MOLECULAR ANALYSIS OF TRANSCRIPTIONAL REPRESSION BY TGIF

描述（由申请人提供）：由TGF β家族信号传导激活的细胞应答是许多发育和增殖事件的基础，包括哺乳动物细胞中的中胚层诱导、背侧化和抗增殖应答。TGIF是一种转录抑制因子，它募集一种包括mSin 3和CtBP在内的一般辅抑制因子的复合物。TGIF与TGF β激活的Smads相互作用，并响应于TGF β信号传导而抑制被TGF β激活的基因的表达。此外，TGIF招募泛素连接酶（Tiul 1），导致Smad 2（TGF β信号传导的关键介质）的泛素化和降解。TGIF的这些功能共同作用，以设定细胞对TGF β的最大转录应答。TGIF还通过特异性类维生素A X受体（RXR）依赖性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基因突变导致前脑无裂畸形，这是一种严重的颅面发育缺陷，其中主要缺陷是腹侧前脑发育失败。已知TGF β和视黄酸信号传导都调节前脑发育，并且这两种途径都与HPE有关。目前尚不清楚TGIF突变是否通过破坏TGF β信号或类维生素A信号而导致HPE。我们将检验TGIF是一种RXR α特异性辅阻遏物的假设，它在配体缺乏的情况下抑制RXR α依赖性基因的表达。RXR α是除视黄酸受体之外的几种核受体的伴侣，使得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依赖性核受体反应被TGIF抑制，以及辅阻遏物募集和核受体泛素化在这种抑制中的作用。我们将确定TGIF和Tiul 1是否通过Tiul 1与PPAR γ的特异性相互作用优先靶向RXR-PPAR γ复合物。最后，我们将测试TGIF是否通过阻断TGF β介导的生长抑制或通过其他TGF β非依赖性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信号传导阻滞细胞周期，导致TGF β反应丧失的突变有助于人类癌症。TGIF在食管肿瘤中扩增，其对TGF β介导的生长抑制更具抗性，表明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.