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Robert G Roeder - One of the best experts on this subject based on the ideXlab platform.
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reconstitution of active human core Mediator Complex reveals a critical role of the med14 subunit
Nature Structural & Molecular Biology, 2014Co-Authors: Murat A Cevher, Yi Shi, Brian T Chait, Sohail Malik, Robert G RoederAbstract:The evolutionarily conserved Mediator Complex is a critical coactivator for RNA polymerase II (Pol II)-mediated transcription. Here we report the reconstitution of a functional 15-subunit human core Mediator Complex and its characterization by functional assays and chemical cross-linking coupled to MS (CX-MS). Whereas the reconstituted head and middle modules can stably associate, basal and coactivator functions are acquired only after incorporation of MED14 into the bimodular Complex. This results from a dramatically enhanced ability of MED14-containing Complexes to associate with Pol II. Altogether, our analyses identify MED14 as both an architectural and a functional backbone of the Mediator Complex. We further establish a conditional requirement for metazoan-specific MED26 that becomes evident in the presence of heterologous nuclear factors. This general approach paves the way for systematic dissection of the multiple layers of functionality associated with the Mediator Complex.
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ccar1 a key regulator of Mediator Complex recruitment to nuclear receptor transcription Complexes
Molecular Cell, 2008Co-Authors: Jeong Hoon Kim, Robert G Roeder, Catherine K Yang, Kyu Heo, Michael R StallcupAbstract:DNA-bound transcription factors recruit many coactivator proteins to remodel chromatin and activate transcription. The Mediator Complex is believed to recruit RNA polymerase II to most protein-encoding genes. It is generally assumed that interaction of Mediator subunits with DNA-binding transcription factors is responsible for Mediator recruitment to promoters. However, we report here that Mediator recruitment by nuclear receptors (NR) requires a coactivator protein, CCAR1 (cell-cycle and apoptosis regulator 1). CCAR1 associates with components of the Mediator and p160 coactivator Complexes and is recruited to endogenous NR target genes in response to the appropriate hormone. Reduction of endogenous CCAR1 levels inhibited hormone-induced expression of endogenous NR target genes, hormone-induced recruitment of Mediator components and RNA polymerase II to target gene promoters, and estrogen-dependent growth of breast cancer cells. Thus, CCAR1 regulates expression of key proliferation-inducing genes. CCAR1 also functions as a p53 coactivator, suggesting a broader role in transcriptional regulation.
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alternative mechanisms by which Mediator subunit med1 trap220 regulates peroxisome proliferator activated receptor γ stimulated adipogenesis and target gene expression
Molecular and Cellular Biology, 2008Co-Authors: Youngwook Cho, Mitsuhiro Ito, Mohamed Guermah, Hong Guo, Teresa Hong, Markus Kalkum, Robert G RoederAbstract:Peroxisome proliferator-activated receptor γ (PPARγ) is a key regulator of transcriptional pathways important for adipogenesis (34). PPARγ−/− mice show a total absence of both brown and white adipose tissue. Furthermore, retrovirus vector-mediated ectopic expression of PPARγ alone can stimulate mouse embryonic fibroblasts (MEFs) to undergo adipogenesis. In such cells, the expression of CCAAT/enhancer-binding protein α (C/EBPα), another key transcriptional regulator of adipogenesis, and adipogenesis markers such as aP2, fatty acid synthase (FAS), and adipsin are induced in a PPARγ-dependent manner. PPARγ 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 the specific target genes important for cell growth, homeostasis, and differentiation (36). These transcription coactivators often exist as multiprotein 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 (40). 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 (7, 26). It is believed to connect transcriptional activators with the RNA polymerase II transcription machinery and appears to be essential for most (17), but not necessarily all (9), 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 α (TRα) from HeLa cells grown in the presence of a TRα ligand (10), and the Complex is similar to or identical with other more recently described Complexes that include the SRB/MED-containing cofactor Complex (SMCC) and the PC2, NAT, mouse Mediator, ARC, CRSP, DRIP, and human Mediator Complexes (26). 