Regulation of PPARgamma and Adipogenesis by Mediator and MED1/TRAP220

PPARgamma和其他核激素受体包括一个DNA结合转录因子的超家族。然而，它们也需要各种转录共激活因子以配体依赖的方式激活对细胞生长、稳态和分化重要的特定靶基因的转录。这些转录辅激活因子通常以多蛋白复合物的形式存在。它们可能通过染色质重塑和组蛋白修饰起作用，在启动子结合的核受体招募后，或在随后的起始前复合物形成或功能（转录起始和延伸）的步骤中起作用。在染色质水平上起作用的转录共激活因子包括atp依赖性染色质重塑复合物和含有（或与之相互作用）组蛋白乙酰转移酶和甲基转移酶的因子。

PPARgamma and other nuclear hormone receptors comprise a superfamily of DNA binding transcription factors. However, they also require various transcriptional coactivators to activate, in a ligand-dependent manner, transcription of specific target genes important for cell growth, homeostasis and differentiation. These transcription coactivators often exist as multi-protein complexes. They may act either through chromatin remodeling and histone modification, after recruitment by promoter-bound nuclear receptors, or at steps involving subsequent preinitiation complex formation or function (transcription initiation and elongation). Transcription coactivators that act at the level of chromatin include ATP-dependent chromatin remodeling complexes and factors that contain (or interact with) histone acetyl transferases and methyltransferases. The Mediator coactivator complex, in contrast to chromatin modifying factors, acts more directly to facilitate promoter recruitment and function of RNA polymerase II and cognate general transcription factors. First identified as a defined complex in yeast, Mediator is evolutionarily conserved and contains approximately 30 subunits. It is believed to connect transcriptional activators with the RNA polymerase II transcription machinery and appears to be essential for most, but not necessarily all, RNA polymerase II transcription. The mammalian Mediator/thyroid hormone receptor-associated protein (TRAP) complex was first isolated through affinity purification of an epitope-tagged thyroid hormone receptor-alpha (TRalpha) from HeLa cells grown in the presence of a TRalpha ligand and is similar or identical to other more recently described complexes including the SRB/MED-containing cofactor complex (SMCC), PC2, NAT, mouse mediator, ARC, CRSP, DRIP and human mediator complexes. These closely related mammalian Mediator complexes have been shown to interact, through distinct subunits, with diverse transcription activators that include nuclear receptors, Sp1, SREBP, NF-kB, p53, VP16 and E1A. The MED1/TRAP220 subunit of the Mediator shows ligand-dependent interactions, through a region containing two nuclear receptor recognition (LXXLL) motifs, with multiple nuclear hormone receptors that include TRalpha, vitamin D receptor (VDR), PPARalpha and gamma, retinoic acid receptor alpha (RARalpha), retinoid X receptor (RXR), farnesoid X receptor, and estrogen receptor alpha and beta. A MED1/TRAP220 LXXLL-dependent interaction of the intact Mediator complex with TRalpha has also been demonstrated. These results have suggested a broad role for the Mediator complex in nuclear receptor function. The mouse MED1/TRAP220 was independently isolated as a PPARgamma interacting protein in yeast 2 hybrid screens and was shown to interact, in a ligand-dependent manner, with PPARgamma. MED1/TRAP220 modestly increased the transcriptional activity on a PPARgamma-responsive reporter and a fragment of MED1/TRAP220 spanning the two LXXLL motifs acted as a dominant-negative repressor, suggesting that MED1/TRAP220 is a coactivator for PPARgamma. We previously showed that the MED1/TRAP220 subunit of the Mediator complex is essential for PPARgamma-stimulated adipogenesis and expression of adipogenesis markers in MEFs, but not for MyoD-stimulated myogenesis. This provided an example of the regulation of cell specific transcription and differentiation events through a distinct Mediator subunit. Further biochemical analyses showed (i) that PPARgamma interacts directly with the purified Mediator complex in a ligand-dependent manner, (ii) that Mediator functions directly as a transcriptional coactivator for PPARgamma on a DNA template containing three copies of the DR1 PPARgamma recognition site in an in vitro transcription system reconstituted with highly purified factors, (iii) that MED1/TRAP220 serves as an essential bridge for the interaction between Mediator complex and PPARgamma in vitro. These data suggested a potential mechanism that may account for the inability of MED1/TRAP220-/- MEFs to undergo PPARgamma-stimulated adipogenesis. However, the precise molecular mechanisms underlying the roles of MED1/TRAP220 and the associated Mediator complex in PPARgamma-stimulated adipogenesis in vivo, and the mechanism by which MED1/TRAP220 and Mediator regulate PPARgamma transcriptional activity, remains unclear. Here, structural and functional analyses of MED1/TRAP220 indicates, surprisingly, that a strong, direct interaction of PPARgamma with Mediator through the LXXLL motifs of MED1/TRAP220 is not required for PPARgamma-stimulated adipogenesis of cultured MEFs, and, further, that PPARgamma target gene expression and recruitment of Mediator to a PPARgamma response element on the aP2 promoter in undifferentiated MEFs do not require MED1/TRAP220. The minimal region required for MED1/TRAP220 function in adipogenesis is mapped to an evolutionarily conserved 530 amino acid N-terminal region that mediates incorporation of MED1/TRAP220 into the Mediator complex. Our data thus suggests the existence of an alternative mechanism, involving other potentially redundant cofactors or intermediate cofactors, by which MED1/TRAP220 and the associated Mediator complex regulate expression of known PPARgamma target genes, as well as the possibility of as yet unidentified genes that require MED1/TRAP220 for expression in adipogenesis.