BCL-6 is a DNA binding protein normally expressed in mature B cells. The ultimate object of the proposed research is to understand at the molecular level how BCL-6 functions in normal cells and in the formation of lymphomas. All diffuse large cell lymphomas contain in the BCL-6 gene, translocations or point mutations, or both, which are likely to cause uncontrolled BCL-6 expression. This is extremely strong circumstantial evidence that BCL-6 plays a central role in the molecular pathogenesis of diffuse large cell lymphoma. To understand the molecular mechanisms of BCL-6 action it is critical both to identify the genes that BCL-6 regulates and to identify and study BCL-6 interacting proteins. In this proposal the applicant focuses on these key unexplored areas of the biology of BCL-6. Once she understands how BCL-6 functions normally, she can begin to unravel the role of BCL-6 in cancer. The applicant proposes two sets of experiments. In the first, she will identify genes that are directly regulated by BCL-6. This will involve generating cell lines expressing functionally inducible BCL-6 fusion proteins. She has shown that fusion of BCL-6 to a modified estrogen receptor hormone binding domain renders the chimeric protein inducible in the presence of a synthetic antiestrogen. She will generate stable cell lines expressing the BCL-6 fusion protein and use the representational difference analysis (RDA) method to screen for RNAs that are differentially expressed in these cell lines as a result of increased BCL-6 activity. She will initially restrict further experimentation to known genes or genes with homology to known genes, as well as genes that show strong BCL-6 regulation. She will then address the role of BCL-6 in cancer by examining whether these targets of BCL-6 are aberrantly regulated in transformed cells that contain BCL-6 translocations. Her second focus will be to identify proteins that interact with BCL-6, using a yeast 'two-hybrid' genetic screen, and then to analyze whether they are important for BCL-6 function. BCL-6 contains a protein-protein interaction motif called the POZ domain. She is particularly interested in identifying potential BCL-6 partner proteins that heterodimerize via the POZ domain. Together, the proposed studies will lead to a much better understanding of how BCL-6 functions in normal cells and ultimately may lead to an explanation of the molecular pathology of BCL-6 in lymphoma.
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