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-κB, p53, VP16, and E1A (reviewed in reference 4). The MED1/TRAP220 subunit of the Mediator Complex shows ligand-dependent interactions, through a region containing two nuclear receptor recognition (LXXLL) motifs, with multiple nuclear hormone receptors that include TRα, vitamin D receptor, PPARγ and PPARα, retinoic acid receptor α (RARα), retinoid X receptor (RXR), farnesoid X receptor, and estrogen receptor α and β (ERα and ERβ) (10, 20, 30, 32, 43, 44). A MED1/TRAP220 LXXLL-dependent interaction between the intact Mediator Complex and TRα has also been demonstrated (25). These results have suggested a broad role for the Mediator Complex in nuclear receptor function. The mouse MED1/TRAP220 subunit was independently isolated as a PPARγ-interacting protein in yeast two-hybrid screens and was shown to interact, in a ligand-dependent manner, with PPARγ. MED1/TRAP220 modestly increased the transcriptional activity with a PPARγ-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 PPARγ (44). We recently showed that the MED1/TRAP220 subunit of the Mediator Complex is essential for PPARγ-stimulated adipogenesis and expression of adipogenesis markers in MEFs, but not for MyoD-stimulated myogenesis (11). This finding provided an example of the regulation of cell-specific transcription and differentiation events through a distinct Mediator subunit. Further biochemical analyses showed (i) that PPARγ interacts directly with the purified Mediator Complex in a ligand-dependent manner, (ii) that Mediator functions directly as a transcriptional coactivator for PPARγ on a DNA template containing three copies of the DR1 PPARγ recognition site in an in vitro transcription system reconstituted with highly purified factors, and (iii) that MED1/TRAP220 serves as an essential bridge for the interaction between Mediator Complex and PPARγ in vitro (11). These data suggested a potential mechanism that may account for the inability of MED1/TRAP220−/− MEFs to undergo PPARγ-stimulated adipogenesis. However, the precise molecular mechanisms underlying the roles of MED1/TRAP220 and the associated Mediator Complex in PPARγ-stimulated adipogenesis in vivo and the mechanism by which MED1/TRAP220 and Mediator regulate PPARγ transcriptional activity remain unclear. Here, structural and functional analyses of MED1/TRAP220 indicate, surprisingly, that a strong, direct interaction of PPARγ with Mediator through the LXXLL motifs of MED1/TRAP220 is not required for PPARγ-stimulated adipogenesis of cultured MEFs and, furthermore, that PPARγ target gene expression and recruitment of Mediator to a PPARγ 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 the incorporation of MED1/TRAP220 into the Mediator Complex. Our data thus suggest the existence of an alternative mechanism, involving other potentially redundant cofactors or intermediate cofactors, by which MED1/TRAP220 and the associated Mediator Complex regulate the expression of known PPARγ target genes, as well as the possibility of as-yet-unidentified genes that require MED1/TRAP220 for expression in adipogenesis.
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the Mediator Complex functions as a coactivator for gata 1 in erythropoiesis via subunit med1 trap220
Proceedings of the National Academy of Sciences of the United States of America, 2006Co-Authors: Melanie Stumpf, Robert G Roeder, Claudia Waskow, Marit Krotschel, Dominic Van Essen, Patrick Rodriguez, Xiaoting Zhang, Boris Guyot, Tilman BorggrefeAbstract:The Mediator Complex forms the bridge between transcriptional activators and RNA polymerase II. Mediator subunit Med1/TRAP220 is a key component of Mediator originally found to associate with nuclear hormone receptors. Med1 deficiency causes lethality at embryonic day 11.5 because of defects in heart and placenta development. Here we show that Med1-deficient 10.5 days postcoitum embryos are anemic but have normal numbers of hematopoietic progenitor cells. Med1-deficient progenitor cells have a defect in forming erythroid burst-forming units (BFU-E) and colony-forming units (CFU-E), but not in forming myeloid colonies. At the molecular level, we demonstrate that Med1 interacts physically with the erythroid master regulator GATA-1. In transcription assays, Med1 deficiency leads to a defect in GATA-1-mediated transactivation. In chromatin immunoprecipitation experiments, we find Mediator components at GATA-1-occupied enhancer sites. Thus, we conclude that Mediator subunit Med1 acts as a pivotal coactivator for GATA-1 in erythroid development.
