MOLECULAR ANALYSIS OF TRANSCRIPTIONAL REPRESSION BY TGIF

描述（由申请人提供）：TGF 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、dorsalization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在内的一般辅抑制因子复合物。TGIF与TGF β激活的Smads相互作用，响应TGF β信号抑制被TGF β激活的基因的表达。此外，TGF募集一种泛素连接酶（Tiul1），导致Smad2的泛素化和降解，Smad2是TGF β信号传导的关键介质。总之，TGF的这些功能决定了细胞对TGF β的最大转录反应。TGIF还通过特定的视黄酮X受体（RXR）依赖的视黄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基因的突变导致全前脑畸形，这是颅面发育的一种严重缺陷，其主要缺陷是腹侧前脑发育失败。TGF β和视黄酸信号通路都调节前脑发育，两者都与HPE有关。目前尚不清楚TGIF突变是否通过破坏TGF β信号或类视黄醛信号导致HPE。我们将验证TGIF是一种RXR α特异性协同抑制因子的假设，它在没有配体的情况下抑制RXR α依赖基因的表达。除了维甲酸受体外，RXR α是几种核受体的伴侣，因此RXR功能的特异性抑制剂将调节许多核受体途径。我们将确定哪些RXR α依赖的核受体反应被TGIF抑制，以及辅抑制子募集和核受体泛素化在这种抑制中的作用。我们将通过Tiul1与PPAR γ的特异性相互作用来确定TGIF和Tiul1是否优先靶向RXR-PPAR γ复合物。最后，我们将测试TGIF是通过阻断TGF β介导的生长抑制，还是通过其他与TGF β无关的手段来调节细胞周期进程。在许多细胞类型中，包括上皮细胞和淋巴细胞，TGF - β信号阻滞细胞周期，导致TGF - β反应丧失的突变导致人类癌症。TGIF在食管肿瘤中扩增，对TGF β介导的生长抑制更有抵抗力，提示TGIF在肿瘤发生中的作用。

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.