MOLECULAR ANALYSIS OF TRANSCRIPTIONAL REPRESSION BY TGIF

描述(申请人提供)：由转化生长因子β家族信号激活的细胞反应是许多发育和增殖事件的基础，包括哺乳动物细胞的中胚层诱导、背化和抗增殖反应。TGIF是一种转录抑制因子，它招募了包括mSin3和CtBP在内的一系列通用辅阻遏子。TGIF与转化生长因子β激活的Smads相互作用，作为对转化生长因子β信号的响应，抑制由转化生长因子β激活的基因的表达。此外，TGIF还招募了泛素连接酶(Tiul1)，导致转化生长因子β信号转导的关键介质Smad2的泛素化和降解。TGIF的这些功能共同作用于设定细胞对转化生长因子β的最大转录反应。TGIF还通过依赖于视黄酸X受体(RXR)的维甲酸反应元件抑制基因表达。然而，这种抑制的机制和受影响的基因范围仍有待确定。人类TGIF基因突变导致全前脑畸形，这是一种严重的颅面发育缺陷，其主要缺陷是腹侧前脑发育失败。已知转化生长因子β和维甲酸信号通路都调节前脑发育，并且这两个通路都与HPE有关。目前尚不清楚TGIF突变是通过干扰转化生长因子β信号还是通过维甲酸信号导致HPE。我们将检验这一假设，即TGIF是一种RXRα特异性辅抑制子，在缺乏配体的情况下抑制RXRα依赖的基因表达。除了维甲酸受体外，RXRα还是几种核受体的伙伴，因此RXR功能的特定抑制剂将调节许多核受体途径。我们将确定哪些依赖RXRα的核受体反应被TGIF抑制，以及辅阻遏子招募和核受体泛素化在这种抑制中的作用。我们将确定TGIF和Tiul1是否通过Tiul1与PPAR伽马的特定相互作用优先靶向RXR-PPAR伽马复合体。最后，我们将测试TGIF是否通过阻断转化生长因子β介导的生长抑制或通过其他非转化生长因子β途径来调节细胞周期进展。在许多细胞类型中，包括上皮细胞和淋巴样细胞，转化生长因子β信号阻断细胞周期，导致转化生长因子β反应丧失的突变导致人类癌症。TGIF在对转化生长因子β介导的生长抑制有较强抵抗力的食道肿瘤中扩增，提示TGIF在肿瘤发生中起一定作用。

DESCRIPTION (provided by applicant): The cellular responses activated by TGF beta family signaling underlie many developmental and proliferative events, including mesoderm induction, dorsalization and antiproliferative responses in mammalian cells. TGIF is a transcriptional represser which recruits a complex of general corepressors, including mSin3 and CtBP. TGIF interacts with TGF beta activated Smads and in response to TGF beta signaling represses expression of genes which are activated by TGF beta. Additionally, TGIF recruits a ubiquitin ligase (Tiul1), resulting in ubiquitination and degradation of Smad2, the critical mediator of TGF beta signaling. Together, these functions of TGIF act to set the maximal transcriptional response of a cell to TGF beta. TGIF also inhibits gene expression via a specific retinoid X receptor (RXR) dependent retinoic acid response element. However, the mechanism of this repression and the range of genes affected remain to be determined. Mutations in the human TGIF gene result in holoprosencephaly, a severe defect of craniofacial development, in which the primary defect is a failure of ventral forebrain development. Both TGF beta and retinoic acid signaling are known to regulate forebrain development, and both pathways are implicated in HPE. It is not known whether TGIF mutations cause HPE by disrupting TGF beta signals or retinoid signaling. We will test the hypothesis that TGIF is an RXR alpha specific corepressor that inhibits RXR alpha dependent gene expression in the absence of ligand. RXR alpha is a partner for several nuclear receptors in addition to retinoic acid receptors, such that a specific inhibitor of RXR function will regulate many nuclear receptor pathways. We will determine which RXR alpha dependent nuclear receptor responses are repressed by TGIF and the role of corepressor recruitment and nuclear receptor ubiquitination in this repression. We will determine whether TGIF and Tiul1 preferentially target RXR-PPAR gamma complexes via a specific interaction of Tiul1 with PPAR gamma. Finally, we will test whether TGIF regulates cell cycle progression by blocking TGF beta mediated growth inhibition, or by other TGF beta independent means. In many cell types, including epithelial and lymphoid cells, TGF beta signaling arrests the cell cycle, and mutations which result in loss of TGF beta responses contribute to human cancer. TGIF is amplified in esophageal tumors, which are more resistant to TGF beta mediated growth inhibition, suggesting a role for TGIF in tumorigenesis.