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To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2006.01.055 Byline: Mohamed Guermah (1), Vikas B. Palhan (1), Alan J. Tackett (2), Brian T. Chait (2), Robert G. Roeder (1) Abstract: We have reconstituted a highly purified RNA polymerase II transcription system containing chromatin templates assembled with purified histones and assembly factors, the histone acetyltransferase p300, and components of the general transcription machinery that, by themselves, suffice for activated transcription (initiation and elongation) on DNA templates. We show that this system mediates activator-dependent initiation, but not productive elongation, on chromatin templates. We further report the purification of a chromatin transcription-enabling activity (CTEA) that, in a manner dependent upon p300 and acetyl-CoA, strongly potentiates transcription elongation through several contiguous nucleosomes as must occur in vivo. The transcription elongation factor SII is a major component of CTEA and strongly synergizes with p300 (histone acetylation) at a step subsequent to preinitiation complex formation. The purification of CTEA also identified HMGB2 as a coactivator that, while inactive on its own, enhances SII and p300 functions. Author Affiliation: (1) Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA (2) Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA Article History: Received 23 August 2005; Revised 9 November 2005; Accepted 22 January 2006 Article Note: (miscellaneous) Published: April 20, 2006

Author(s): Kai Ge [1]; Mohamed Guermah [1]; Chao-Xing Yuan [1, 3]; Mitsuhiro Ito [1]; Annika E. Wallberg [1]; Bruce M. Spiegelman [2]; Robert G. Roeder (corresponding author) [1] The TRAP [...]

Target gene activation by nuclear hormone receptors, including estrogen receptors (ERs), is thought to be mediated by a variety of interacting cofactors. Here we identify a number of nuclear extract-derived proteins that interact with immobilized ER ligand binding domains in a 17/[beta]-estradiol-dependent manner. The most prominent of these are components of the thyroid hormone receptor-associated protein (TRAP)/Mediator coactivator complex, which interacts with ER[alpha] and ER[beta] in both unfractionated nuclear extracts and purified form. Studies with extracts from [TRAP220.sup.-/-] fibroblasts reveal that these interactions depend on TRAP220, a TRAP/Mediator subunit previously shown to interact with ER and other nuclear receptors in a ligand-dependent manner. The physiological relevance of the in vitro interaction is documented further by the isolation of an ER[alpha]-TRAP/Mediator complex from cultured cells expressing an epitope-tagged ER[alpha]. Finally, the complete TRAP/Mediator complex is shown to enhance ER function directly in a highly purified cell-free transcription system. These studies firmly establish a direct role for TRAP/Mediator, through TRAP220, in ER function.

An analysis of the human transcription initiation factor, TFIID, composed of the TATA-binding polypeptide (TBP) and TBP-associated factors (TAFs) revealed that TAF(sub II)80 interacts with TAF(sub II)250, TAF(sub II)31, TAF(sub II)20, and TAP while TAF(sub II)80 interacts with TRIIEalpha and TRIIFalpha. The mutation of TAF(sub II)80 indicated three distinct interaction domains falling within regions conserved in human TAF(sub II)80, Drosophila TAF(sub II)60, and yeast TAF(sub II)60. This process of isolating these cDNAs are discussed.

Human TFIID contains the TATA-binding protein (TBP) and several TBP-associated factors (hTAFs) that have been shown to play important roles, within TFIID, both in core promoter recognition and as coactivators. Here we show that the human TAFII43 (TAF8) is an integral component of a functional TFIID and an apparent ortholog to the recently reported mouse TBN, which is essential for early embryonic mouse developmental events. Significantly, we also show that TAF8 is dramatically induced and sequestered within TFIID upon differentiation of 3T3-L1 preadipocytes to adipocytes, whereas the expression of all other TAFs tested is slightly reduced. Moreover, when ectopically expressed, the histone fold domain of TAF8 acts as a dominant-negative mutant and selectively inhibits 3T3-L1 adipogenic differentiation. Furthermore TAF8 acts as a positive regulator of adipogenesis and reverses the inhibitory effect of its histone fold. These data suggest a selective role for TAF8 in a specific cell differentiation process(es). [Copyright &y& Elsevier]

Examines the role of thyroid hormone receptor-associated proteins (TRAP) transcription coactivator complex in adipogenesis. Interaction between TRAP220 with peroxisome proliferator-activated receptorΓ2; Relevance of TRAP in physiological processes; Ability to increase the transcription factors of peroxisomeproliferator-activated receptorΓ.

