Thomas D. Gilmore - US grants

Rev-T is an avian retrovirus that specifically transforms early lymphoid cells in vivo and in vitro. The oncogene of Rev-T is v- rel; v-rel encodes the transforming protein p59v-rel. v-rel is derived from a normal cellular gene c-rel, and is highly related to the Drosophila dorsal gene. We are interested in the mechanism by which v-rel transforms lymphoid cells, and the reason why it does not transform fibroblast cells. Using linker insertion and in vivo mutagenesis we intend to isolate v-rel mutants that are temperature-sensitive for transformation of lymphoid cells. These mutants will be used in experiments designed to determine if p59v-rel is, or is asssociated with, a protein kinase activity important for its transforming function. If kinase activity does not seem to be important for the v-rel transforming function we will attempt to determine a pertinent biochemical function of p59v-rel using protein purified from bacterial cells expressing the v-rel protein. We will also determine the sites of phosphorylation and the identity of a second nuclear directing function in p59v- rel, and the importance of these sequences for the transforming function using site-directed mutagenesis. The avian protein p59v- rel is toxic in murine fibroblasts. We will determine the v-rel sequences responsible for this toxicity, and whether a non-toxic avian rel protein can transform murine lymphoid cells. If not, we will attempt to isolate a murine c-rel cDNA clone, and by in vitro mutagenesis we will attempt to create a dominant murine rel transforming gene. Using DNA transfer techniques, we will attempt to isolate genes from lymphoid cells that will allow transformation of fibroblast cells. Finally, by making rel-dorsal hybrid genes in retroviral vectors we will determine whether the sequence homology between these two genes also reflects a functional homology between the two proteins. The proposed studies are intended to further our understanding of retroviral transforming processes, and may have relevance to lymphoid tumor development and other normal developmental processes in general.

DESCRIPTION: (Adapted from investigator's abstract) The Rel/NF-kB family of transcription factors controls a number of genes involved in key cellular processes, such as proliferation, immune and inflammatory responses, and apoptosis. Expression of mutant Rel transcription factors has also been associated with several animal and human cancers. This application explores the mechanism by which an avian retrovirus malignantly transforms and immortalizes avian lymphoid cells, as a model for the molecular basis of certain human lymphoid cancers that are caused by mutant Rel transcription factors. Specific functions of v-Rel that are important for its ability to transform cells will be investigated. These include the ability of mutant Envelope amino acids to endow v-Rel with an N-terminal transactivation domain, the effect of phosphorylation on transaction by v-Rel, and the effect of C0terminal sequences on cytoplasmic localization of c-Rel. the ability of anti-apoptosis proteins in the Bcl-2 family to cooperate in v-Rel-induced oncogenesis will be investigated by creating vectors for the expression of weakly oncogenic Rel proteins and Bcl-2 family members. In a third project, the protein Trip6, which was isolated in a two-hybrid screen with v-Rel, will be characterized. Specifically, requirements for transactivation and subcellular localization of Trip6 will be explored by creating Trip6 knockout mice and cell lines. Finally, cell lines deficient in specific Rel/NF-kappaB family members will be characterized, and these cell lines will be used to analyze the contribution of specific Rel/NJ-kappaB complexes to distinct physiological processes, such as adhesion, growth control, and apoptosis. A long-term goal of this project will be to develop mammalian model systems for studying Rel-mediated oncogenesis, in order to more closely mimic human cancers that involve alterations in Rel transcription factor function.

DESCRIPTION (provided by applicant): The Rel/NF-kappaB family of transcription factors is mis-regulated in a number of human cancers, and this proposal focuses on systems that model this signaling pathway in cancer. In particular, the human REL gene (encoding c-Rel or REL) is amplified or mutated in several rather common human lymphoid cell cancers, especially diffuse B-cell lymphomas and Hodgkin's lymphoma. Nevertheless, there are few in vitro models for REL-induced lymphomagenesis, and there are no specific inhibitors of REL protein function. Thus, one primary focus of this proposal is to use a model system developed in this laboratory to further understand the mechanism by which human REL malignantly transforms chicken lymphoid cells by identifying REL mutants with altered transforming activity and proteins that can interact with REL transactivation sequences required for transformation. Furthermore, using fusion proteins and co-expression systems, the inhibition of REL-induced transformation by the human estrogen receptor will be investigated and REL target genes will be identified. In two related Aims, attempts will be made to develop mouse model systems for REL-induced oncogenesis, and in collaborative studies with Dr John A Porco (Chemistry Department and Center for Chemical Methodology & Library Development at Boston University), first-stage chemical inhibitors of REL protein function will be developed using a yeast-based reporter gene assay. Finally, mouse cell lines in which the absence of RelA is associated with the transformed state will be further characterized. As such, this proposal describes exploratory research that seeks to develop models to validate the REL transcription factor as a therapeutic target in certain human lymphoid cell cancers, and, as such, the information derived from this work may have prognostic or therapeutic significance for the treatment of specific human diseases.

