Elizabeth Jane Taparowsky - US grants

The progression of a normal cell to a fully malignant tumor is a multistep process involving events of initiation, promotion and progression. The "activation" of a diverse of cellular genes, called oncogenes, is the molecular basis behind several of these events. Two such oncogenes, ras and myc, are activated in certain tumor cell lines and can cooperate to transform primary rodent cells in culture. We have tested whether ras and myc cooperate to cause the transformation of C3H10T1/2 cells, an established cell line of embryonic origin. Myc transfected C3H10T1/2 cells do not form transformed foci; C3H10T1/2 cells transfected with ras show a low incidence of transformation (80 foci/Mug ras). Interestingly, the frequency of focus formation can be increased 15 fold when C3H10T1/2 cells are co-transfected with ras and myc. In addition, comparisons based on morphology and on growth rates in soft agar reveal that ras-myc foci are dramatically different from ras foci. The proposed study will examine the contributions of the ras and myc oncogene products, as separate agents and in cooperation with each other, to the transformation of C3H10T1/2 cells. We will establish (1) the parameters governing ras transformation and how these parameters change when ras and myc cooperate to cause a distinctly different phenotype, (2) the role of the myc protein in the cooperation event and whether the timing of myc expression is critical to that role and (3) the structural domains of the myc protein involved in cooperation with gas and whether these overlap with the structural domains governing the nuclear localization of the myc protein, the DNA binding properties of the myc protein or the ability of the myc protein to transform avian cells. The data obtained from this investigation will form the core of future studies aimed at identifying exogenous and/or endogenous cellular factors that participate with the ras and myc oncogene products in the cascade of events leading to in vivo tumor formation.

DESCRIPTION: The functional characterization of tissue-specific transcription factors that regulate hematopoietic cell growth and lineage development will provide molecular targets for the design of strategies to manipulate the leukemic phenotype. B-ATF is a new basic leucine zipper (bZIP) protein that is expressed in a tissue-specific manner and is induced rapidly in human B cells following infection with Epstein-Barr virus (EBV) and the expression of the EBV-encoded transactivator protein, EBNA-2. B-ATF is a nuclear protein that heterodimerizes with members of the Jun family of oncoproteins to bind to AP-1 target DNA sites. In cultured cells engineered to overexpress B-ATF, Jun, and Fos, transcription of an AP-1 responsive reporter gene is repressed. This suggests that B-ATF functions as a negative regulator of AP-1, a transcription complex that normally is associated with the induction of cellular growth. Based on these observations, it remains unclear why EBNA-2, a viral latency gene product required for B cell immortalization and transformation, triggers the expression of B-ATF. To explore further the relationship between EBV and B-ATF, the cis-trans regulatory mechanisms responsible for enhanced expression of the human B-ATF gene by EBNA-2 will be investigated. To establish a functional role for B-ATF in B cells, experiments will be performed to assess the impact of B-ATF on the biochemical and biological function of the AP-1 transcription factor complex and on the activities of an additional B-ATF-interacting protein (BIP-13) that is expressed preferentially in human B cells. The requirement for spatial and temporal control of B-ATF expression in vivo will be investigated using in situ hybridization to fully characterize the B-ATF gene expression pattern in embryonic and adult mice. Transgenic technology will be used to establish if perturbation of B-ATF expression adversely impacts the development of the whole animal. The PI anticipates that these studies will elucidate the role of B-ATF in B cell growth and development and provide the information needed to assess the feasibility of using this new bZIP protein as a target in the management of B cell malignancies.

DESCRIPTION (provided by applicant): Activator Protein-1 (AP-1) consists of dimerizing basic leucine zipper transcription factors and plays an essential role in cell proliferation, cell survival and cell death. The most well characterized AP-1 family members, the Fos and Jun proteins, are nuclear targets of all major intracellular signaling pathways. As a result, in vivo approaches to control the activities of the Fos:Jun heterodimer would find clinical applications in disease states as diverse as cancer and neurodegenerative disorders. BATF and the highly related protein, JDP1, are unique among AP-1 family members in that they form high affinity dimers with Jun that show the same DNA binding preference as Fos:Jun dimers, yet display no ability to activate gene transcription. Thus, these proteins possess the properties of naturally occurring, negative regulators of AP-1 that could be exploited to control AP-1 activity in vivo. Transgenic mouse models of tissue-specific expression of wild type and variant BATF proteins will be employed to examine the biological consequences of BATF-mediated modulation of AP-1 activity in vivo. Target genes that are regulated by BATF containing AP-1 complexes will be identified using a novel approach combining chromatin immunoprecipitation with AP-1 promoter microarray screening. Mice in which BATF and JDP1 have been functionally inactivated will reveal the importance of these proteins to mammalian growth and development and will be used to profile gene expression changes associated with the absence of BATF-mediated AP-1 regulation in selected tissues. Lastly, as a first step toward identifying signaling pathways that could be exploited for strategies to manipulate the levels of BATF expression in vivo, the regulatory elements and associated transcription factors that control BATF gene expression will be characterized. Inducible BATF expression in cultured cells will be used to validate BATF target genes identified in mice and to explore the role (if unknown) of the proteins encoded by these genes in the cellular phenotypes associated with BATF expression. Our short-term goal is to fully characterize the impact of BATF on AP-1 activity in cells, with the long-term goal of using the properties of this unique AP-1 family member (or molecular strategies based on the properties of BATF) to control the aberrant activity of AP-1 that is frequently associated with human disease.

