Jun/Fos heterodimer functions as the transcriptional activator AP1

Rel/NF-kappaB transcription factors and IkappaBalpha (Drosophila homolog: Cactus) function in an autoregulatory network. Avian IkappaBalpha transcription is increased in response to both c-Rel and v-Rel. IkappaBalpha transcription is synergistically stimulated by Rel (Drosophila homolog: Dorsal) and AP-1 factors (c-Fos and c-Jun). A 386 bp region of the IkappaBalpha promoter (containing two NF-kappaB and one AP-1 binding sites) is necessary and sufficient for response to both Rel factors alone or Rel factors in conjunction with the AP-1 proteins. In addition, an imperfect NF-kappaB binding site is found to overlap the AP-1 binding site. Mutation of either of the NF-kappaB binding sites or the AP-1 binding site dramatically decreases the response of the IkappaBalpha promoter to Rel proteins alone or Rel and AP-1 factors. Overexpression of c-Rel results in the formation of DNA binding complexes associates with the imperfect NF-kappaB binding site which overlaps the AP-1 site. v-Rel associated with the imperfect NF-kappaB site stronger than c-Rel, and overexpression of v-Rel also results in the formation of a v-Rel containing complex bound to a consensus AP-1 site (Kralova, 1996).

Transcription factor AP-1 is constituted by the products of the various fos and jun genes. AP-1 activity is modulated by second messengers and appears to involve post-translational modifications of FOS and JUN. It has been shown that phosphorylation mediated by glycogen synthase kinase 3 (the mammalian homolog of shaggy) is involved in negative regulation of c-Jun DNA-binding function in vitro. Two forms of GSK-3 decrease DNA-binding activity as well as transcriptional activation elicited by c-Jun in vivo. Similarly, the other members of the Jun family, JunB, JunD and v-Jun, are negatively regulated by GSK-3 in vivo, although to a slightly lesser extent than c-Jun. The Shaggy protein of Drosophila shares homology with the mammalian GSK-3. The product of the sgg gene can function as negative regulators of Jun/AP-1 (de Groot, 1993).

An important first step in the chromatin remodelling process is the initial binding of a transcriptional activator to a nucleosomal template. An investigation sought to determine the ability of AP-1, the Fos/Jun heterodimer, to interact with its cognate binding site located in the promoter region of the mouse fos-related antigen-2 ( the fra-2 promoter), when this site was reconstituted into a nucleosome. Two different nucleosome assembly systems were employed to assemble either principally non-acetylated or acetylated nucleosomes. Fos/Jun heterodimer interactive capability differs markedly with either an acetylated or an unacetylated nucleosome: Fos/Jun binds to an unacetylated nucleosome with only a 4- to 5-fold reduction in DNA binding affinity as compared with naked DNA. Strikingly, the binding of Fos/Jun to a single high-affinity site incorporated into an acetylated nucleosome results in the complete disruption of nucleosomal structure without histone displacement. This disruption is sufficient to facilitate the subsequent binding of a second transcription factor. It is suggested that the disruption reported here, which is not energy dependent, involves a change in the conformation of a nucleosome produced by acetylated histones H3 and H4. This change of conformation alters the nucleosome structure sufficiently to modify the DNAseI sensitivity of the DNA segment (Ng, 1997).

Jun transcriptional targets

JUN and ETS (Drosophila homologs: Pointed and Yan) are required for the regulation of Tumor Necrosis Factor. Specific binding of ETS and JUN to their respective elements has been demonstrated by competition analysis as well as by supershift assays. As shown by promoter deletion analysis, these two binding sites are essential for both basal promoter activity and responsiveness to the phorbol ester phorbol 12-myristate 13-acetate (Kramer, 1995).

