TAF7 (TAFII55) plays a role in the transcription activation by c-Jun (original) (raw)
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Cloning, expression, and spectroscopic studies of the Jun leucine zipper domain
European Journal of Biochemistry, 1994
Association of the human c-Jun and c-Fos proteins depends upon interactions involving their leucine zipper domains. We are interested in elucidating the tertiary structure of the Jun and Fos leucine zipper domains with a view to understanding the precise intermolecular interactions which govern the affinity and specificity of interaction in these proteins, which have the unusual capacity to form either homodimeric or heterodimeric zipper pairs. With this goal in mind, we have developed a bacterial expression system for the efficient production of both unlabelled and isotopically labelled c-Jun leucine zipper domain. A synthetic junLZ gene was created by annealing, ligation, and polymerase-chain-reaction amplification of overlapping synthetic oligonucleotides which comprised 132 bp of coding sequence encompassing residues Arg276-Asn314 of cJun plus a total of five engineered non-native residues at the N-and C-termini. The junLZ gene was cloned into the pGEX-2T vector from which recombinant c-Jun leucine zipper domain (rJunLZ; 46 residues, 5.1 kDa) was overexpressed (~1 5 % total cell protein) in Escherichia coli as a fusion protein of 31.4 kDa, consisting of rJunLZ fused to the carbuxy-terminal portion of Schistosorna japonicum glutathione Stransferase. Two markedly different expression strategies have been devised which allow purification of rJunLZ from the soluble or inclusion-body fraction of induced cells. We have used these strategies to produce unlabelled and uniformly 15N-labelled rJunLZ for NMR studies which, in combination with circular dichroic measurements, reveal that rJunLZ most likely forms a symmetric coiled-coil of parallel a-helices. We also present I5N-NMR chemical shift assignments for the backbone and sidechain amide nitrogens of rJunLZ, which should assist in determination of a high-resolution structure of the homodimeric Jun leucine zipper using heteronuclear three-dimensional NMR spectroscopy.
The mammalian Jun proteins: redundancy and specificity
Oncogene, 2001
The AP-1 transcription factor is composed of a mixture of homo-and hetero-dimers formed between Jun and Fos proteins. The dierent Jun and Fos family members vary signi®cantly in their relative abundance and their interactions with additional proteins generating a complex network of transcriptional regulators. Thus, the functional activity of AP-1 in any given cell depends on the relative amount of speci®c Jun/Fos proteins which are expressed, as well as other potential interacting proteins. This diversity of AP-1 components has complicated our understanding of AP-1 function and resulted in a paucity of information about the precise role of individual AP-1 members in distinct cellular processes. We shall discuss recent studies which suggest that dierent Jun and Fos family members may have both opposite and overlapping functions in cellular proliferation and cell fate.
Molecular and cellular biology, 1994
NF-IL6 and AP-1 family transcription factors are coordinately induced by interleukin-6 (IL-6) in a cell-type-specific manner, suggesting that they mediate IL-6 signals in the nucleus. We show that the basic leucine zipper (bZIP) region of NF-IL6 mediates a direct association with the bZIP regions of Fos and Jun in vitro. This interaction does not depend on the presence of their cognate recognition DNA elements or the posttranslational modification of either partner. NF-IL6 homodimers can bind to both NF-IL6 and AP-1 sites, whereas Fos and Jun cannot bind to most NF-IL6 sites. Cross-family association with Fos or with Jun alters the DNA binding specificity of NF-IL6 and reduced its binding to NF-IL6 sites. NF-IL6 isoforms that differ in the site of translation initiation have distinct transcriptional activities. Activation of a reporter gene linked to the NF-IL6 site by NF-IL6 is repressed by Fos and by Jun in transient transfection assays. Thus, association with AP-1 results in repr...
