REGULATION OF MIS TYPE II RECEPTOR AND TARGET GENES

MIS II 型受体和靶基因的调控

• 批准号: 6513201
• 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 2004-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6513201
• 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; 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.

Mullerian抑制物质，TGF-Beta超家族的成员 生长和分化因子是由Sertoli细胞在 胚胎发育，是正常生殖需要的 雄性胚胎的发育。 MIS的信号活动是 穆勒风管的回归，子宫的前体，输卵管 管和上阴道。 在成人睾丸中也产生了MIS 由Sertoli细胞和在卵巢中由颗粒细胞确切的 角色仍有待探索。 基于体外和体内 证据，我们的假设是通过其通过其信号传递的信号。 需要保持杂音丝氨酸/苏氨酸激酶受体才能维持 性腺的生殖能力并防止增生生长。 该提议的目的是（i）了解发展的，细胞的发展 具体和性二态分子机制调节 Leydig细胞中MIS II型受体（MISRII）的表达 细胞和颗粒细胞在发育的不同阶段 并在穆勒管周围的间充质细胞中 胚胎发育和（ii）发现其表达的靶基因 由MIS信号转导调节。 由于我们已经将Misrii基因与无tata的启动子和 已经确定了表达内源性不良的细胞系，我们现在有 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/ 记者在MIS II型受体表达细胞中的构造，然后 使用核进行DNase I足迹和凝胶转移分析 从这些细胞中制备的提取物，以确定顺式作用DNA 必要和足够的元素进行转录，并检查 TFII-I在启动络合物组装中的作用。 序列 确定的将用于反式作用的寡聚学纯化 与这些序列结合的因素。 我们还将永生 经历凋亡和消退的穆勒式管道间充质细胞 响应MIS提供识别的单元线 MIS的下游，转录调控的靶基因 参与这个重要的过程。 我们将通过 研究我们可能期望受到调节的候选基因。 我们将 还与两个R2C细胞进行减法杂交 误以为，表达受体，我们最近从中构建了一个 cDNA库。 这些研究发现的信息将有助于 为了理解MIS如何启动凋亡并导致G1逮捕 并且可以利用这些分子机制来控制 已知会反应MIS的肿瘤，例如人类卵巢癌。