Our research has diversified into two main areas: 1) How viruses infect and disseminate in skin; 2) How microbial and environmental factors play a role in the development of metabolic syndrome. Our prior research addressed how human cells senesce, leading to aging, and how they become immortal, leading to cancer, with a particular interest in on how human papillomaviruses transform cells. Our expertise in cell immortalization and cell culture techniques has allowed us develop 3D cell culture models that recapitulate human tissue for our research.
1) How viruses infect and disseminate in skin. Collaborative studies were recently initiated with Wendy Maury’s lab to examine how Ebola virus (EBOV) infects and transmits through human skin. We found that EBOV can infect and replicate in different skin cell populations. We are currently working to understand the course of infection in skin, what specific receptors are being utilized by EBOV in skin cells, and what role skin infection plays in transmission and pathogenesis.
2) How microbial and environmental factors play a role in the development of metabolic syndrome. Our success with immortalizing human preadipocytes (pre-fat) cells has led to studies on how environmental and bacterial toxins cause or exacerbate type II diabetes through effects on fat tissue. We found that dioxin-like polychlorinated biphenyls (PCBs), which are persistent organic pollutants, can disrupt adipogenesis (i.e. the development of functional fat cells) through activation of the aryl hydrocarbon receptor (AhR). This causes a proinflammatory response and inhibits master regulatory genes involved in adipogenesis. Endogenous microbial-derived tryptophan metabolites are also able to activate AhR. Studies are underway to determine the mechanism by which AhR activation disrupts adipogenesis and to develop 3D cultures and in vivo genetic models to assess the role of AhR in the development of metabolic syndrome.
The eukaryotic nucleus is highly organized to facilitate the regulation of gene expression. This organization includes the localization of chromosomes into discrete nuclear territories that are enriched in different classes of transcription factors, as well as the definition of independent structural domains within chromosomes that constrain interactions between transcriptional control elements and target promoters. To understand these processes, our laboratory uses Drosophila melanogaster as a model organism, employing multi-dimensional experimental approaches that include genetics, biochemistry and molecular biology. Two major research areas are represented in the laboratory. First, we investigate how proteins located in the nuclear envelope and underlying lamina contribute to chromosome organization and gene expression. Second, we study the properties of DNA elements called insulators that are used to define independent structural and functional domains within chromosomes.
The nuclear lamina is an extensive protein network comprised of lamins and several classes of lamin interacting proteins. Our studies focus on the family of LEM domain proteins that share a protein interaction domain that establishes a link between the nuclear lamina and chromosomes. Alterations in LEM domain proteins cause several human diseases, including Emery-Dreifuss muscular dystrophy and Buschke-Ollendorf syndrome. Mechanisms responsible for these diseases are intriguing as these nuclear lamina proteins are globally expressed, yet pathologies are often tissue-specific. We are determining the role of the LEM domain proteins in regulation of genes involved in tissue differentiation and development. Our studies will provide insights into the molecular mechanisms contributing to human diseases associated with laminopathies.
Insulators are a conserved class of DNA elements that define chromatin domains of independent gene function. Insulators block interactions between enhancers and silencers when placed between these elements and a promoter. This property suggests that insulators are favorable candidates for improving the design of gene therapy vectors, as insulators have the ability to prevent cross-regulatory interactions between vector genes and the regulatory elements in the host genome. Our studies are directed at identifying novel insulators and defining the molecular mechanism by which insulators establish independent functional domains.
Human cancers arise through a multi-step genetic process that involves the loss of tumor suppressor genes and activation of proto-oncogenes. However, it is challenging to identify the causal mutations amid the large number of genetic and epigenetic changes typically found in human tumors. Mouse models of human cancer have been useful in testing the contribution of specific mutations in oncogenesis. Unfortunately, single gene mouse models often do not translate well when compared to human cancer. It is thought that these models lack a sufficient number of mutations to generate aggressive tumors typically seen in human cancer patients. Insertional mutagenesis provides many advantages over single gene mouse models. The most common insertional mutagen used in mouse models of cancer is the murine leukemia virus. In these models, retroviral infection of hematopoietic cells leads to mutation when the provirus integrates into the host cell genome. Several rounds of retroviral infection followed by clonal expansion eventually leads to the development of leukemia/lymphoma in the mouse. This multi-step process is similar to what occurs in human cancer and produces tumors that are genetically heterogeneous. In retrovirally-induced tumors the provirus marks the site of mutation and provides a sequence tag that can be used to efficiently identify the mutated genes. Several labs have identified over 150 candidate cancer genes by cloning more than 2,000 proviral integrations sites from a variety of retrovirally-induced mouse models of leukemia and lymphoma (available at http://genome2.ncifcrf.gov/RTCGD). Unfortunately, retroviral infection of mice produces mammary and hematopoietic malignancies primarily. However, Sleeping Beauty (SB), an engineered cut-and-paste transposon system, has demonstrated activity in a variety of tissues in the mouse. The system consists of two parts—the transposase enzyme provided in trans and the DNA transposon vector. When these elements are present in the same cell, SB transposase binds to sites in the transposon and mediates excision from the donor site and integration to a random TA dinucleotide in the genome. Previous work has shown that transposons can generate de novo mutations when they integrated into the mouse genome. Recent work has modified the SB system as a somatic cell mutagen to induce tumor formation in mice. This work describes the first nonviral insertional mutagen capable of inducing tumors in vivo. In this regard, SB has several advantages over retroviruses in their ability to perform screens for cancer genes mainly because SB mutagenesis can be controlled by simply by regulating SB transposase expression. Current efforts are focused on generating tissue specific models of cancer in mice by directing transposase expression to specific sites in the mouse.
Alternation of generations in land plants; Vegetative phase change in maize
All land plants progress through a life cycle that alternates between two multicellular generations, the haploid gametophyte and the diploid sporophyte. In ferns, seedless vascular plants of the Monilophyte clade, both generations are free living. In addition to the normal life cycle using meiosis to generate spores and sexual union to form zygotes, in nature many fern species can switch from one generation to another asexually. In the asexual pathways, a gametophyte is generated from sporophytic cells without meiosis and a sporophyte is generated from gametophytic cells without fertilization, respectively. The model fern Ceratopteris richardii does not reproduce asexually in nature but both pathways can be induced in the laboratory using specific culture conditions. The independence of the two generations in ferns and the ease of switching from one generation to the other through the asexual pathways offer a system suitable for studying how each generation is initiated. This developmental plasticity of crossing generation barriers, i.e., meiosis and fertilization, is not unique to ferns and is manifested in the complex pathways leading to apomixis in some seed plants. My lab has identified genes potentially important in the asexual pathways in C. richardii. We are interested in learning how the functions of these genes are evolved between the fern and the seed plant Arabidopsis.
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