Fos-Jun dimerization promotes interaction of the basic region with TFIIE-34 and TFIIF
Molecular and cellular …, 1996
The regulation of RNA polymerase II-mediated transcription involves both direct and indirect interactions among regulatory proteins and the general transcription factors (GTFs) that assemble at TATA-containing promoters. Here we show that the oncogenic transcription factors Fos and Jun make direct physical contacts with three proteins of the basal transcription apparatus, TFIIE-34 (TFIIE-), TFIIF-30 (RAP30), and TFIIF-74 (RAP74). The interactions among the activator proteins and these three GTFs were not detected with other transcription factors, including some bZIP protein family members. Both coimmunoprecipitation and protein blotting experiments demonstrated that the interactions were strongly favored by dimerization of Fos and Jun and that they involved the basic region and basic region-proximal domain of both proteins. Mutations within the DNA-binding domains of Fos and Jun abolished binding to GTFs, although the presence of DNA was not required for the association. Surprisingly, only a single basic region in the context of a protein dimer was sufficient for the interaction. Squelching of AP-1-dependent transcription in vitro by an excess of Fos-Jun dimers was relieved by the addition of TFIIE, indicating that it is a direct functional target of Fos and Jun. These results suggest that dimerization induces a conformational alteration in the basic region of Fos and Jun that promotes an association with TFIIE-34 and TFIIF, thus contributing to transcription initiation.
Proceedings of the National Academy of Sciences, 1992
Transcription factor c-Jun appears to be a nuclear target of the Ras-induced signal transduction pathway. In fact, some experiments show that transforming forms of the Ras protein cooperate with Jun in transcriptional activation mediated by an AP-1 site and others indicate that the two oncoproteins cooperate in cellular transformation. Although it is likely that intracellular signaling systems activated by Ras might act directly on c-Jun by inducing specific phosphorylation, it is unclear how c-Jun participates in the transformation process. Here, we present results obtained with a LexA-Jun zipper fusion that lacks both the transcriptional activation domains and the basic region of the DNA-binding domain of c-Jun and contains only the intact leucine-zipper domain. This fusion product has a dominant negative effect on the transcriptional activation elicited by phorbol esters, c-Jun, c-Fos, Ras and E1A on an AP-1-responsive site. An analogous LexA-Fos zipper fusion has similar effects...
Biochemical and Biophysical Research Communications, 2010
Leucine zippers, structural motifs typically comprised of five successive heptads of amino acids with a signature leucine at every seventh position, play a central role in the dimerization of bZIP family of transcription factors and their subsequent binding to the DNA promoter regions of target genes. Herein, using analytical laser scattering (ALS) in combination with isothermal titration calorimetry (ITC), we study the effect of successive C-terminal truncation of leucine zippers on the dimerization and energetics of binding of bZIP domains of Jun transcription factor to its DNA response element. Our data show that all five heptads are critical for the dimerization of bZIP domains and that the successive C-terminal truncation of residues leading up to each signature leucine significantly compromises the binding of bZIP domains to DNA. Taken together, our study provides novel insights into the energetic contributions of leucine zippers to the binding of bZIP domains of Jun transcription factor to DNA.
Biochemical and Biophysical Research Communications, 2008
Jun and Fos are components of the AP1 family of transcription factors and bind to the promoters of a diverse multitude of genes involved in critical cellular responses such as cell growth and proliferation, cell cycle regulation, embryonic development and cancer. Here, using the powerful technique of isothermal titration calorimetry, we characterize the thermodynamics of heterodimerization of leucine zippers of Jun and Fos. Our data suggest that the heterodimerization of leucine zippers is driven by enthalpic forces with unfavorable entropy change at physiological temperatures. Furthermore, the basic regions appear to modulate the heterodimerization of leucine zippers and may undergo at least partial folding upon heterodimerization. Large negative heat capacity changes accompanying the heterodimerization of leucine zippers are consistent with the view that leucine zippers do not retain a-helical conformations in isolation and that the formation of the native coiled-coil a-helical dimer is attained through a coupled folding-dimerization mechanism.
