Regulation of PPARgamma and Adipogenesis by Mediator and MED1/TRAP220

介体和 MED1/TRAP220 对 PPARgamma 和脂肪生成的调节

• 批准号: 7734166
• 负责人: Kai Ge
• 金额: $ 16.5万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7734166
• 关键词: Accounting; Affinity Chromatography; Amino Acids; Binding; Biochemical; Cells; Chromatin; Chromatin Remodeling Factor; Complex; DNA; DNA Binding; DR1 gene; Data; Diabetes Mellitus; Dominant-Negative Mutation; Embryo; Epitopes; Estrogen Receptor alpha; Event; Fatty acid glycerol esters; Fibroblasts; Gene Expression; Gene Targeting; General Transcription Factors; Generations; Genes; Genetic Transcription; Hela Cells; Histones; Homeostasis; Human; Hybrids; In Vitro; Lead; Ligands; Maps; Mediating; Mediator of activation protein; Methyltransferase; Modification; Molecular; Morbidity - disease rate; Mus; N-terminal; NF-kappa B; Non-Insulin-Dependent Diabetes Mellitus; Nuclear Hormone Receptors; Nuclear Receptors; Obesity; PPAR alpha; PPAR gamma; Pathway interactions; Proprotein Convertase 2; Proteins; RNA Polymerase II; RXR; Regulation; Reporter; Reporting; Response Elements; Risk Factors; Role; Site; System; TP53 gene; TRAP Complex; Thyroid hormone receptor alpha; Tissues; Transcription Coactivator; Transcription Initiation; Transferase; Undifferentiated; VP 16; Vitamin D3 Receptor; Yeasts; cell growth; chromatin remodeling; cofactor; in vivo; lipid biosynthesis; mortality; myogenesis; novel strategies; obesity treatment; promoter; receptor; receptor function; reconstitution; retinoic acid receptor alpha; transcription factor

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.

PPARGamma和其他核激素受体组成DNA结合转录因子超家族。然而，它们也需要各种转录辅助激活因子以配体依赖的方式激活特定靶基因的转录，这些基因对细胞生长、动态平衡和分化至关重要。这些转录共激活因子通常以多蛋白复合体的形式存在。它们可能在被启动子结合的核受体募集后通过染色质重塑和组蛋白修饰发挥作用，或者在涉及随后的预起始复合体形成或功能(转录起始和延伸)的步骤中起作用。作用于染色质水平的转录共激活因子包括依赖于ATP的染色质重塑复合体以及含有(或与之相互作用)组蛋白乙酰转移酶和甲基转移酶的因子。 与染色质修饰因子相比，介体共激活物复合体更直接地促进启动子的招募和RNA聚合酶II和同源一般转录因子的功能。Mediator首先被确定为酵母中的一个已定义的复合体，它在进化上是保守的，包含大约30个亚基。它被认为连接转录激活物和RNA聚合酶II转录机制，似乎对大多数但不一定是所有的RNA聚合酶II转录是必不可少的。哺乳动物介体/甲状腺激素受体相关蛋白(TRAP)复合体最初是通过亲和纯化一个表位标记的甲状腺激素受体-α(TRpha)从HeLa细胞中分离出来的，该复合体与最近描述的其他复合体相似或相同，包括SRB/MED-含辅因子复合体(SMCC)、PC2、NAT、小鼠介体、ARC、CRSP、水滴和人类介体复合体。这些密切相关的哺乳动物介体复合体已被证明通过不同的亚基与不同的转录激活因子相互作用，包括核受体Sp1、SREBP、NF-kB、P53、VP16和E1a。 介体的MED1/TRAP220亚基通过一个包含两个核受体识别(LxxLL)基序的区域与多种核激素受体相互作用，这些核激素受体包括TRpha、维生素D受体(VDR)、PPARpha和Gamma、维甲酸受体α(RARpha)、维甲酸X受体(RXR)、法尼醇X受体以及雌激素受体α和β。还证明了完整的介体复合体与TRpha之间的相互作用依赖于MED1/TRAP220 LxxLL。这些结果表明，介体复合体在核受体功能中具有广泛的作用。在酵母2杂交筛选中，小鼠MED1/TRAP220被独立地分离为PPARGamma相互作用蛋白，并被证明以配体依赖的方式与PPARGamma相互作用。MED1/TRAP220适度增加了PPAR-Gamma反应报告基因的转录活性，而横跨两个LxxLL基序的MED1/TRAP220片段起到了显性-负抑制的作用，表明MED1/TRAP220是PPARGamma的共激活因子。 我们之前已经证明，介体复合体的MED1/TRAP220亚单位对于PPAR-γ刺激的脂肪生成和MEF中脂肪生成标志物的表达是必不可少的，但对MyoD刺激的肌肉生成不是必需的。这为通过不同的中介亚基调控细胞特异性转录和分化事件提供了一个例子。进一步的生化分析表明：(I)PPARGamma以配体依赖的方式直接与纯化的介体复合体相互作用，(Ii)在由高纯度因子重组的体外转录系统中，介体直接作为PPARGamma的转录共激活因子在含有三个拷贝的DR1 PPARGamma识别位点的DNA模板上发挥作用，(Iii)MED1/TRAP220是介体复合体与PPARGamma体外相互作用的重要桥梁。这些数据提示了MED1/TRAP220-/-MEF不能进行PPAR-γ刺激的脂肪生成的潜在机制。然而，MED1/TRAP220和相关的中介复合体在体内PPAR-γ刺激的脂肪生成中作用的确切分子机制，以及MED1/TRAP220和Mediator调控PPARGamma转录活性的机制仍不清楚。 在这里，对MED1/TRAP220的结构和功能分析表明，令人惊讶的是，PPARGamma通过MED1/TRAP220的LxxLL基序与介体的强烈、直接的相互作用不是PPARGamma刺激的培养MEF成脂所必需的，此外，在未分化的MEF中，PPARGamma的靶基因表达和介体招募到aP2启动子上的PPARGamma反应元件不需要MED1/TRAP220。MED1/TRAP220在脂肪形成中所需的最小区域被映射到进化上保守的530个氨基酸的N-末端区域，该区域介导MED1/TRAP220进入中介复合体。因此，我们的数据表明存在另一种机制，涉及其他潜在的冗余辅助因子或中间辅助因子，通过该机制，MED1/TRAP220和相关的中介复合体调节已知的PPAR伽马靶基因的表达，以及在脂肪形成过程中可能存在需要MED1/TRAP220表达的未知基因。