Regulation of Salivary Glands-Specific Cystatin S

描述：我们的长期目标是以胱抑素S基因为范例，了解唾液腺发育过程中组织特异性转录的调控。我们之前证明了这个基因是独一无二的，因为它是细胞类型和唾液腺特有的，受激素和自主神经系统的调节，在出生后发育的特定阶段表达。此外，成年大鼠的半胱氨酸氨基转移酶S基因的表达虽然被关闭，但可以通过b受体介导的机制来诱导。半胱氨酸氨基转移酶S基因的系统发育“足迹”显示，在1.9kb的5‘-侧翼序列中，至少有3种类型的DNA结合“模块”，包括一个激素模块(图23)。在激素模块中，已经确定了在所有已知的唾液腺特异基因的5‘侧翼序列中发现的三个假定的“域”，这些基因已经被测序。在胱抑素S基因的I、II和III区中，有一个位于序列元件II和III之间的(GT)27区。此外，一个新的GR/PR(糖皮质激素/孕激素，Cys GRE)反应元件位于元件I和II之间，其中含有一个Hmx3(同源盒)结合位点和一个雌激素反应元件(ERE)。在cystatin S基因的启动子区域还包含一个潜在的CREB/AP-1结合位点和另外两个潜在的糖皮质激素反应元件(GRDS)。为了确定哪些顺式元件具有功能并负责组织特异性调控胱抑素S基因，我们建立了两个转基因小鼠模型。前者表达绿色荧光蛋白报告基因上游1.9kb的胱抑素S基因，包含所有转录因子结合模块、结构域I、II、III和GREs、Hmx3结合位点、Cre/AP-1和ERE等顺式作用元件；另一个表达的转基因基因5‘侧翼区1kb，包括激素模块(结构域I、II和III、GRE、Hmx3结合位点和ERE)和其他顺式作用元件。结合细胞培养的研究，这两个模型和我们建议建立的额外的转基因小鼠，将增加我们对顺式作用的了解，将增加我们对顺式作用元件和转录因子的理解，这些转录因子决定了胱抑素S基因的唾液腺特异性和发育调节表达。我们的两个特异目标是：1)鉴定对胱抑素S基因的组织特异性表达和发育调控起重要作用的顺式作用元件；2)鉴定和鉴定调控胱抑素S基因的组织特异性表达和发育调控的反式作用因子。

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.