MOLECULAR ANALYSIS OF TRANSCRIPTIONAL REPRESSION BY TGIF

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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 (TiuH), 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 RXRfunction 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 Tiull preferentially target RXR-PPARgamma complexes via a specific interaction of Tiull 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.