Linda Sealy - US grants

Enhancers are a class of cis-acting regulatory sequences that can activate transcription in a directionally-independent manner over long distances. Although first identified in viral genomes, they have been implicated in the cell-type or tissue-specific control of an increasing number of cellular genes. Accumulating evidence suggests that the action of enhancers involves their interaction with specific protein factors that can confer both positive and negative regulation of enhancer activity. The objectives of this research will be to identify and characterize protein factors which interact with the enhancer of an avian retrovirus, Rous Sarcoma Virus (RSV). This enhancer is constiutively active in essentially all cell types, and it is anticipated that an understanding of the molecular mechanisms responsible for its activation of transcription will be generally applicable to other enhancers. Recently, using sensitive assays designed to identify sequence-specific DNA binding proteins in crude nuclear extracts, at least two proteins (EFI, EFII) have been detected in avian cell extracts that specifically bind to different nucleotide sequences in the RSV LTR enhancer. Site-directed mutagenesis and the construction of synthetic enhancers will be employed to further evaluate the functional significance of EFI and EFII (and possible additional RSV enhancer factors) in mediating LTR enhancer activity in vivo. Purification of these factors by newly developed affinity chromatography methods will permit their identification and further characterization. Specific plans include attempting to reproduce RSV LTR enhancer in a soluble transcription system in vitro so that the mechanisms by which these proteins act can be further evaluated. We will also exploit recently developed in vitro chromatin assembly systems to evaluate the influence of RSV LTR enhancer factors on chromatin organization and transcription in vitro. Finally, we will attempt to obtain cDNA clones for EFI and EFII so that we can establish their primary structure and utilize site-specific mutagenesis to establish structural and functional relationships. Acquiring enhancer factor cDNA clones will also enable these proteins to be overexpressed in bacteria, potentially providing large quantities of material for biochemical and biophysical studies of an important class of regulatory proteins that would otherwise be unfeasible.

The long term objective of our research is to understand the molecular mechanisms by which growth promoting signals at the cell surface lead to alterations in gene expression in the nucleus. We are focusing on the transcriptional regulatory sequences located in the long terminal repeat (LTR) of the avian retrovirus, Rous sarcoma virus (RSV). These sequences contain a strong enhancer and promotor that are constitutively active in many cell types; however, the transcriptional activity of the RSV LTR is rapidly induced 3 to 5-fold when quiescent cells re exposed to serum or individual growth factors such as EGF. We wish to establish the molecular basis for the rapid responsiveness of the RSV LTR to mitogenic stimulation. Two CArG boxes are present within the 200 bp LTR enhancer and promotor. This sequence motif has been shown to be a pivotal cis acting element mediating the rapid transcriptional induction of a family of "early response" genes, including cfos, Egr1, Egr2 and cytoskeletal actin. Multiple nuclear proteins have been shown to interact with the CArG box upstream of the cfos promotor, including the well characterized SRF. However, the molecular details by which the CArG box responds to mitogenic stimulation are not known. We have found that the promotor-proximal CArG box (called EFIIIa) in the RSV LTR does not respond rapidly to EGF or other mitogens, despite the fact that it binds the SRF with high affinity. We have identified a new nuclear protein, EFIV, which binds with high affinity to cfos SRE but not EFIIIa DNA. A high affinity binding site for EFIV is also present adjacent to the second CArG box (EFIIb) in the LTR enhancer. We plan to determine if the second LTR CArG box, in conjunction with the EFIV binding site, mediates the rapid transcriptional response of the LTR to mitogenic stimulation. The involvement of other LTR sequences will also be ascertained. We plan to examine the possible role of EFIV in the rapid activation of both the LTR and cfos SRE. This will be in conjunction with our efforts to characterize the molecular mechanisms responsible for the rapid induction by EGF and other mitogens of specific LTR cis elements and their cognate transcription factors. Once the transcription factor(s) which are nuclear target(s) for mitogenic signaling pathways have been identified, we will analyze the immediate post receptor events required to elicit the activation of these specific transcription factor(s) upon exposure of cells to polypeptide mitogens such as EGF. Transcription of early response genes is one of the earliest known nuclear responses to growth factor treatment, and it is thought that many of the products of early response genes regulate subsequent transcriptional events which are necessary to commit cells to traverse the cell cycle and divide. Consequently, elucidating the mechanisms responsible for the transcriptional induction of early response genes is critical to understanding the pathways regulating proliferation in normal cells and the defects in this regulatory network which result in neoplastic transformation.