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a subunit of the Mediator Complex regulates vertebrate neuronal development
Proceedings of the National Academy of Sciences of the United States of America, 2006Co-Authors: Xiaoqun Wang, Robert G Roeder, Nan Yang, Etsuko Uno, Su GuoAbstract:The unique profiles of gene expression dictate distinct cellular identity. How these profiles are established during development is not clear. Here we report that the mutant motionless (mot), identified in a genetic screen for mutations that affect neuronal development in zebrafish, displays deficits of monoaminergic neurons and cranial sensory ganglia, whereas expression of the pan-neuronal marker Hu is largely unperturbed; GABAergic and subsets of cranial motor neurons do not appear to be deficient. Positional cloning reveals that mot encodes Med12, a component of the evolutionarily conserved Mediator Complex, whose in vivo function is not well understood in vertebrates. mot/med12 transcripts are enriched in the embryonic brain and appear distinct from two other Mediator components Med17 and Med21. Delivery of human med12 RNA into zebrafish restores normality to the mot mutant and, strikingly, leads to premature neuronal differentiation and an increased production of monoaminergic neuronal subtypes in WT. Further investigation reveals that mot/med12 is necessary to regulate, and when overexpressed is capable of increasing, the expression of distinct neuronal determination genes, including zash1a and lim1, and serves as an in vivo cofactor for Sox9 in this process. Together, our analyses reveal a regulatory role of Mot/Med12 in vertebrate neuronal development.
Joan W Conaway - One of the best experts on this subject based on the ideXlab platform.
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subunit architecture and functional modular rearrangements of the transcriptional Mediator Complex
Cell, 2014Co-Authors: Kuanglei Tsai, Ronald C Conaway, Joan W Conaway, Shigeo Sato, Chieri Tomomorisato, Francisco J AsturiasAbstract:Summary The multisubunit Mediator, comprising ∼30 distinct proteins, plays an essential role in gene expression regulation by acting as a bridge between DNA-binding transcription factors and the RNA polymerase II (RNAPII) transcription machinery. Efforts to uncover the Mediator mechanism have been hindered by a poor understanding of its structure, subunit organization, and conformational rearrangements. By overcoming biochemical and image analysis hurdles, we obtained accurate EM structures of yeast and human Mediators. Subunit localization experiments, docking of partial X-ray structures, and biochemical analyses resulted in comprehensive mapping of yeast Mediator subunits and a complete reinterpretation of our previous Mediator organization model. Large-scale Mediator rearrangements depend on changes at the interfaces between previously described Mediator modules, which appear to be facilitated by factors conducive to transcription initiation. Conservation across eukaryotes of Mediator structure, subunit organization, and RNA polymerase II interaction suggest conservation of fundamental aspects of the Mediator mechanism.
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the Mediator Complex and transcription elongation
Biochimica et Biophysica Acta, 2013Co-Authors: Ronald C Conaway, Joan W ConawayAbstract:Abstract Background Mediator is an evolutionarily conserved multisubunit RNA polymerase II (Pol II) coregulatory Complex. Although Mediator was initially found to play a critical role in the regulation of the initiation of Pol II transcription, recent studies have brought to light an expanded role for Mediator at post-initiation stages of transcription. Scope of review We provide a brief description of the structure of Mediator and its function in the regulation of Pol II transcription initiation, and we summarize recent findings implicating Mediator in the regulation of various stages of Pol II transcription elongation. Major conclusions Emerging evidence is revealing new roles for Mediator in nearly all stages of Pol II transcription, including initiation, promoter escape, elongation, pre-mRNA processing, and termination. General significance Mediator plays a central role in the regulation of gene expression by impacting nearly all stages of mRNA synthesis. This article is part of a Special Issue entitled: RNA polymerase II Transcript Elongation.
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origins and activity of the Mediator Complex
Seminars in Cell & Developmental Biology, 2011Co-Authors: Ronald C Conaway, Joan W ConawayAbstract:The Mediator is a large, multisubunit RNA polymerase II transcriptional regulator that was first identified in Saccharomyces cerevisiae as a factor required for responsiveness of Pol II and the general initiation factors to DNA binding transactivators. Since its discovery in yeast, Mediator has been shown to be an integral and highly evolutionarily conserved component of the Pol II transcriptional machinery with critical roles in multiple stages of transcription, from regulation of assembly of the Pol II initiation Complex to regulation of Pol II elongation. Here we provide a brief overview of the evolutionary origins of Mediator, its subunit composition, and its remarkably diverse collection of activities in Pol II transcription.
