Eukaryotic transcription factors regulate gene expression through specific protein-DNA interactions involving cis-acting promoter and enhancer elements.
The aim of this grant application is to investigate the biochemical basis of B-cell specific regulation of immunoglobulin gene expression. Genetic and biochemical approaches will be used to characterize a representative transcription factor -- the Oct-2 protein -- and its conserved DNA recognition element -- the octamer site -- in the immunoglobulin promoter and enhancer. The Oct-2 protein belongs to the newly recognized and highly conserved class of homeodomain proteins -- POU motif -- involved in the specification of cell fate in eukaryotic development. The Oct-2/octamer system thus provides a general model for protein-DNA and protein-protein interactions involved in the control of tissue-specific gene expression. Accordingly, biochemical rules for specificity and protein folding obtained in the proposed studies of Oct-2 are likely to have general relevance to a wide class of regulatory interactions in eukaryotic cell biology. Our objectives are organized in three parts. (1) As a baseline, the wild- type Oct-2 POU-homeodomain will be overexpressed in E. coli, purified and characterized with respect to domain organization and DNA-binding. Sequence-specificity will be evaluated by the random DNA selection method. (2) Comparative studies will be conducted of mutant POU-homeodomains constructed by site-directed mutagenesis; design of mutants will be guided by homology with other POU and homeotic proteins in order to define structure-function relationships. (3) To provide a more general genetic analysis, a prokaryotic model system in E. coli will be constructed for the selection of defined Oct-2 dependent phenotypes; i.e., a synthetic operon, based on the lac and lambda repressor-operator paradigm, will be designed to provide selectable markers regulated by the Oct-2/octamer system. The full power of bacterial genetics may thus be applied to Oct-2 for the mutational analysis of DNA recognition and protein folding. It is anticipated that this approach will be of general utility in the analysis of human transcription factors, providing a rigorous foundation for their future characterization as potential pharmacologic targets, with application in the present case to B-cell lymphomas and leukemias.
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| Botfield, M C; Jancso, A; Weiss, M A (1994) An invariant asparagine in the POU-specific homeodomain regulates the specificity of the Oct-2 POU motif. Biochemistry 33:8113-21 |
| Jancso, A; Botfield, M C; Sowers, L C et al. (1994) An altered-specificity mutation in a human POU domain demonstrates functional analogy between the POU-specific subdomain and phage lambda repressor. Proc Natl Acad Sci U S A 91:3887-91 |
| Sivaraja, M; Botfield, M C; Mueller, M et al. (1994) Solution structure of a POU-specific homeodomain: 3D-NMR studies of human B-cell transcription factor Oct-2. Biochemistry 33:9845-55 |
| Botfield, M C; Jancso, A; Weiss, M A (1992) Biochemical characterization of the Oct-2 POU domain with implications for bipartite DNA recognition. Biochemistry 31:5841-8 |