The primary aim of our research is to define the molecular mechanisms by which the Rous sarcoma virus (RSV) long terminal repeat (LTR) enhancer activates transcription in a wide variety of eukaryotic cells. We have chosen to study the RSV LTR because this retrovirus appears to have been extremely adept at coopting cellular transcriptional machinery to promote its own expression. The enhancer and promotor elements located in the U3 region of the RSV LTR are among the strongest transactivating sequences identified in eukaryotic cells. Their potency and widespread function in many different cell types suggests that the transcription factors mediating LTR enhancer/promotor function will be essential proteins that normally play critical roles in regulating cellular gene expression. Consequently, understanding LTR enhancer function may provide valuable insights into the complex transcriptional regulatory network governing cell growth and/or differentiation that when altered, leads to neoplastic transformation. The molecular picture of the RSV LTR enhancer that has emerged from our studies over the past four years certainly supports this prediction. The major players mediating LTR enhancer function are a ubiquitously expressed, multi-component CCAAT-binding complex (CBC), the serum response factor (SRF), one or more members of the C/EBP family of transcription factors, and Myc, most likely with its partner Max. We propose that the LTR enhancer can be viewed as a bipartite structure. Transactivation is mediated by the distal one/third of the enhancer which binds C/EBP-related factors or Myc/Max, depending upon the relative balance of these transcription factors in the host cell. The CCAAT- binding complex and SRF, binding to the proximal two/thirds of the enhancer, create a specific protein-DNA architecture which conducive to high level enhancer function because it brings the more distally bound transactivators and basal transcription initiation machinery into close proximity. Research designed to test the validity of this view will be focused on (1) defining the protein composition of the multi-component CBC through reconstitution with recombinant proteins, (2) characterizing the RNA and DNA binding properties of a putative member of this complex, EFI/A, as well as pursuing state-of-the-art biophysical techniques to analyze the protein-DNA structure created when two CBCs and SRF bind to the proximal segment of the LTR enhancer in vitro, (3) positive identification of the C/EBP-related EFII factors via microprotein sequencing, including confirmation of preliminary data suggesting EFIIa is LIP and (4) further analysis of Myc and Max binding to the EFII cis element in vitro and potentially, in vivo. Information gained in these areas will provide a better understanding of how these various factors work together to achieve very high levels of transcriptional activation mediated by the LTR enhancer. At the same time, valuable information on the function of several critical regulators of cellular gene expression can be expected.

DESCRIPTION (provided by applicant): The basic leucine zipper transcription factor, C/EBPbeta, is critical for growth and differentiation of the mammary gland. Epithelial cell proliferation in early pregnancy and differentiation at late pregnancy are severely impaired in C/EBPbeta null mice, which fail to lactate. Alternative translation of the intronless C/EBPbeta gene produces three different protein isoforms. C/EBPbeta-1 and -2 are transactivators, whereas C/EBPbeta-3 lacks a transactivation domain and inhibits transcription. Although differences between C/EBPbeta-1 and -2 have received little attention, our studies show that they are functionally distinct. Ectopic C/EBPbeta-2 expression transforms mammary epithelial cells (MECs) in vitro. The cells become anchorage independent, undergo an epithelial to mesenchymal transition (EMT), and gain invasive growth characteristics. In contrast, C/EBPbeta-1 expression blocks the invasive growth of metastatic breast cancer cell lines in culture. In this application we propose to examine the mechanistic basis underlying the functional dichotomy in C/EBPbeta-1 vs. -2 expression. These two transactivators differ by only 21 N-terminal amino acids present in C/EBPbeta-1, but absent from C/EBPbeta-2. We hypothesize that C/EBPbeta-1 and -2 undergo isoform-specific posttranslational modifications essential for their different functions. In support of this hypothesis, we have recently shown that C/EBPbeta-1, but not C/EBPbeta-2, is conjugated to the small ubiquitin-like modifier proteins, SUMO2 and SUMO3. We will determine whether SUMO-conjugation is necessary for the inhibition of invasive growth by C/EBPbeta-1. We will also investigate whether SUMO2,3 modification is required for protein-protein interactions with the Swi/Snf chromatin remodeling complex and/or affects the localization of C/EBPbeta-1 is subnuclear speckles. In contrast, C/EBPbeta-2 is targeted by multiple protein kinases, some of which are downstream of Ras activation. Indeed, C/EBPbeta has been shown to be an essential target of oncogenic Ras signaling in skin tumorigenesis and NIH 3T3 transformation. We will examine the requirement for C/EBPbeta-2 phosphorylation by ERKI/2 and p90Rsk-2 in mammary epithelial cell transformation. In human breast cancer, Ras-dependent signaling pathways are often activated by alterations in receptor tyrosine kinases. Therefore, we plan to determine whether receptor tyrosine kinase activation will synergize with C/EBPbeta-2 in transforming mammary epithelial cells. Because C/EBPbeta-2 promotes, whereas C/EBPbeta-1 inhibits, the invasive growth of MECs, misregulated expression of these two transactivator isoforms could contribute to growth and metastasis in breast cancer. Interestingly, C/EBPbeta-2 is undetectable in normal human breast tissue (obtained from reduction mammoplasty) where only C/EBPbeta-1 is expressed. However, 60% of primary breast tumors examined showed a high level of C/EBPbeta-2 expression, and moderate expression was detected in another 30% of the samples. Moreover, all breast cancer cell lines in culture express C/EBPbeta-2, but none express C/EBPbeta-1. We have recently generated mice carrying an MMTV-driven C/EBPbeta-2 transgene, and significantly, virgin females exhibit precocious, hyperplastic mammary gland development. We will continue to study these animals to determine if females with hyperplasia go on to develop neoplasia and/or have accelerated development of metastatic carcinoma when crossed with other mouse models of breast cancer. Once cancer cells have metastasized, breast cancers are largely incurable even with state-of-the-art approaches. These studies will provide important insights into how misregulated C/EBPbeta isoform expression may contribute to the development of metastatic mammary carcinoma.