[unreadable] DESCRIPTION (provided by applicant): Intracellular signaling culminates in gene expression changes that are mediated by transcription factors such as activator protein-1 (AP-1). AP-1 is a widely expressed transcription complex consisting of dimerizing basic leucine zipper (bZIP) proteins. AP-1 functions in cell growth, differentiation, survival and apoptosis, and deregulated AP-1 activity figures prominently in human diseases from cancer to neurodegenerative disorders. Transcription factors are major targets for therapeutic intervention, and in vivo strategies designed to modulate AP-1 activity would be a significant advance in many clinical situations. BATF, and the highly related JDP1 protein, are unique AP-1 family members that dimerize with the central components of AP-1 - the Jun proteins - and inhibit AP-1 activity. The objective of this application is to define the roles of these AP-1 inhibitors in vivo and to test the hypothesis that these roles are linked to the regulation of specific AP-1 target genes. In Aim 1, genetically engineered mouse models expressing epitope-tagged BATF and JDP1 at endogenous levels will be used for defining the precise expression patterns of these proteins and for identifying genes directly bound by BATF using chromatin immunoprecipitation (ChIP) coupled with the screening of novel AP-1 promoter microarrays. BATF null, JDP1 null and double null mice will be used to assess how the loss of AP-1 inhibition mediated by these proteins affects development and to profile the full spectrum of gene expresson changes that characterize the BATF null phenotype. Aim 2 will use mass spectral analysis to identify and quantify discreet phosphorylation events that regulate the DNA binding and dimerization properties of BATF and influence its efficacy as an AP-1 inhibitor. Lastly, in Aim 3, the M1 cell system will be used to further investigate the function of BATF by following BATF-mediated regulation of target genes and by positioning BATF within the signaling network that controls the differentiation of these cells. These studies have the short-term goal of establishing the function of BATF and JDP1 in vivo and will generate the information necessary to address our long-term goals of using these AP-1 inhibitors for transcription-targeted therapies. [unreadable] [unreadable] Relevance: The aberrant cell growth that is a feature of many human diseases often is linked to deregulated gene expression mediated by proteins such as the AP-1 family of transcription factors. BATF and JDP1 have been identified as AP-1 family members that negatively regulate gene expression. The goal of this research is to fully characterize the biological activities of these proteins as a first step towards establishing their utility in AP-1 targeted therapies. [unreadable] [unreadable] [unreadable]

Cancer Center Support Grant; cell growth; NCI Center for Cancer Research; programs; Universities

PROJECT SUMMARY BATF is the founding member of the BATF family of basic leucine zipper proteins and a major contributor to the function of AP-1 transcription factor complexes in immune cells. Batf null (Batf KO) mice are viable, yet display a number of phenotypes that position the protein within pathways critical for the regulation of inflammation, autoimmunity, host protection against pathogens and the prevention of lymphoproliferative disorders. As a result, there is enormous interest in using BATF, its regulators, and its effector genes in targeted strategies to control these health concerns. However, in order to assess which strategy to pursue, it is critical to identify targetable determinants that control BATF function with an emphasis on those determinants that discriminate between BATF function in different cellular contexts. This proposal is designed to address this gap in our knowledge. What is known is that BATF forms heterodimers with the JUN AP-1 proteins and that JUN:BATF dimers regulate genes by binding classic AP-1 DNA, or by recruiting IRF transcription factors to composite DNA elements called AICE. Both target gene activation and repression are associated with BATF expression in cells, but the precise manner by which these functions are distinguished has not been fully described. Additionally, there is evidence that BATF proteins can impact gene expression in the absence of DNA binding and that two phosphorylation events alter BATF target gene regulation by impacting its DNA binding and dimerization properties. The goal of this proposal is to test the hypothesis that phosphorylation of BATF selectively modulates BATF protein function in vivo. In Aim 1, BATF proteins engineered to mimic phosphorylation events and containing additional mutations that disrupt association with IRF4 and EGR2, will be assayed for DNA binding properties and for the ability to influence transcription using both AP-1 and AICE regulated genes. In Aim 2, the behaviors characterized in Aim 1 will be tested for their function in three, different, Batf-dependent transcription programs by examining BATF variants for their ability to rescue Batf KO T cell phenotypes. Completion of these aims will address our immediate goals of defining how naturally-occurring post-translational modifications impact the molecular properties of BATF and influence their competency to direct specific T cell functions. Results from this R21 will set the stage for future work to identify the kinase/phosphatase network(s) directing these BATF modifications in vivo and to profile how the cellular transcriptome responds following BATF modification. Reversible phosphorylation is among the most successful approaches for targeted drug design. A small molecule approach aimed at manipulating BATF post-translational modifications to inhibit, or augment, BATF function in vivo, will be a valuable tool for the management of immune system disorders.