Human TAF_II130 Is a Coactivator for NFATp

Abstract

NFATp is one member of a family of transcriptional activators that regulate the expression of cytokine genes. To study mechanisms of NFATp transcriptional activation, we established a reconstituted transcription system consisting of human components that is responsive to activation by full-length NFATp. The TATA-associated factor (TAF II ) subunits of the TFIID complex were required for NFATp-mediated activation in this transcription system, since TATA-binding protein ( Transcription is a highly regulated process of RNA synthesis, the initiation of which is a primary control point in gene expression. In eukaryotes, genes encoding mRNA are transcribed by RNA polymerase II, a multiprotein enzyme. Reconstitution of an in vitro transcription system with purified RNA polymerase II proved to be insufficient for RNA synthesis from promoters, propelling the identification of a phylogenetically conserved set of general transcription factors (TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH) that are necessary for basal transcription (reviewed in reference 45). The assembly of RNA polymerase II and the general transcription factors on promoter DNA leads to the formation of stable nucleoprotein complexes (preinitiation complexes) that are transcriptionally competent in vitro. Preinitiation complex formation can be regulated by the action of activators and repressors. The activities of these distinct regulatory proteins can promote (activators) or repress (repressors) the recruitment of general transcription factors and RNA polymerase II onto promoters, resulting in promoter-specific augmentation or suppression of transcription.Eukaryotic organisms have evolved elaborate mechanisms for the deployment of RNA polymerase II onto specialized promoter elements that can modulate basal promoter strength. Many RNA polymerase II promoters contain one or more of three identified core promoter elements that direct accurate transcription initiation (5): the TATA box, the initiator element, and the downstream promoter element. In a temporal scheme, TFIID is the first basal factor to bind to a core promoter, serving as a nucleation center for the rest of the transcriptional apparatus (60). The architecture of TFIID is also phylogenetically conserved, and it appears that TFIID complexes in yeast, Drosophila melanogaster, and humans have a similar complement of subunits (6). TFIID is a sequence-specific DNA binding general transcription factor consisting of TATA-binding protein (TBP) and 10 to 13 TATA-associated factors (TAF II s) that together can recognize the TATA box, initiator, and downstream promoter elements.

Inducer-Specific Enhanceosome Formation Controls Tumor Necrosis Factor Alpha Gene Expression in T Lymphocytes

“…Mutation of the NFAT/Ets-binding sites at Ϫ117 and Ϫ180 and the Ϫ170 Sp1 site also all decrease the transcriptional activation of the gene VOL. 22,2002 DISTINCT ENHANCEOSOMES CONTROL TNF-␣ TRANSCRIPTION 2621 by ionophore and virus but to a comparatively greater extent by virus (Fig. 2B).…”

Dynamic Glycosylation of the Transcription Factor CREB: A Potential Role in Gene Regulation

“…Following centrifugation, the agarose was washed twice with buffer H (20 mM HEPES pH 7.9, 100 mM KCl, 0.2 mM EDTA, 5 mM MgCl 2 , 0.5 mM DTT, 0.1% NP-40, 20% glycerol, 0.1 mg/ml BSA) 9 containing 50 mM imidazole and twice with buffer H. The agarose was then incubated with [ 35 S]-labeled TAF II 130 lysate (0.13 volumes) in buffer H (2.5 volumes) for 4 h at 4 o C with mixing. 10,11 Following centrifugation, the agarose was washed five times with buffer H containing 50 mM imidazole. Samples were boiled in SDS-PAGE loading dye and resolved by SDS-PAGE.…”

“…9 Binding assays were performed with minor modifications to published procedures. 10,11 Proteins (50-100 µg CREB purified from Sf9 cells; 100-200 µg OGT purified from Sf9 cells as a control) were incubated with Ni-NTA agarose for 4-5 h at 4 o C with mixing. Following centrifugation, the agarose was washed twice with buffer H (20 mM HEPES pH 7.9, 100 mM KCl, 0.2 mM EDTA, 5 mM MgCl 2 , 0.5 mM DTT, 0.1% NP-40, 20% glycerol, 0.1 mg/ml BSA) 9 containing 50 mM imidazole and twice with buffer H. The agarose was then incubated with [ 35 S]-labeled TAF II 130 lysate (0.13 volumes) in buffer H (2.5 volumes) for 4 h at 4 o C with mixing.…”

Cytohesin Binder and Regulator Augments T Cell Receptor-induced Nuclear Factor of Activated T Cells·AP-1 Activation through Regulation of the JNK Pathway

“…The NFAT luciferase reporter construct pGL3-NFAT 3 -luciferase (pNFAT-Luc), consisting of three direct copies of the murine IL-4 high affinity NFAT site (region Ϫ82 to Ϫ64 of the murine IL-4 promoter) (23,24) was a generous gift of Dr. James A. Goodrich (University of Colorado, Boulder, CO). The NFAT-IFN luciferase reporter construct was a generous gift of Dr. A. Altman (La Jolla Institute for Allergy and Immunology, La Jolla, CA) and was described previously (25).…”