DESCRIPTION (provided by applicant): The goal of this IMSD Proposal is to increase the number of students in graduate training who contribute to diversity in the biomedical research endeavor and promote their successful completion of the PhD degree. Our focus is upon students who have very strong letters of recommendation in terms of their research talents, but who because of weaker scores (GPA, GRE) might not gain admission into a strong biomedical graduate program. We describe a broad recruiting approach and a rigorous admission strategy. The students are extensively mentored throughout their time in the program to enable them to reach their full potential in a relatively short time period. They take an extensive and thorough didactic course in their first summer, along with a lab course designed to expose them to cutting edge technologies. This has the advantage of making these students highly competitive once they begin their rotations. The students are mentored very closely. This includes meetings with the program faculty for tutoring and advising on a weekly basis. In addition they participate fully in all of our mentoring activities for all first year biomedical graduate students. This includes a weekly IMPACT session wherein small groups of students meet with a faculty member to discuss any and all issues pertaining to graduate education, as well as a weekly FOCUS session wherein a small group of students meet with a faculty member to analyze and discuss a recent paper from the literature. Here the goal is to become entirely comfortable with gaining information from the literature rather than from a textbook. To enhance oral presentation as well as critical thinking skills, students participate in the IMSD journal club each spring. Finally, and in the long run most important, the IMSD students rotate through faculty laboratories as they choose the lab for their thesis research. We provide a performance-based program so that students exit the IMSD program after a one or two year period, depending on individual progress. So far all of our students are in good standing and progressing well, with one exception as a consequence of a serious illness.

Project Summary This is a new application for a short term (summer, 10 week) training program in cardiovascular science for under-represented, undergraduate scholars. We propose a unique training experience that builds upon existing collaboration with a core group of Historically Black Colleges and Universities (HBCUs) and the American Heart Association/American Stroke Association (AHA/ASA) that seeks to create a new paradigm promoting the pathway to health science careers and aspirational life choices for a cadre of under-represented undergraduate scholars. The proposed program provides an intellectually, socially and culturally rewarding experience to engage scholars at multiple levels. We leverage the expertise Vanderbilt University to cultivate academic knowledge and research skills and AHA/ASA's public health leadership position to provide an integrated training program against a social determinants of health backdrop. We will provide a 10 week research intensive immersion for a cadre of 10 students, expanding to 15 in years 4 and 5. We have a pool of distinguished mentors with diverse interests related to cardiovascular science and disease. The leadership team is composed of senior individuals with overlapping expertise in cardiovascular disease, mentoring, and community engagement. In addition to an intense, mentored research experience in the area of cardiovascular science, we will offer unique perspectives and resources, provide a productive framework to fully understand health inequities, and foster invaluable connections among scholars and their mentors and communities. We posit that students on the path to professions in science, medicine, healthcare or public policy must also be trained on real-life community engagement and cultural competency. They must be aware and knowledgeable of the forces shaping the health disparities so prevalent in the nation, in addition to the social determinants of health that determine the nature and severity of those disparities. Our overall goal is ambitious and innovative; we seek to create a cadre of students that will ultimately become the integrators needed to connect science and systems of health care with underrepresented communities through their academic and professional credentials, ability to build and retain trust, and cultural competence skills. We will fulfill this vision through: an intensive academic research and training immersion experience, with mentoring as a central component; regular opportunities to interact with their peers and acquire the core values of collaboration, teamwork and personal enrichment; and exposure to AHA/ASA initiatives targeted to address health inequities through public awareness, education, policy and social change. Together, we will thus provide an exciting and comprehensive experience to minority scholars poised to make career-defining, life-changing decisions with future implications for the biological, behavioral, and social determinants of health across the nation.

