REGULATION OF MIS TYPE II RECEPTOR AND TARGET GENES

MIS II 型受体和靶基因的调控

• 批准号: 2896740
• 负责人: JOSE M. TEIXEIRA
• 金额: $ 11.97万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-04-01 至 2003-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2896740
• 关键词: DNA footprinting; Leydig cells; Mullerian duct inhibiting substance; Sertoli cells; affinity chromatography; apoptosis; biological signal transduction; cell growth regulation; cell line; complementary DNA; cyclins; embryo /fetus cell /tissue; embryogenesis; gel mobility shift assay; gene expression; genetic promoter element; genetic regulatory element; granulosa cell; hormone receptor; hormone regulation /control mechanism; laboratory rat; receptor expression; subtraction hybridization; transcription factor; transforming growth factors

Mullerian Inhibiting Substance, a member of the TGF-beta superfamily of growth and differentiation factors, is produced by Sertoli cells during embryonal development and is required for normal reproductive development in male embryos. The signal activity of MIS is the regression of the Mullerian duct, the precursor of the uterus, fallopian tubes and upper vagina. MIS is also produced both in the adult testis by Sertoli cells and in the ovary by granulosa cells, where its exact role remains to be fully explored. Based on in vitro and in vivo evidence, it is our hypothesis that signal transduction by MIS, via its heteromeric serine/threonine kinase receptor, is required to maintain reproductive competence of the gonad and to prevent hyperplastic growth. The goal of this proposal is to (I) understand the developmental, cell- specific and sexually dimorphic molecular mechanisms regulating the expression of the MIS type II receptor (MISrII) in Leydig cells, Sertoli cells, and granulosa cells during different stages of the development and also in the mesenchymal cells surrounding the Mullerian duct during embryonal development and (II) to uncover target genes whose expression is regulated by MIS signal transduction. Since we have cloned the MISrII gene with its TATA-less promoter and have identified cell lines expressing endogenous MISrII, we now have the tools to study expression of MISrII and its downstream target genes. To analyze the MISrII promoter, we will express chimeric promoter/ reporter constructs in MIS type II receptor expressing cells, then perform DNase I footprinting and gel shift analysis, with nuclear extracts prepared from those cells, to determine the cis-acting DNA elements necessary and sufficient for transcription and to examine the role of TFII-I in assembly of the pre-initiation complex. Sequences identified will be used for oligoaffinity purification of trans-acting factors which bind to those sequences. We will also immortalize the Mullerian duct mesenchymal cells which undergo apoptosis and regression in response to MIS to provide cell lines in which to identify downstream, transcriptionally regulated target genes of MIS participating in this important process. We will approach this by studying candidate genes that we might expect to be regulated. We will also perform subtractive hybridization with both R2C cells which respond to MIS, express the receptor, and from which we recently constructed a cDNA library. Information uncovered by these studies will contribute to our understanding of how MIS initiates apoptosis and causes G1 arrest and that these molecular mechanisms can be harnessed for the control of tumors known to respond to MIS, such as human ovarian cancer.

米勒抑制物质，转化生长因子-β超家族成员 生长和分化因子是由支持细胞在 胚胎发育，是正常生殖所必需的 男性胚胎的发育。管理信息系统的信号活动是 子宫的前驱--输卵管的苗勒管退行性变 输卵管和上阴道。MI也是在成人睾丸中产生的 在支持细胞和卵巢颗粒细胞中，它的确切位置 这一作用仍有待充分挖掘。基于体外和体内 证据，这是我们的假设，由管理信息系统的信号转导，通过其 异构体丝氨酸/苏氨酸激酶受体，需要维持 提高性腺的生殖能力，防止增生性生长。 这项建议的目标是(I)了解发育中的细胞- 特定的和性别二态的分子机制调节 MISrII受体在支持间质细胞中的表达 细胞和颗粒细胞在发育的不同阶段 在米勒管周围的间质细胞中也是如此。 胚胎发育和(Ii)发现其表达的靶基因 是由信息系统信号转导调节的。 由于我们已经克隆了MISrII基因及其非TATA启动子，并且 已经鉴定出表达内源性MISrII的细胞系，我们现在有了 研究MISrII及其下游靶基因表达的工具。 为了分析MISrII启动子，我们将表达嵌合启动子/ 报告构建在MISII型受体表达细胞中，然后 进行DNase I足迹和凝胶位移分析，与核 从这些细胞中提取，以确定顺式作用的DNA 转录所必需的和充分的要素，并检查 TFII-I在预引发复合体组装中的作用。数列 鉴定出将用于反式作用的寡亲和纯化 与这些序列相结合的因子。我们也将不朽的 苗勒管间充质细胞的凋亡和退化 响应于管理信息系统提供在其中识别 管理信息系统下游转录调控的靶基因 参与这一重要进程。我们将通过以下方式实现这一点 研究我们可能预期会受到调控的候选基因。我们会 也可以与两个R2C细胞进行消减杂交， 来表达受体，我们最近从它构建了一个 C DNA文库。这些研究发现的信息将有助于 对于我们对MIS如何启动细胞凋亡和导致G1期停滞的理解 并且这些分子机制可以被用来控制 已知对管理信息系统有反应的肿瘤，如人卵巢癌。