Transcriptional regulation by transforming growth factor beta (TGF-beta) is a complex process which is likely to involve cross talk between different DNA responsive elements and transcription factors to achieve maximal promoter activation and specificity. This work has uncovered a concurrent requirement for two discrete responsive elements in the regulation of the c-Jun promoter: one, a binding site for a Smad3-Smad4 complex and the other an AP-1 binding site. The two elements are located 120 bp apart in the proximal c-Jun promoter, and each is able to independently bind its corresponding transcription factor complex. The effects of independently mutating each of these elements are nonadditive; disruption of either sequence results in complete or severe reductions in TGF-beta responsiveness. This simultaneous requirement for two distinct and independent DNA binding elements suggests that Smad and AP-1 complexes function synergistically to mediate TGF-beta-induced transcriptional activation of the c-Jun promoter (Wong, 1999).

Biosynthesis of tumor necrosis factor-alpha (TNF-alpha) is carried out predominantly by cells of the monocytic lineage. This study examined the role of various cis-acting regulatory elements in the lipopolysaccharide (LPS) induction of the human TNF-alpha promoter in cells of monocytic lineage. In one region [-182 to -37 base pairs (bp)] the TNF-alpha promoter possesses enhancer elements that are required for optimal transcription of the TNF-alpha gene in response to LPS. Two regions were identified: region I (-182 to -162 bp) contains an overlapping Sp1/Egr-1 site, and region II (-119 to -88) contains CRE and NF-kappaB (designated kappaB3) sites. The following were all found to bind to the CRE site: unstimulated THP-1, CRE-binding protein and, to a lesser extent, c-Jun complexes. LPS stimulation increases the binding of c-Jun-containing complexes. In addition, LPS stimulation induces the binding of cognate nuclear factors to the Egr-1 and kappaB3 sites, which were identified as Egr-1 (Drosophila homolog Stripe) and p50/p65, respectively. The CRE and kappaB3 sites in region II together confer strong LPS responsiveness to a heterologous promoter, whereas individually they fail to provide transcriptional activation. Increasing the spacing between the CRE and the kappaB3 sites completely abolishes LPS induction, suggesting a cooperative interaction between c-Jun complexes and p50/p65. These studies indicate that maximal LPS induction of the TNF-alpha promoter is mediated by concerted participation of at least two separate cis-acting regulatory elements (Yao, 1997).

The c-jun proto-oncogene encodes a component of the mitogen-inducible immediate-early transcription factor AP-1 and has been implicated as a positive regulator of cell proliferation and G1-to-S-phase progression. Fibroblasts derived from c-jun-/- mouse fetuses exhibit a severe proliferation defect and undergo a prolonged crisis before spontaneous immortalization. The cyclin D1- and cyclin E-dependent kinases (CDKs) and transcription factor E2F are poorly activated, resulting in inefficient G1-to-S-phase progression. Furthermore, the absence of c-Jun results in elevated expression of the tumor suppressor gene p53 and its target gene, the CDK inhibitor p21, whereas overexpression of c-Jun represses p53 and p21 expression and accelerates cell proliferation. Surprisingly, protein stabilization, the common mechanism of p53 regulation, is not involved in up-regulation of p53 in c-jun-/- fibroblasts. Rather, c-Jun regulates transcription of p53 negatively by direct binding to a variant AP-1 site in the p53 promoter. Importantly, deletion of p53 abrogates all defects of cells lacking c-Jun in cell cycle progression, proliferation, immortalization, and activation of G1 CDKs and E2F. These results demonstrate that an essential, rate-limiting function of c-Jun in fibroblast proliferation is negative regulation of p53 expression, and establish a mechanistic link between c-Jun-dependent mitogenic signaling and cell-cycle regulation (Schreiber, 1999).

