Regulation of Salivary Glands-Specific Cystatin S

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DESCRIPTION: Our long-term goal is to understand regulation of tissue specific transcription during salivary gland development using the cystatin S gene as the paradigm. We previously showed this gene to be unique in that it is cell type- and salivary gland-specific, is regulated by hormones and by the autonomic nervous system, and is expressed at a specific stage of postnatal development. Furthermore, cystatin S gene expression, though turned off in adult rats, can be induced by b-receptor mediated mechanisms. Phylogenetic "footprinting" of the cystatin S gene reveals that are at least 3 types of DNA binding "modules" located in the 1.9 kilobase (kb) 5'-flanking sequence, including a hormone module (Fig. 23). Within the hormone module, three putative "domains" have been identified that are found in the 5'-flanking sequence of all known salivary gland-specific genes that have been sequenced. In the cystatin S gene these "domains", I, II and III, include a (GT)27 region located between sequence elements II and III. In addition, a novel GR/PR (glucocorticoid/progesterone, Cys GRE) responsive element, harboring within it an Hmx3 (homeobox) binding site and an estrogen responsive element (ERE) is located between elements I and II. The promoter region of the cystatin S gene also contains a potential CREB/AP-1 binding site, and two other potential glucocorticoid responsive elements (GRDs). To determine which of the cis-elements are functional and responsible for tissue specific regulation of the cystatin S gene, we generated two transgenic mouse models. One expresses 1.9 kb of the 5'-flanking region of the cystatin S gene, upstream of the green fluorescent protein (GFP) reporter gene and contains all of the transcription factor binding "modules, "domains" I, II, and III, and GREs, Hmx3 binding site, CRE/AP-1 and ERE and other cis-acting elements, and the second expressing transgene with 1 kb of the 5'-flanking region contains 3 modules including the "hormone module" (domains I, II, and III, a GRE, Hmx3 binding site, and an ERE) and other cis-acting elements. In conjunction with studies in cell culture, these two models and the additional transgenic mice we propose to generate, will increase our understanding of the cis-acting the additional transgenic mice we propose to generate, will increase our understanding of the cis-acting elements, and transcription factors that determine the salivary gland-specific and developmentally regulated expression of the cystatin S gene. Our two Specific Aims are to:1) identify cis-acting DNA elements that are important for tissue-specific expression and developmental regulation of the cystatin S gene, and 2) identify and characterize trans-acting factors regulating tissue-specific expression and developmental regulation of the cystatin S gene.