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function and regulation of the Mediator Complex
Current Opinion in Genetics & Development, 2011Co-Authors: Ronald C Conaway, Joan W ConawayAbstract:Over the past few years, advances in biochemical and genetic studies of the structure and function of the Mediator Complex have shed new light on its subunit architecture and its mechanism of action in transcription by RNA polymerase II (pol II). The development of improved methods for reconstitution of recombinant Mediator subassemblies is enabling more in-depth analyses of basic features of the mechanisms by which Mediator interacts with and controls the activity of pol II and the general initiation factors. The discovery and characterization of multiple, functionally distinct forms of Mediator characterized by the presence or absence of the Cdk8 kinase module have led to new insights into how Mediator functions in both Pol II transcription activation and repression. Finally, progress in studies of the mechanisms by which the transcriptional activation domains (ADs) of DNA binding transcription factors target Mediator have brought to light unexpected Complexities in the way Mediator participates in signal transduction.
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the mammalian Mediator Complex and its role in transcriptional regulation
Trends in Biochemical Sciences, 2005Co-Authors: Ronald C Conaway, Joan W Conaway, Shigeo Sato, Chieri Tomomorisato, Tingting YaoAbstract:Mediator is an essential component of the RNA polymerase II general transcriptional machinery and plays a crucial part in the activation and repression of eukaryotic mRNA synthesis. The Saccharomyces cerevisiae Mediator was the first to be defined and is a high molecular mass Complex composed of >20 distinct subunits that performs multiple activities in transcription. Recent studies have defined the subunit composition and associated activities of mammalian Mediator, and revealed a striking evolutionary conservation of Mediator structure and function from yeast to man.
Ronald C Conaway - One of the best experts on this subject based on the ideXlab platform.
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subunit architecture and functional modular rearrangements of the transcriptional Mediator Complex
Cell, 2014Co-Authors: Kuanglei Tsai, Ronald C Conaway, Joan W Conaway, Shigeo Sato, Chieri Tomomorisato, Francisco J AsturiasAbstract:Summary The multisubunit Mediator, comprising ∼30 distinct proteins, plays an essential role in gene expression regulation by acting as a bridge between DNA-binding transcription factors and the RNA polymerase II (RNAPII) transcription machinery. Efforts to uncover the Mediator mechanism have been hindered by a poor understanding of its structure, subunit organization, and conformational rearrangements. By overcoming biochemical and image analysis hurdles, we obtained accurate EM structures of yeast and human Mediators. Subunit localization experiments, docking of partial X-ray structures, and biochemical analyses resulted in comprehensive mapping of yeast Mediator subunits and a complete reinterpretation of our previous Mediator organization model. Large-scale Mediator rearrangements depend on changes at the interfaces between previously described Mediator modules, which appear to be facilitated by factors conducive to transcription initiation. Conservation across eukaryotes of Mediator structure, subunit organization, and RNA polymerase II interaction suggest conservation of fundamental aspects of the Mediator mechanism.
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the Mediator Complex and transcription elongation
Biochimica et Biophysica Acta, 2013Co-Authors: Ronald C Conaway, Joan W ConawayAbstract:Abstract Background Mediator is an evolutionarily conserved multisubunit RNA polymerase II (Pol II) coregulatory Complex. Although Mediator was initially found to play a critical role in the regulation of the initiation of Pol II transcription, recent studies have brought to light an expanded role for Mediator at post-initiation stages of transcription. Scope of review We provide a brief description of the structure of Mediator and its function in the regulation of Pol II transcription initiation, and we summarize recent findings implicating Mediator in the regulation of various stages of Pol II transcription elongation. Major conclusions Emerging evidence is revealing new roles for Mediator in nearly all stages of Pol II transcription, including initiation, promoter escape, elongation, pre-mRNA processing, and termination. General significance Mediator plays a central role in the regulation of gene expression by impacting nearly all stages of mRNA synthesis. This article is part of a Special Issue entitled: RNA polymerase II Transcript Elongation.
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origins and activity of the Mediator Complex
Seminars in Cell & Developmental Biology, 2011Co-Authors: Ronald C Conaway, Joan W ConawayAbstract:The Mediator is a large, multisubunit RNA polymerase II transcriptional regulator that was first identified in Saccharomyces cerevisiae as a factor required for responsiveness of Pol II and the general initiation factors to DNA binding transactivators. Since its discovery in yeast, Mediator has been shown to be an integral and highly evolutionarily conserved component of the Pol II transcriptional machinery with critical roles in multiple stages of transcription, from regulation of assembly of the Pol II initiation Complex to regulation of Pol II elongation. Here we provide a brief overview of the evolutionary origins of Mediator, its subunit composition, and its remarkably diverse collection of activities in Pol II transcription.