Tissue factor (TF) is induced in THP-1 cells stimulated with lipopolysaccharide (LPS). DNase I footprinting identifies six sites of protein-DNA interaction between -383 and the cap site that varies between control and induced extracts. Four footprints show qualitative differences in nuclease sensitivity. Footprints I (-85 to -52) and V (-197 to -175) are induction-specific and localize to regions of the promoter that mediate serum, phorbol ester, partial LPS response (-111 to +14), and the major LPS-inducible element (-231 to -172). Electrophoretic mobility shift assays with the -231 to -172 probe demonstrate JunD and Fos binding in both control and induced nuclear extracts; however, binding of c-Jun is only detected following LPS stimulation. Antibody inhibition studies implicate binding of Ets-1 or Ets-2 to the consensus site between -192 and -177, a region that contains an induction-specific footprint. The proximal region (-85 to -52), containing the second inducible footprint, binds Egr-1 following induction. These data suggest that LPS stimulation of THP-1 cells activate binding of c-Jun, Ets, and Egr-1 to the TF promoter and implicates these factors in the transcriptional activation of TF mRNA synthesis (Groupp, 1997).

Tumor necrosis factor alpha (TNF alpha) is a key regulatory cytokine whose expression is controlled by a complex set of stimuli in a variety of cell types. The monocyte/macrophage-enriched nuclear transcription factor C/EBPbeta plays an important role in the regulation of the TNF alpha gene in myelomonocytic cells. Abundant evidence suggests that other transcription factors participate as well. Interactions between C/EBPbeta and c-Jun, a component of the ubiquitously expressed AP-1 complex have been analyzed. In phorbol myristate acetate (PMA)-treated Jurkat T cells, which does not possess endogenous C/EBPbeta, expression of c-Jun by itself has relatively little effect on TNF alpha promoter activity. However, the combination of C/EBPbeta and c-Jun is synergistic, resulting in greater than 130-fold activation. This effect requires both the leucine zipper and DNA binding domains, but not the transactivation domain, of c-Jun, plus the AP-1 binding site centered 102/103 bp upstream of the transcription start site in the TNF alpha promoter. To determine if C/EBPbeta and c-Jun might cooperate to regulate the cellular TNF alpha gene in myelomonocytic cells, an examination was made of U937 cells that possess endogenous C/EBPbeta, which were stably transfected with either wild-type c-Jun or the transactivation domain deletion mutant (TAM-67). U937 cells expressing either ectopic wild-type c-Jun or TAM-67 secreted over threefold more TNF alpha than the control line, in response to PMA plus lipopolysaccharide. Transient transfection of the U937 cells expressing TAM-67 suggests that TAM-67 binding to the -106/-99-bp AP-1 binding site cooperates with endogenous C/EBPbeta in the activation of the -120 TNF alpha promoter-reporter. DNA binding assays using oligonucleotides derived from the TNF alpha promoter suggested that C/EBPbeta and c-Jun interact in vitro and that the interaction may be DNA dependent. These data demonstrate that the TNF alpha gene is regulated by the interaction of the ubiquitous AP-1 complex protein c-Jun and the monocyte/macrophage-enriched transcription factor C/EBPbeta and that this interaction contributes to the expression of the cellular TNF alpha gene in myelomonocytic cells. This interaction is unique in that it does not require the c-Jun transactivation domain; it provides new insight into the cell-type-specific regulation of the TNF alpha gene (Zagariya, 1998).

In cooperation with an activated ras oncogene, the site-dependent AP-1 transcription factor c-Jun transforms primary rat embryo fibroblasts (REF). Although signal transduction pathways leading to activation of c-Jun proteins have been extensively studied, little is known about c-Jun cellular targets. c-Jun-upregulated cDNA clones homologous to the tenascin-C gene (see Drosophila Tenascin major) have been identified by differential screening of a cDNA library from REF. This tightly regulated gene encodes a rare extracellular matrix protein involved in cell attachment and migration and in the control of cell growth. Transient overexpression of c-Jun induces tenascin-C expression in primary REF and in FR3T3, an established fibroblast cell line. Surprisingly, tenascin-C synthesis is repressed after stable transformation by c-Jun, as compared to that in the nontransformed parental cells. As assessed by using the tenascin-C (-220 to +79) promoter fragment cloned in a reporter construct, the c-Jun-induced transient activation is mediated by two binding sites: one GCN4/AP-1-like site, at position -146, and one NF-kappaB site, at position -210. As demonstrated by gel shift experiments and cotransfections of the reporter plasmid and expression vectors encoding the p65 subunit of NF-kappaB and c-Jun, the two transcription factors bind and synergistically transactivate the tenascin-C promoter. Two other extracellular matrix proteins, SPARC and thrombospondin-1, are c-Jun targets. Thus, these results strongly suggest that the regulation of the extracellular matrix composition plays a central role in c-Jun-induced transformation (Mettouchi, 1997).

