Regulation of Salivary Glands-Specific Cystatin S

唾液腺特异性胱抑素 S 的调节

• 批准号: 6823909
• 负责人: PHYLLIS Ann SHAW
• 金额: $ 37.29万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-05-01 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6823909
• 关键词: animal genetic material tag; cell differentiation; cell growth regulation; developmental genetics; gene expression; gene induction /repression; genetic promoter element; genetically modified animals; glycoproteins; in situ hybridization; laboratory rat; nucleic acid sequence; protease inhibitor; protein structure function; saliva; salivary glands; secretory protein; site directed mutagenesis; tissue /cell culture; transcription factor

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.

产品说明：我们的长期目标是了解在唾液腺发育过程中，使用半胱氨酸蛋白酶抑制剂S基因作为范例的组织特异性转录调控。我们以前表明，这个基因是独特的，因为它是细胞类型和唾液腺特异性的，是由激素和自主神经系统调节，并在出生后发育的特定阶段表达。此外，半胱氨酸蛋白酶抑制剂S基因的表达，虽然关闭在成年大鼠，可以诱导b-受体介导的机制。半胱氨酸蛋白酶抑制剂S基因的系统发育“足迹”揭示了至少3种类型的DNA结合“模块”位于1.9 kb的5 '-侧翼序列中，包括激素模块（图23）。在激素模块中，已经鉴定了三个推定的“结构域”，它们存在于所有已知的唾液腺特异性基因的5 '侧翼序列中。在半胱氨酸蛋白酶抑制剂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。此外，一种新的GR/PR（糖皮质激素/孕酮，Cys GRE）反应元件，其内含有Hmx 3（同源框）结合位点和雌激素反应元件（ERE）位于元件I和II之间。半胱氨酸蛋白酶抑制剂S基因的启动子区还含有潜在的CREB/AP-1结合位点和另外两个潜在的糖皮质激素反应元件（GRD）。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节，我们产生了两个转基因小鼠模型。一种表达胱抑素S基因的1.9 kb的5 ′侧翼区，位于绿色荧光蛋白（GFP）报告基因的上游，并含有所有的转录因子结合“模块“，结构域I、II和III，以及GRES、Hmx 3结合位点、CRE/AP-1和ERE以及其它顺式作用元件，具有1 kb的5 ′-侧翼区的第二表达转基因含有3个模块，包括“激素模块”（结构域I、II和III，GRE、Hmx 3结合位点和ERE）和其它顺式作用元件。结合细胞培养的研究，这两种模型和我们提出的额外的转基因小鼠，将增加我们对我们提出的额外的转基因小鼠的顺式作用的理解，将增加我们对顺式作用元件的理解，以及决定唾液腺特异性和发育调控的半胱氨酸蛋白酶抑制剂S基因表达的转录因子。我们的两个具体目标是：1）确定cis-acting DNA元件，这是重要的组织特异性表达和发育调控的胱抑素S基因，和2）确定和表征反式作用因子调节组织特异性表达和发育调控的胱抑素S基因。