REGULATION OF MIS TYPE II RECEPTOR AND TARGET GENES

苗勒管抑制物质，TGF-β超家族的成员， 生长和分化因子，由支持细胞在 胚胎发育和正常生殖所需 男性胚胎的发育。 MIS的信号活动是 子宫输卵管前体苗勒管退化 输卵管和阴道上部 MIS也产生于成人睾丸和 支持细胞和卵巢中的颗粒细胞， 作用有待充分探讨。 基于体外和体内 证据，这是我们的假设，信号转导的MIS，通过其 异聚丝氨酸/苏氨酸激酶受体，需要维持 生殖能力的性腺和防止增生性生长。 本提案的目标是（I）了解发育、细胞- 特异性和性二态性的分子机制， MIS II型受体（MISrII）在睾丸间质细胞中的表达， 细胞和颗粒细胞在发育的不同阶段 以及苗勒管周围的间充质细胞， 胚胎发育和（II）揭示其表达的靶基因 受MIS信号转导的调节。 由于我们已经克隆了MISrII基因及其无TATA启动子， 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/ 在MIS II型受体表达细胞中构建报告基因，然后 进行DNA酶I足迹和凝胶位移分析， 从这些细胞中提取，以确定顺式作用DNA 必要和充分的转录和检查的要素 TFII-I在前起始复合物组装中的作用。 序列 鉴定的寡亲和纯化将用于反式作用的 与这些序列结合的因子。 我们还将使 经历凋亡和退化的苗勒管间充质细胞 以提供细胞系，在其中鉴定 下游转录调控的MIS靶基因 参与这一重要进程。 我们将通过以下方式处理这一问题： 研究我们可能期望被调控的候选基因。 我们将 我还与两种R2C细胞进行消减杂交， 到MIS，表达受体，我们最近构建了一个 cdna文库 这些研究所揭示的信息将有助于 我们对MIS如何启动细胞凋亡并导致G1期阻滞的理解 这些分子机制可以用来控制 已知对MIS有反应的肿瘤，如人卵巢癌。

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.