Cyclin A plays an essential role in the G1 to S phase transition of the cell cycle. The expression of cyclin A is restrained during G0 and G1, but steeply induced at the G1/S boundary. Analysis of the rat cyclin A promoter elements with the 5' sequential deletion derivatives of the promoter fused to the luciferase cDNA indicate that the ATF/CRE motif primary determines the of inducibility at G1/S. Gel shift analysis of the complex formed at the ATF/CRE site indicates that the complex was not formed with the G0/G1 cell extract, but maximally formed with the late-G1 cell extract. The complex is supershifted by anti-JunD antibody; Western blot analysis of the immune complexes prepared with anti-JunD antibody reveals the presence of ATF2, suggesting heterodimerization of JunD with ATF2. The cyclin A promoter in a reporter plasmid is activated nearly 10-fold in quiescent rat 3Y1 cells by cotransfection with the expression of plasmids encoding ATF2 and Jun family members. In contrast, cotransfection with the ATF4 expression plasmid suppresses the promoter activation mediated by ATF2 and Jun family members. The expression of Jun family members during G1 to S progression is induced biphasically in early and late G1 and the level of JunD increases markedly at the G1/S, while that of ATF family members is gradually increased along with the G1 to S progression. These results indicate that the cyclin A promoter activity is regulated, at least in part, by relative amounts of the ATF and Jun family members (Shimizu, 1998).

The cell cycle inhibitor protein p21WAF1/Cip1 (p21) is a critical downstream effector in p53-dependent mechanisms of growth control and p53-independent pathways of terminal differentiation. The transforming growth factor-beta pathway-specific Smad3 and Smad4 proteins transactivate the human p21 promoter via a short proximal region, which contains multiple binding sites for the ubiquitous transcription factor Sp1. The Sp1-occupied promoter region mediates transactivation of the p21 promoter by c-Jun and the related proteins JunB, JunD, and ATF-2. Gel electrophoretic mobility shift assays show that this region does not contain a binding site for c-Jun. In accordance with the DNA binding data, c-Jun is unable to transactivate the p21 promoter when overexpressed in the Sp1-deficient Drosophila-derived SL2 cells. Coexpression of c-Jun and Sp1 in these cells results in a strong synergistic transactivation of this promoter. In addition, a chimeric promoter consisting of six tandem high affinity Sp1-binding sites fused with the CAT gene is transactivated by overexpressed c-Jun in HepG2 cells. The above data suggest functional cooperation between c-Jun and Sp1. Physical interactions between the two factors have been demonstrated in vitro by using GST-Sp1 hybrid proteins expressed in bacteria and in vitro transcribed-translated c-Jun. The region of c-Jun mediating interaction with Sp1 maps within the basic region leucine zipper domain. In vivo, functional interactions between c-Jun and Sp1 have been demonstrated using a GAL4-based transactivation assay. Overexpressed c-Jun transactivates only a chimeric promoter consisting of five tandem GAL4-binding sites when coexpressed with GAL4-Sp1-(83-778) fusion proteins in HepG2 cells. By utilizing the same assay, it was found that the glutamine-rich segment of the B domain of Sp1 (Bc, amino acids 424-542) is sufficient for c-Jun-induced transactivation of the p21 promoter. In conclusion, these data support a mechanism for superactivation of Sp1 by c-Jun that is based on physical and functional interactions between these two transcription factors on the human p21 and possibly other Sp1-dependent promoters (Kardassis, 1999).