Examines the role of the target gene activation by nuclear hormone receptors in the interacting cofactors. Identification of the nuclear extract-derived proteins; Composition of the thyroid hormones; Activation of the ligand binding domain.

Mediator is a general coactivator complex connecting transcription activators and RNA polymerase II. Recent work has shown that the nuclear receptor-interacting MED1/TRAP220 subunit of Mediator is required for peroxisome proliferator-activated receptorγ (PPARγ)-stimulated adipogenesis of mouse embryonic fibroblasts (MEFs). However, the molecular mechanisms remain undefined. Here, we show an intracellular PPARγ-Mediator interaction that requires the two LXXLL nuclear receptor recognition motifs on MED1/TRAP220 and, furthermore, we show that the intact LXXLL motifs are essential for optimal PPARγ function in a reconstituted cell-free transcription system. Surprisingly, a conserved N-terminal region of MED1/TRAP220 that lacks the LXXLL motifs but gets incorporated into Mediator fully supports PPARγ-stimulated adipogenesis. Moreover, in undifferentiated MEFs, MED1/TRAP220 is dispensable both for PPARγ-mediated target gene activation and for recruitment of Mediator to a PPAR response element on the aP2 target gene promoter. However, PPARγ shows significantly reduced transcriptional activity in cells deficient for a subunit (MED24/TRAP100) important for the integrity of the Mediator complex, indicating a general Mediator requirement for PPARγ function. These results indicate that there is a conditional requirement for MED1/TRAP220 and that a direct interaction between PPARγ and Mediator through MED1/TRAP220 is not essential either for PPARγ-stimulated adipogenesis or for PPARγ target gene expression in cultured fibroblasts. As Mediator is apparently essential for PPARγ transcriptional activity, our data indicate the presence of alternative mechanisms for Mediator recruitment, possibly through intermediate cofactors or other cofactors that are functionally redundant with MED1/TRAP220. [ABSTRACT FROM AUTHOR]

Mediator is a general coactivator complex connecting transcription activators and RNA polymerase II. Recent work has shown that the nuclear receptor-interacting MED1/TRAP220 subunit of Mediator is required for peroxisome proliferator-activated receptor gamma (PPARgamma)-stimulated adipogenesis of mouse embryonic fibroblasts (MEFs). However, the molecular mechanisms remain undefined. Here, we show an intracellular PPARgamma-Mediator interaction that requires the two LXXLL nuclear receptor recognition motifs on MED1/TRAP220 and, furthermore, we show that the intact LXXLL motifs are essential for optimal PPARgamma function in a reconstituted cell-free transcription system. Surprisingly, a conserved N-terminal region of MED1/TRAP220 that lacks the LXXLL motifs but gets incorporated into Mediator fully supports PPARgamma-stimulated adipogenesis. Moreover, in undifferentiated MEFs, MED1/TRAP220 is dispensable both for PPARgamma-mediated target gene activation and for recruitment of Mediator to a PPAR response element on the aP2 target gene promoter. However, PPARgamma shows significantly reduced transcriptional activity in cells deficient for a subunit (MED24/TRAP100) important for the integrity of the Mediator complex, indicating a general Mediator requirement for PPARgamma function. These results indicate that there is a conditional requirement for MED1/TRAP220 and that a direct interaction between PPARgamma and Mediator through MED1/TRAP220 is not essential either for PPARgamma-stimulated adipogenesis or for PPARgamma target gene expression in cultured fibroblasts. As Mediator is apparently essential for PPARgamma transcriptional activity, our data indicate the presence of alternative mechanisms for Mediator recruitment, possibly through intermediate cofactors or other cofactors that are functionally redundant with MED1/TRAP220.