This Undergraduate Research Opportunity Program (UROP) at Boston University awards Summer Undergraduate Research Fellowships (SURF) to undergraduate students, especially those from underrepresented minority groups. Ten students will be provided support for a 10-week research experience each year. The main objectives of the program are to 1) provide students with a research experience that takes advantage of modern cross-disciplinary approaches in the STEM disciplines; 2) train students in research methods, scientific ethics, and the scientific culture; and 3) encourage underrepresented minority students to continue their education through graduate school. Applications from well-qualified students within all STEM disciplines (Science, Technology, Engineering and Mathematics) including Computer Science are welcome. The research projects available to students use cross-disciplinary approaches to biological problems. Chemical, computational, and engineering methods are applied to developmental, molecular and neurobiological scientific questions. As a result, students will acquire in-depth knowledge of a number of related areas and learn the varied skills needed to integrate information from several disciplines. Exposure to a diverse set of projects will also aid the student in determining potential research areas they might like to pursue in graduate school. Students will be matched to research teams consisting of a faculty mentor and a graduate and undergraduate student within the mentor's lab. Students will give two oral presentations during the summer and will return to Boston University in the fall semester to present the results of their research in poster format at the university's Undergraduate Research Symposium. Additionally, they will attend weekly summer workshops on scientific publication, graduate school admissions, and scientific ethics. Social events designed to integrate students into the research environment are included throughout the summer experience. Research stipends, housing and travel are provided. More information is available from Professor Mary Erskine, Director, and Jeremy Goodman, Assistant Director, UROP, 617-353-2020, urop@bu.edu, or by visiting http://www.bu.edu/surf.

The survival of all organisms depends on their ability to manage environmental stress, whether caused by physical (e.g., heat, oxygen, chemicals) or biological (e.g., pathogens) insult. Many animals, including insects and vertebrates, respond to a wide range of environmental stresses by activating a conserved molecular pathway, called the NF-kappaB transcription factor signal transduction pathway. Although the NF-kappaB pathway is critical for many higher animal stress response systems, its evolutionary origins are obscure. In the current project, genes encoding many key proteins similar to the human NF-kappaB pathway have been identified and cloned in a simple marine organism, the starlet sea anemone Nematostella vectensis (phylum Cnidaria).

This project involves interdisciplinary research between a molecular evolutionary biologist (Finnerty) and a cell & molecular biologist (Gilmore) that seeks to gain insights into the ancestral function of the NF-kappaB signaling pathway, and the biological and ecological significance of a gene variant of NF-kappaB that exists in wild populations of Nematostella. Specifically, this research is investigating how NF-kappaB functions in simple marine animals, and three major experimental aims will be accomplished. (1) NF-kappaB signaling proteins are being characterized in the sea anemone Nematostella using a variety of molecular, cellular, and whole animal assays. (2) Genes regulated by NF-kappaB and stressors that can activate NF-kappaB and its target genes will be identified in Nematostella. (3) The functional and selective consequences of gene variants of the NF-kappaB protein of Nematostella will be determined.

Broader Impacts
The research will lead to a better understanding of the evolutionary origins of the stress response pathways of contemporary animals, and a better understanding of how these pathways can evolve in response to environmental change (climatic shifts, new physical/chemical insults, novel pathogens). The research program will have three major broader impacts. It will provide interdisciplinary training in cell and molecular biology, evolutionary biology, genomics, and developmental biology to students at the PhD and undergraduate levels, including students from groups that are under-represented in science. It will augment three heavily utilized Internet databases that serve a broad research community: StellaBase (http://stellabase.org); The Nematostella Web Resource (http://nematostella.org); and Rel/NF-kappaB Transcription Factors (http://www.nf-kb.org). It will contribute to ongoing marine conservation efforts by identifying molecular markers for environmental stressors. Such information may better enable conservation scientists to monitor the biological effects of environmental threats impacting indicator species in key coastal habitats, such as the coastal estuaries inhabited by the sea anemone Nematostella.