The effects of the pituitary adenylase cyclate-activating peptides (PACAP) 27 and 38 on proenkephalin (PENK) gene transcription were examined in PC12 (rat pheochromocytoma) cells using transient transfection assays. Both ligands stimulate PENK gene transcription in a dose-dependent manner, with an apparent ED50 close to 5 x 10(-11) M. Inactivation of cAMP dependent-protein kinase (PKA) with a dominant inhibitory mutant strongly reduces PACAP-stimulated PENK transcription. Using reporter genes driven by either the minimal TPA-responsive element (TRE: TGACTCA) or cAMP-responsive element (CRE: TGACGTCA), it has been shown that the two PACAPs activate transcription through both regulatory sequences. These effects could result from direct post-translational activation of Jun and CREB, as shown using GAL4-Jun or GAL4-CREB fusion proteins. Expression of a dominant inhibitory mutant of CREB decreases by 60% the response to PACAP, suggesting that CREB is implicated in PENK transactivation. Similarly, expression of c-fos antisense RNA reduces by 80% the stimulatory effects of PACAP. Taken together, these results indicate that PACAP stimulates PENK transcription by members of both the AP1 and the CREB families. However, AP1 by itself is not sufficient to increase PENK transcription, as insulin-like growth factor 1 (IGF1), which stimulates AP1 activity but not cAMP production, is unable to stimulate PENK transcription. These results indicate a cooperative effect of AP1 and CREB on PENK transcription (Monnier, 1998).

Smad proteins transduce signals for transforming growth factor-beta (TGF-beta)-related factors. Smad proteins activated by receptors for TGF-beta form complexes with Smad4. These complexes are translocated into the nucleus and regulate ligand-induced gene transcription. 12-O-tetradecanoyl-13-acetate (TPA)-responsive gene promoter elements (TREs) are involved in the transcriptional responses of several genes to TGF-beta. AP-1 transcription factors, composed of c-Jun and c-Fos, bind to and direct transcription from TREs, which are therefore known as AP1-binding sites. Smad3 interacts directly with the TRE and Smad3 and Smad4 can activate TGF-beta-inducible transcription from the TRE in the absence of c-Jun and c-Fos. Smad3 and Smad4 also act together with c-Jun and c-Fos to activate transcription in response to TGF-beta, through a TGF-beta-inducible association of c-Jun with Smad3 and an interaction of Smad3 and c-Fos. These interactions complement interactions between c-Jun and c-Fos, and between Smad3 and Smad4. This mechanism of transcriptional activation by TGF-beta, through functional and physical interactions between Smad3-Smad4 and c-Jun-c-Fos, shows that Smad signaling and MAPK/JNK signaling converge at AP1-binding promoter sites (Zhang, 1998).

T lymphocytes undergo apoptosis in response to a variety of stimuli, including exposure to UV radiation and gamma-irradiation. While the mechanism by which stress stimuli induce apoptosis is not well understood, the induction of Fas ligand (FasL) gene expression by environmental stress stimuli is dependent on c-Jun N-terminal kinase (JNK) activation. Using inducible dominant-active (DA) JNK kinase kinase (MEKK1) expression in Jurkat cells, a specific MEKK1-regulated response element has been mapped to positions -338 to -316 of the Fas ligand (FasL) promoter. Mutation of this response element abrogates MEKK1-mediated FasL promoter activation and interferes in stress-induced activation of the promoter. Activator protein 1 (AP-1) binding proteins [namely, activating transcription factor 2 (ATF2) and c-Jun], bind to the MEKK1 response element. Transient transfection of interfering c-Jun and ATF2 mutants, which lack the consensus JNK phosphorylation sites, abrogates the transcriptional activation of the FasL promoter, demonstrating the involvement of c-Jun and ATF2 in the regulation of the FasL promoter. Taken together, these data indicate that MEKK1 and transcription factors regulated by the JNK pathway play a role in committing lymphocytes to undergo apoptosis by inducing FasL expression via a novel response element in the promoter of that gene (Faris, 1998).