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function and regulation of the Mediator Complex
Current Opinion in Genetics & Development, 2011Co-Authors: Ronald C Conaway, Joan W ConawayAbstract:Over the past few years, advances in biochemical and genetic studies of the structure and function of the Mediator Complex have shed new light on its subunit architecture and its mechanism of action in transcription by RNA polymerase II (pol II). The development of improved methods for reconstitution of recombinant Mediator subassemblies is enabling more in-depth analyses of basic features of the mechanisms by which Mediator interacts with and controls the activity of pol II and the general initiation factors. The discovery and characterization of multiple, functionally distinct forms of Mediator characterized by the presence or absence of the Cdk8 kinase module have led to new insights into how Mediator functions in both Pol II transcription activation and repression. Finally, progress in studies of the mechanisms by which the transcriptional activation domains (ADs) of DNA binding transcription factors target Mediator have brought to light unexpected Complexities in the way Mediator participates in signal transduction.
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the mammalian Mediator Complex and its role in transcriptional regulation
Trends in Biochemical Sciences, 2005Co-Authors: Ronald C Conaway, Joan W Conaway, Shigeo Sato, Chieri Tomomorisato, Tingting YaoAbstract:Mediator is an essential component of the RNA polymerase II general transcriptional machinery and plays a crucial part in the activation and repression of eukaryotic mRNA synthesis. The Saccharomyces cerevisiae Mediator was the first to be defined and is a high molecular mass Complex composed of >20 distinct subunits that performs multiple activities in transcription. Recent studies have defined the subunit composition and associated activities of mammalian Mediator, and revealed a striking evolutionary conservation of Mediator structure and function from yeast to man.
Amna Mhamdi - One of the best experts on this subject based on the ideXlab platform.
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the arabidopsis Mediator Complex subunit 8 regulates oxidative stress responses
The Plant Cell, 2021Co-Authors: Jordi Denecker, Katrien Van Der Kelen, Patrick Willems, Robin Pottie, Su Yin Phua, Matthew A Hannah, Didier Vertommen, Frank Van Breusegem, Amna MhamdiAbstract:Signaling events triggered by hydrogen peroxide (H2O2) regulate plant growth and defense by orchestrating a genome-wide transcriptional reprogramming. However, the specific mechanisms that govern H2O2-dependent gene expression are still poorly understood. Here, we identify the Arabidopsis Mediator Complex subunit MED8 as a regulator of H2O2 responses. The introduction of the med8 mutation in a constitutive oxidative stress genetic background (catalase-deficient, cat2) was associated with enhanced activation of the salicylic acid pathway and accelerated cell death. Interestingly, med8 seedlings were more tolerant to oxidative stress generated by the herbicide methyl viologen (MV) and exhibited transcriptional hyperactivation of defense signaling, in particular salicylic acid- and jasmonic acid-related pathways. The med8-triggered tolerance to MV was manipulated by the introduction of secondary mutations in salicylic acid and jasmonic acid pathways. In addition, analysis of the Mediator interactome revealed interactions with components involved in mRNA processing and microRNA biogenesis, hence expanding the role of Mediator beyond transcription. Notably, MED8 interacted with the transcriptional regulator NEGATIVE ON TATA-LESS, NOT2, to control the expression of H2O2-inducible genes and stress responses. Our work establishes MED8 as a component regulating oxidative stress responses and demonstrates that it acts as a negative regulator of H2O2-driven activation of defense gene expression.
Sohail Malik - One of the best experts on this subject based on the ideXlab platform.
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reconstitution of active human core Mediator Complex reveals a critical role of the med14 subunit
Nature Structural & Molecular Biology, 2014Co-Authors: Murat A Cevher, Yi Shi, Brian T Chait, Sohail Malik, Robert G RoederAbstract:The evolutionarily conserved Mediator Complex is a critical coactivator for RNA polymerase II (Pol II)-mediated transcription. Here we report the reconstitution of a functional 15-subunit human core Mediator Complex and its characterization by functional assays and chemical cross-linking coupled to MS (CX-MS). Whereas the reconstituted head and middle modules can stably associate, basal and coactivator functions are acquired only after incorporation of MED14 into the bimodular Complex. This results from a dramatically enhanced ability of MED14-containing Complexes to associate with Pol II. Altogether, our analyses identify MED14 as both an architectural and a functional backbone of the Mediator Complex. We further establish a conditional requirement for metazoan-specific MED26 that becomes evident in the presence of heterologous nuclear factors. This general approach paves the way for systematic dissection of the multiple layers of functionality associated with the Mediator Complex.
