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和其他核激素受体包括DNA结合转录因子的超家族。但是，他们还需要各种转录共激活因子以配体依赖性的方式激活特定靶基因对细胞生长，稳态和分化重要的特定靶基因。这些转录共激活因子通常以多蛋白质复合物而存在。它们可以通过染色质的重塑和组蛋白修饰，在启动子结合的核受体募集后，或在涉及随后的预上络合物形成或功能的步骤中起作用（转录和伸长率）。作用于染色质水平的转录共激活剂包括依赖ATP的染色质重塑络合物以及包含（或与）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，介体共激活剂复合物更直接地起作用，以促进RNA聚合酶II的启动子募集和功能和认知一般转录因子。首先被鉴定为酵母中定义的复合物，介体在进化上是保守的，并包含大约30个亚基。据信它可以将转录激活剂与RNA聚合酶II转录机械连接起来，并且对于大多数（但不一定都是RNA聚合酶II转录）似乎是必不可少的。首先，通过纯度纯化表位式标记的甲状腺激素受体 - α（TRALPHA），从tralpha配体的存在下与其他更复杂的复合物相似或相同的复合物，首先分离出哺乳动物介体/甲状腺激素受体相关蛋白（TRAP）复合物，首先通过亲和力纯化表erpage-coft（包括Smcc）的形式（包括Smcc），从NAT，小鼠介体，弧，CRSP，滴水和人类介体配合物。这些密切相关的哺乳动物介体复合物已通过不同的亚基与包括核受体，SP1，SREBP，NF-KB，NF-KB，P53，VP16和E1A的不同转录激活剂相互作用。 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,以及雌激素受体α和β。也已证明了完整的中介复合物与Tralpha的Med1/Trap220 LXXLL依赖性相互作用。这些结果表明，介质复合物在核受体功能中起着广泛的作用。小鼠MED1/TRAP220在酵母2杂交筛选中独立分离为Ppargamma相互作用蛋白，并显示出与ppargamma相互作用的相互作用。 MED1/TRAP220适度地增加了PPARGAMMA响应报告基因的转录活性和Med1/Trap220的片段，跨越了两个LXXLL模式，表明Med1/Trap220是Ppargamma的共激活器。 我们先前表明，介体复合物的MED1/TRAP220亚基对于MEF中的ppargamma刺激的脂肪形成和脂肪形成标记的表达至关重要，但对于Myod刺激的肌发生不是必不可少的。这提供了一个通过不同的介体亚基调节细胞特异性转录和分化事件的示例。进一步的生化分析表明（i）表明，ppargamma以配体依赖性方式直接与纯化的介体复合物相互作用，（ii），（ii），介体在DNA模板上直接用作ppargamma的转录共激活剂，该DNA模板包含三个副本，该模板包含三个副本，该模板包含三个副本的DR1 PPARGAMMA识别位点，高度纯化了米中的ppargammified 20（II均具有纯正的替补）。作为体外介体复合物和ppargamma之间相互作用的必不可少的桥梁。这些数据提出了一种潜在的机制，该机制可能解释了Med1/Trap220 - / - MEF无法经历Ppargamma刺激的脂肪形成。然而，Med1/Trap220和相关的介体复合物在pPargamma刺激的体内脂肪形成中的作用和相关的介体复合物以及Med1/Trap220和介体调节ppargamma转录活性的机制的确切分子机制。 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未分化的MEF不需要MED1/TRAP220。脂肪形成中MED1/TRAP220功能所需的最小区域映射到进化保守的530个氨基酸N末端区域，该区域将Med1/Trap220介导在介体中心中。因此，我们的数据表明存在一种替代机制，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和相关的介体复合物调节已知的PPARGAMMA靶基因的表达，以及需要AS需要Med1/Trap220的未识别基因以在脂肪生成中表达的可能性。