Regulation of Salivary Glands-Specific Cystatin S

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。我们之前的研究表明，这种基因是独特的，因为它是细胞类型和唾液腺特异性的，受激素和自主神经系统的调节，并在出生后发育的特定阶段表达。此外，胱抑素S基因表达虽然在成年大鼠中被关闭，但可以通过b受体介导的机制诱导。胱抑素S基因的系统发育“足迹”显示，位于1.9千碱基（kb） 5'侧的序列中至少有3种DNA结合“模块”，包括激素模块（图23）。在激素模块中，已经确定了三个假定的“结构域”，它们位于所有已知的唾液腺特异性基因的5'-侧翼序列中。在胱抑素S基因中，这些“结构域”I、II和III包括位于序列元件II和III之间的（GT）27区域。此外，一个新的GR/PR（糖皮质激素/孕激素，Cys GRE）响应元件，其内部包含一个Hmx3（同源盒）结合位点和一个雌激素响应元件（ERE）位于元件I和II之间。胱抑素S基因的启动子区域还包含一个潜在的CREB/AP-1结合位点，以及另外两个潜在的糖皮质激素响应元件（GRDs）。为了确定哪些顺式元件是功能性的，并负责胱抑素S基因的组织特异性调控，我们建立了两个转基因小鼠模型。一个表达1.9 kb的胱抑素S基因5′侧区，位于绿色荧光蛋白（GFP）报告基因的上游，包含所有转录因子结合“模块”、“结构域”I、II和III，以及GREs、Hmx3结合位点、CRE/AP-1和ERE等顺式作用元件；第二个表达1 kb的5′侧区转基因包含3个模块，包括“激素模块”（结构域I、II和III，一个GRE、Hmx3结合位点，1个GFP、Hmx3结合位点）。和一个ERE)和其他顺式作用元件。结合细胞培养的研究，这两种模型和我们拟产生的其他转基因小鼠，将增加我们对顺式作用的其他转基因小鼠的理解，将增加我们对顺式作用元件和转录因子的理解，这些元件和转录因子决定了唾液特异性和发育调节的胱抑素S基因的表达。我们的两个具体目标是：1)鉴定对胱抑素S基因组织特异性表达和发育调控重要的顺式作用DNA元件，2)鉴定和表征调节胱抑素S基因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.