats with STZ-induced diabetes, as demonstrated using DNA microarrays. Inhibition of tyrosine protein phosphatases is believed to be a major cause of the insulin-like effects of V. Our results implicate the interaction of V with cellular oxidation-reduction reactions as being important in the anti-diabetic mechanism of V complexes. A new project in the lab studies the mode of action of medicinal plant mixtures used by the native healers of Peru.