The overall goal of the Fisk-Vanderbilt R25 Bridge to the Biomedical PhD Program (R25-BMP) is to provide research experiences, coursework and professional development training to assure at least 70% of trainees transition to PhD granting programs at Vanderbilt or other institutions nationally, and more than 80% of those who transition to the PhD complete the degree. This proposal adds a number of innovative strategies to the original R25 programming, based on lessons learned, including: 1) direct linkage of the Biomedical Track in the Fisk Master?s phase to Vanderbilt?s Interdisciplinary Graduate Program, specifically the embedded Vanderbilt Initiative for Maximizing Student Development (IMSD) Program, linked to 16 possible bio-medically relevant PhD-granting programs; 2) embedding incoming Master?s trainees in IMSD student led data/journal clubs the Fall of their first year; 3) participation in the Bio-regulation course, required of all VU biomedical PhD programs, during year two of the Master?s for PhD-level credit for students who earn a B or higher; 4) National Networking Liaisons at research focused institutions where our Biomedical Bridge trainees have transitioned previously and/or whose research areas align with the Master?s phase research, and 5) IMSD peer mentors connected to Master?s phase trainees from the start of their first year.

The Initiative for Maximizing Student Development at Vanderbilt University (V-IMSD) seeks to continue advancing diversity and inclusion in our PhD graduate programs. V-IMSD is firmly grounded in a successful 15 year history of the Vanderbilt IMSD in promoting diversity, so that we have become a top producer of PhDs awarded to historically underrepresented students. We know that diversity in all its forms, including a diversity of individuals, thoughts, and approaches, will be essential to adequately prepare the next generation of scientific investigators and realize the potential of biomedical research. And yet we also know that the benefits of diversity are only fully realized through inclusion. Thus, V-IMSD will serve as an essential catalyst for creating a more inclusive training environment. Reflecting the inextricable link between diversity and inclusion, the purpose of this training program is to ensure that students from diverse backgrounds and identities not only acquire the knowledge and skills to pursue successful careers in biomedical research and related areas, but do so in an environment that provides a sense of belonging, where they are comfortable bringing their authentic self to the training experience and empowered to reach their full potential. We plan to continue the IMSD programming that has led to substantial gains in diversity. Our current programming covers five key competencies: research excellence, oral and written communication skills, leadership and management, teaching skills, and responsible conduct of research. Going forward, V-IMSD will expand programming to cover additional competencies such as study design and reproducibility, and quantitative skills. Students will matriculate into V-IMSD as first and second year students, staying associated with the program until degree completion. This enables the development of a community of scholars modeled on Wegner's ?community of practice.? Through (1) joint enterprises, such as Data Club, Journal Club and Writing group, (2) mutual engagement, in the form of networking, peer mentoring and social/recreational events, and (3) shared accomplishments via the annual town hall, newsletter, and social media tools, we build a community that offers the mentoring and psychosocial support needed for the success of diverse students. While working to sustain the level of diversity we have achieved in our biomedical PhD programs, we plan to take the training experience to the next level with a focus on improving the inclusivity of our training environment, providing mentee education, or ?mentoring up? to help mentees engage with agency in their mentoring relationships, expanding quantitative and computational training, especially for trainees who are not comfortable with these skill sets, leveraging our career development resources in a more UR-centric way, and developing strategic partnerships with the discipline-specific T32 training programs to ensure all students benefit from V-IMSD programming and our leadership efforts on inclusion, particularly focusing on mentorship that is effective and intentionally responsive to different identities.