A Research Experience for Undergraduates (REU) Site award has been made to Boston University (BU) that will provide research training for ten students, for ten weeks during the summers of 2013-2016. The program is coordinated by the BU Undergraduate Research Opportunity Program (UROP), and approximately 35 faculty from the Biology Department and related programs serve as research mentors. Molecular, cellular, ecological, genomics, evolutionary, and computational research methods are used by students and mentors to address biological problems, primarily as they relate to the effects of environmental influences on gene expression or function. Students perform full-time intensive research as part of a research team consisting of a faculty member and a graduate student or post-doctoral mentor. In addition to research, students attend weekly summer workshops on topics including scientific publication, professional presentations, career opportunities in science, graduate school admissions, and scientific ethics. Students give several oral presentations during the summer and return to BU in the fall semester to present at the Annual Undergraduate Research Symposium. Social events designed to integrate students into the research environment are included. After completion of the program, students will be tracked to determine continued interest in research and career paths. Research stipends, housing and travel are provided. Students are selected based on academic record, letters of recommendation, potential for outstanding research in the program?s research area, and matching interests with research mentor, with special focus on students from underrepresented minority groups or smaller colleges. The program?s recruitment efforts consist of traditional "hard copy" formats, on-line advertising, and networking activities with previous students and at national research conferences. Information about the program outcome will be assessed by various means, including use of an REU common assessment tool. More information is available by visiting http://www.bu.edu/surf/program/, or by contacting the PI (Dr. Tom Gilmore, gilmore@bu.edu) or the Program Manager (Ms. Tina Fresta, urop@bu.edu).

The conserved NF-κB transcription factor signal transduction pathway controls innate immune responses in a variety of organisms, but also controls developmental cell and tissue fates in certain invertebrate and vertebrate systems. NF-κB carries out its biological effects by controlling the expression of genes that program developmental fate. This research will investigate how NF-κB functions in basal marine animals. The results of the proposed research will lead to a better understanding of the evolutionary underpinnings of a key molecular pathway (NF-κB) that is used by a variety of organisms for immunity, development and stress responses. It will also provide an understanding of how such signaling pathways can evolve and adapt to direct novel biological outcomes through development or in response to environmental changes. The planned studies will have four major broader impacts for society. First, the award will support interdisciplinary training in cell and molecular biology, evolutionary biology, genomics, computational biology, and developmental biology to students at the PhD, Masters and undergraduate levels, including students from groups underrepresented in science. Such training will enhance the scientific competence of the STEM field workforce. Results form the project will augment heavily utilized Internet databases that serve a broad research community. The investigators will also develop additional transcriptomic and genomic resources that will be valuable for other researchers studying invertebrate animal. Finally, data from these studies will contribute to ongoing marine conservation and management efforts by identifying molecular mechanisms underlying basal marine organism development and adaptation. Such information may better enable scientists to assess biological effects of environmental threats impacting indicator species in key coastal habitats, such as estuaries inhabited by the studied model organism, the sea anemone Nematostella vectensis.

This award supports interdisciplinary research involving an invertebrate model organism, the sea anemone Nematostella vectensis (Nv), in comparative developmental and molecular studies. The research will provide insights into the function of the NF-κB signaling pathway in cnidarian development and the biological significance of a functional polymorphism in NF-κB that exists in wild populations of Nv. Four major experimental aims will be accomplished. (1) To elucidate upstream NF-κB signaling pathways, additional signaling proteins will be characterized in Nv using molecular, cellular, and whole animal assays. For example, the tissue and developmental expression patterns of IκB Kinase and Toll-like Receptor proteins will be determined using immunohistochemistry. (2) The derived role of NF-κB in the development of a phylum-specific cell type, the cnidocyte, will be investigated using combined molecular and biological approaches. (3) Target genes of NF-κB that play roles in developmental cell fate or other physiological processes will be computationally predicted and experimentally validated. (4) The functional and biological consequences of polymorphisms in the NF-κB protein of Nv will be determined using a protein DNA binding-site microarray technology, as well as biological analyses. Results from the studies will be made available through peer-reviewed scientific publications, presentations at meetings, and through relevant WWW-sites, including: StellaBase (stellabase.org); The Nematostella Web Resource (nematostella.org); and NF-κB Transcription Factors (www.nf-kb.org).