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dynamic regulation of pol ii transcription by the mammalian Mediator Complex
Trends in Biochemical Sciences, 2005Co-Authors: Sohail Malik, Robert G RoederAbstract:Mammalian Mediator is a key coactivator that enables transcriptional activators to regulate transcription by RNA polymerase II (pol II). Like the yeast Complex to which it is phylogenetically related, it contains up to 30 subunits. These subunits are organized as a tightly associated core sub-Complex, which associates with several groups of subunits that might constitute distinct modules. Although the Complex seems to be universally required at all genes, specific subunits are dedicated to regulation of distinct expression programs via interactions with relevant gene-specific transcriptional activators. These interactions, in conjunction with dynamic effects of the core Complex on pol II and the general transcription factors, lead to activation of transcription at the target gene. In addition, the compositional Complexity of the Mediator allows for assimilation of other diverse signals such as those emanating from repressors and other coactivators.
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structural and functional organization of trap220 the trap Mediator subunit that is targeted by nuclear receptors
Molecular and Cellular Biology, 2004Co-Authors: Sohail Malik, Mohamed Guermah, Chaoxing Yuan, Weizhen Wu, Soichiro Yamamura, Robert G RoederAbstract:The TRAP/Mediator Complex serves as a coactivator for many transcriptional activators, including nuclear receptors such as the thyroid hormone receptor (TR) that targets the TRAP220 subunit. The critical but selective function of TRAP220 is evidenced by the embryonic lethal phenotype of Trap220−/− mice and by the observation that Trap220−/− fibroblasts (isolated before embryonic death) are impaired in specific nuclear receptor-dependent pathways. Here we have used a biochemical and genetic approach to understand the basis of specificity in TRAP220 function. We show that Trap220−/− cells possess a TRAP/Mediator Complex that is relatively intact and compromised in its ability to support TR-dependent, but not VP16-dependent, transcription in vitro. Transfection studies using TRAP220 mutants revealed that the N terminus of TRAP220 is necessary and sufficient for stable association with the TRAP/Mediator Complex and, further, that TRAP220-dependent TR function in transfected cells requires both of the NR boxes that contain the LXXLL motif implicated in nuclear receptor binding. Similarly, an analysis of isolated TRAP/Mediator Complexes with mutations in either or both of the two NR boxes confirmed a critical role for them in in vitro coactivator function. The implications of these observations are discussed in terms of our present understanding of coactivator function.
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isolation and functional characterization of the trap Mediator Complex
Methods in Enzymology, 2003Co-Authors: Sohail Malik, Robert G RoederAbstract:Publisher Summary This chapter describes the methods developed for isolating functionally active preparations of the TRAP/Mediator Complex and for testing them in cell-free assay systems. The TRAP/Mediator Complex was initially isolated from HeLa cells stably expressing FLAG-tagged thyroid hormone receptor (TRα). The propagation of this cell-line in the presence of the cognate ligand (thyroid hormone), but not in its absence, allowed the isolation (by subsequent affinity chromatography utilizing the FLAG tag) of a multiprotein Complex (TRAP) associated with TRα. It is easier to isolate the human TRAP/Mediator by affinity purification from extracts of HeLa cells that stably express a FLAG-tagged version of an integral TRAP/Mediator subunit. The experimental systems presented in the chapter provide important insights into some of the mechanisms by which nuclear receptors activate their target genes. They establish the dominant role of the TRAP/Mediator coactivator, especially as it pertains to transcription from DNA templates. As the emphasis in the transcription field shifts to the study of distinct genes, it is likely that more natural templates–that is, chromatin templates carrying their physiological arrangements of regulatory elements (e.g., promoters andenhancers) will increasingly be used in in vitro experiments. The availability of pure TRAP/Mediator, together with the assays utilizing purified factors (to which new cofactors can be added) and with unfractionated extracts (from which cofactors can be selectively removed and resupplied), should provide a foundation for this next phase of investigation.