1989
fra-1 encodes a serum-inducible protein (Fra-1) that is antigenically related to Fos. We have characterized Fra-1 expression in serum-stimulated cells using antibodies raised against several regions of this protein. Fra-1, expressed transiently in COS cells or in serum-stimulated rat fibroblasts, undergoes extensive post-translational modification, primarily by phosphorylation of serine residues. It is present in both the nucleus and the cytoplasm and participates in a protein complex with Jun. Using proteins synthesized in reticulocyte lysates, we have shown that Fra-1, like Fos, binds to the AP-1 recognition element cooperatively with Jun. A truncated Fra-1 protein that contains the leucine zipper region but not an adjacent basic amino acid domain, complexes with Jun in vitro but fails to bind AP-1 oligonucleotides. These results demonstrate that Fra-1 contributes to the DNA-binding activity ascribed to transcription factor AP-1.
Journal of Biological Chemistry, 2001
Heterodimeric transcription regulatory proteins can bind to palindromic recognition elements in two opposite orientations. We have developed a gel-based fluorescence resonance energy transfer (gelFRET) assay for quantifying heterodimer orientation preferences. Fos-Jun heterodimers bind in opposite orientations to AP-1 sites with different flanking sequences. The effects of individual amino acid and base pair substitutions on heterodimer binding orientation were quantified. Base pairs at positions ±6 and ±10 relative to the center of the AP-1 site were the principal determinants of Fos-Jun binding orientation. Amino acid residues of opposite charge adjacent to the basic regions of Fos and Jun had independent effects on heterodimer orientation. Exchange of these amino acid residues between the bZIP domains of Fos and Jun reversed the binding orientation. Heterodimers formed by full-length Fos and Jun exhibited the same changes in binding orientation in response to amino acid and base pair substitutions. The preferred orientation of heterodimer binding affected the stability of Fos-Jun-NFAT1 complexes at composite regulatory elements. Changes in heterodimer orientation preference altered the transcriptional activity and the promoter selectivity of Fos-Jun-NFAT1 complexes. Consequently, the orientation of Fos-Jun binding can influence transcriptional activity by altering cooperative interactions with other transcription regulatory proteins. by guest on March 13, 2016 http://www.jbc.org/ Downloaded from Ramirez-Carrozzi and Kerppola 3 Regulation of the transcriptional activities of the myriad of genes in mammalian genomes requires combinatorial interactions among multiple transcription regulatory proteins within promoter and enhancer regions (reviewed in 1). Whether the regulation occurs at the level of chromatin remodeling, coactivator recruitment, or assembly of the transcription machinery, specification of a unique target in the genome requires cooperation among multiple transcription factors (2-5). The cooperative interactions among many transcription regulatory proteins require juxtaposition of contact surfaces that are either part of, or tightly coupled to the DNA binding domain (6-9). Interactions among such proteins generally require a specific spacing and orientation of the DNA recognition sequences to allow cooperative complex formation. Many mammalian transcription regulatory proteins form heterodimers that recognize palindromic DNA sequence elements. Such heterodimers can potentially bind to their recognition sequences in either of two opposite orientations. Heterodimers that bind in opposite orientations can differ in their interactions with transcription factors that bind to adjacent regulatory elements. The binding orientations of such heterodimers may be controlled by recognition of asymmetric base pairs flanking the palindromic core sequence element, or through interactions with other transcription factors (8,10-12). Promoter elements are classically defined based on their position-and orientationdependent transcriptional activities. The orientation of heterodimer binding to asymmetric regulatory elements can influence their transcriptional activities (12-15). The mechanisms whereby the orientation of heterodimer binding influences transcriptional activity remain to be characterized. Fos and Jun are members of the bZIP family of transcription