This REU Site award to Boston University, located in Boston, MA, will support the training of 10 students for 10 weeks during each summer of 2017- 2020. Participants will conduct research experiments in the area of cell and molecular biology focusing on how changes in gene expression control biological processes, and they will be mentored in laboratories of faculty researchers in the Biology Department. Projects may involve investigating how transcription factors and gene expression changes play a role in development, differentiation, and the recognition of specific DNA sites. Students will receive training in conducting experiments, analyzing data, interpreting results, the oral presentation of scientific data, laboratory safety, and the ethical and responsible conduct of research, and will participate in a variety of educational, networking, and social activities with the larger Boston University summer research community. Participants will be selected by the Principal Investigator and potential faculty mentors after completion of an online application pool. Special emphasis will be placed on the recruitment of students from ethnic groups underrepresented in STEM research fields and from institutions (e.g., community colleges, non-PhD granting) without extensive access to research opportunities.

It is anticipated that a total of 40 students, primarily from schools with limited research opportunities, will be trained in this program. Students will learn how research is conducted, and many will present the results of their work at scientific conferences.

A common web-based assessment tool used by all REU Site programs funded by the Division of Biological Infrastructure will be used to determine the effectiveness of the training program. Students will be tracked after participation in the program in order to determine their career paths. There is special emphasis on the training of students who will choose research career paths in academia or industry, and it is anticipated that many students will pursue advanced degrees in the biological sciences. Students will be asked to respond to an automatic email sent via the NSF reporting system. More information about the program is available by visiting http://www.bu.edu/surf, or by contacting the PI (Dr. Thomas Gilmore at surf@bu.edu).

This research will provide increased knowledge of the molecular factors enabling different hosts (especially bats vs. humans) to tolerate the recently emergent SARS-Cov-2 coronavirus. This project will use bat cell line models to investigate differences in cellular proteins which lead to differences in immunity profiles of cells from the different species. The proposed research will identify proteins that are differentially active in different cell line models, and the effects that immune-stimulating factors and SARS-CoV-2 proteins have on host cell protein activities. Results from this research could lead to new insights into why and how certain viruses, in particular SARS-CoV-2, can be tolerated in some species (e.g., bats) but not others (e.g., humans), and could provide information for the emergence of future viruses that can cause pandemics in animal and human populations. The interdisciplinary research team comprises three experienced lead scientists with expertise in complementary areas of research. In addition, the research will provide training to two graduate students who will be able to pursue careers in related areas in academia, the private sector, or government.

The goal of this research is to further knowledge of the molecular processes that underlie species-specific infection of cells with SARS-CoV2. This research will provide insights into transcription factor-based differences between host cells of bat origin, which can tolerate SARS-CoV2 infection in a non-pathogenic manner, vs human and other bat cells that do not show this tolerance. The research involves the collaboration of three PIs with complementary approaches that they will bring to this project. Five major experimental goals will be addressed: 1) characterization of transcriptional and nuclear proteomic profiles that distinguish different bat cell lines; 2) use of a novel protein-binding microarray to profile active transcription factors among these cell lines; 3) characterization of candidate immunity transcription factors, such as NF-?B and IRFs, that are likely different between SARS-CoV2 tolerant and susceptible cell lines; 4) effects of SARS-CoV2 gene products on identified transcription factors in the various cell lines; and 5) potential for replication of novel SARS-CoV2 tracer viruses in different bat cell lines. The proposed research will increase knowledge of SARS-SoV-2 biology. In addition, it investigates the hypothesis that bat cells have evolved molecular immunity pathways that promote or lead towards tolerance (perhaps even symbiosis) of bats with coronaviruses. Such information may lead to the identification of therapeutics for humans and animals against coronaviruses. This RAPID award is made by the Physiological and Structural Systems Cluster in the BIO Division of Integrative Organismal Systems, using funds from the Coronavirus Aid, Relief, and Economic Security (CARES) Act.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.