Salivary Gland Gene Transcription and Organogenesis

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The goal of the proposed research is to identify salivary gland transcription factors that establish and maintain the developmental program specific for the submandibular gland. In accord with other developing organ systems, it is proposed that many transcription factors that mediate cell-specific gene expression in the mature gland will also play a regulatory role in the process of organogenesis. Therefore, the plan is to begin (Specific Aim 1) by identifying the DNA-binding transcription factors that mediate the submandibular activity of the transcriptional enhancers of the kallikrein gene family, which are products of the differentiated ductal cells of the submandibular gland. This will require defining minimal functional enhancers and then mapping nuclear protein binding sites by DNase I foot-printing in vitro and computer-based searches for known consensus sequences of transcription factor families. The relevance of the footprints and consensus binding sites will be confirmed by analyzing the effects of mutating the sites on the activity of the enhancer. Candidate DNA-binding transcription factors will be identified by screening known factors whose expression patterns suggest they may play a role in salivary gland gene expression and by identifying additional members of factor families present in the mature and developing submandibular glands by an RT-PCR protocol (Specific Aim 2). Individual submandibular factors will be matched with potential enhancer binding sites through the known consensus binding sequences of the family to which they belong. The factor identification will be verified by testing whether the nucleotide sequence requirements for factor binding match the sequence requirements for activity of the element in the enhancer and whether the factor can activate a reporter gene through the element in transfected cells. Finally, the role of candidate factors will be tested by forced expression of dominant-negative and constitutively active chimeric forms of the factors in submandibular rudiments capable of morphogenesis in culture (Specific Aim 3).