factors and bind to palindromic AP-1 regulatory elements. The X-ray crystal structure of the bZIP domains of Fos and Jun bound to the AP-1 site revealed that the heterodimer could bind to the recognition element in both orientations (16). In solution, Fos-Jun heterodimers bind to different AP-1 sites in opposite preferred orientations (11,17,18). The orientation of heterodimer binding is affected by sequences flanking the palindromic by guest on March 13, 2016 http://www.jbc.org/ Downloaded from Ramirez-Carrozzi and Kerppola 4 core AP-1 recognition element and amino acid residues adjacent to the basic regions of Fos and Jun (11). No direct contacts between the amino acid residues adjacent to the basic regions of Fos-Jun and the base pairs flanking the AP-1 site are observed in the X-ray crystal structure (16). Thus, the orientation of Fos-Jun binding appears to be controlled by indirect recognition of differences in DNA structure between flanking sequences on opposite sides of the AP-1 site. Fos and Jun activate different genes in different cell types and in response to different extracellular signals (19). These differences in Fos-Jun regulatory specificity are mediated at least in part by cooperative interactions with structurally unrelated transcription factors. Fos-Jun heterodimers can physically and functionally interact with members of the NFAT, Ets, Smad, and nuclear hormone receptor transcription factor families (9,20,21). The interaction between Fos-Jun and NFAT1 (NFATp, NFATc2) has been characterized in greatest detail. Cooperative binding by Fos-Jun and NFAT1 to composite regulatory elements in cytokine gene promoters requires a specific orientation of Fos-Jun binding (8,10). The X-ray crystal structure of the Fos-Jun-NFAT1 complex at the ARRE2 element shows a specific contact interface between NFAT1 and one face of the leucine zipper (7). The interaction with NFAT1 can reverse the orientation of heterodimer binding (8). However, the preferred orientation of Fos-Jun binding may influence the stability or the transcriptional activity of Fos-Jun-NFAT1 complexes. We have investigated the structural basis and the functional significance of the opposite orientations of Fos-Jun heterodimer binding at different AP-1 sites. To compare the effects of individual base pairs and amino acid residues on heterodimer orientation, we developed an approach for determination of the free energy of Fos-Jun heterodimer reorientation at different AP-1 sites. The functional consequences of opposite orientations of Fos-Jun heterodimer binding were examined by comparing the stabilities and transcriptional activities of Fos-Jun-NFAT1 complexes formed by heterodimers with opposite orientation preferences. by guest on March 13, 2016 http://www.jbc.org/ Downloaded from Ramirez-Carrozzi and Kerppola 5 Experimental Procedures
A functional interaction between ATF7 and TAF12 that is modulated by TAF4
Oncogene, 2005
The ATF7 proteins, which are members of the cyclic AMP responsive binding protein (CREB)/activating transcription factor (ATF) family of transcription factors, display quite versatile properties: they can interact with the adenovirus E1a oncoprotein, mediating part of its transcriptional activity; they heterodimerize with the Jun, Fos or related transcription factors, likely modulating their DNA-binding specificity; they also recruit to the promoter a stress-induced protein kinase (JNK2). In the present study, we investigate the functional relationships of ATF7 with hsTAF12 (formerly hsTAF II 20/15), which has originally been identified as a component of the general transcription factor TFIID. We show that overexpression of hsTAF12 potentiates ATF7-induced transcriptional activation through direct interaction with ATF7, suggesting that TAF12 is a functional partner of ATF7. In support of this conclusion, chromatin immunoprecipitation experiments confirm the interaction of ATF7 with TAF12 on an ATF7-responsive promoter, in the absence of any artificial overexpression of both proteins. We also show that the TAF12-dependent transcriptional activation is competitively inhibited by TAF4. Although both TAF12 isoforms (TAF12-1 and -2, formerly TAF II 20 and TAF II 15) interact with the ATF7 activation region through their histone-fold domain, only the largest, hsTAF12-1, mediates transcriptional activation through its N-terminal region.