The transcription factor AP-1, composed of Jun and Fos pro teins, is a major target of mitogen-activated signal transduction pathways. However, little is known about AP-1 function in normal cycling cells. The quantity and the phosphorylation state of the c-Jun and JunB proteins vary at the M-G1 transition. Phosphorylation of JunB by the p34cdc2-cyclin B kinase is associated with lower JunB protein levels in mitotic and early G1 cells. In contrast, c-Jun levels remain constant while the protein undergoes N-terminal phosphorylation, increasing its transactivation potential. Since JunB represses and c-Jun activates the cyclin D1 promoter, these modifications of AP-1 activity during the M-G1 transition could provide an impetus for G1 progression by a temporal increase in cyclin D1 transcription. These findings constitute a novel example of a reciprocal connection between transcription factors and the cell cycle machinery (Bakiri, 2000).

The decrease in the concentration of JunB relative to c-Jun and the N-terminal phosphorylation of c-Jun at the beginning of the G1 phase could result in increased AP-1 activity and the induction of genes that respond better to c-Jun. A number of putative AP-1 target genes are known. Among them, cyclin D1 is interesting since it is synthesized early in the G1 phase and is required for progression to S phase. c-Jun has also been reported to induce cyclin D1 transcription in transient transfection assays. The human cyclin D1 transcriptional control sequences have been cloned and shown to contain different regulatory elements, including a TRE site and a CRE site, located 935 and 52 bp upstream of the initiation site, respectively. In exponentially cycling cells, transcriptional activation by c-Jun has been shown to be mediated essentially by these two sites, and Jun, Fos and ATF proteins might be part of a complex bound to this site. Using cyclin D1 promoter constructs containing both sites for transient transfection in HeLa cells, different responses to JunB and c-Jun were observed. While c-Jun strongly activates the cyclin D1 reporter, JunB repressed it in a dose-dependent manner. In contrast, JunB reproducibly activates a collagenase promoter construct containing the canonical TRE element, although this activation is weak compared with c-Jun. Furthermore, the strong activation by c-Jun is inhibited by co-expression of JunB. However, the inhibition of the cyclin D1 promoter is more pronounced than that of the collagenase promoter. When c-Jun N-terminal phosphorylation was mimicked by using a c-Jun protein in which serines 63 and 73 are replaced by aspartic acid, the ability of c-Jun to activate cyclin D1 transcription increases. In contrast, replacing these residues with leucines, which blocks c-Jun phosphorylation, strongly decreases its transcriptional activation of the cyclin D1 promoter. This c-JunLL protein exhibites activity similar to JunD, which is a weak activator of cyclin D1. Finally, c-Jun and JunB seem to affect the same sites on the cyclin D1 promoter since only the mutation of both the proximal CRE and the distal TRE could abolish the effect of the two proteins (Bakiri, 2000).

Another important point with respect to the effect of AP-1 on transcription is that, in the same cellular system and depending on the promoter context, two opposite effects of JunB have been observed: JunB-ER activation reproducibly induces a collagenase reporter construct while it represses the cyclin D1 reporter and RNA. This is an interesting example of differential regulation of two target genes by the same AP-1 component. JunB, like c-Jun, can form both active and inactive AP-1 dimers, depending not only on the nature of its partner (Fos, Jun or ATF) but also on the targeted promoter sequence. Since the AP-1-responsive elements in cyclin D1 and in the collagenase promoters are different, the opposing effects of JunB on these two genes could be explained by differences in cooperation with other transcription factors. The fact that JunB can activate or inhibit transcription depending on the promoter and partner context may extend to other genes and could contribute to the complexity of the genetic response to AP-1 (Bakiri, 2000).

Cooperation between nuclear factor of activated T cells (NFAT: Drosophila homolog: CG11172) and AP-1 (Fos-Jun) proteins on composite NFAT-AP-1 DNA elements constitutes a powerful mechanism for signal integration of the calcium and protein kinase C/Ras pathways in the regulation of gene expression. NFAT can induce expression of certain genes in T cells without the need for cooperative recruitment of Fos and Jun. Using NFAT1 mutant proteins that are unable to interact with Fos-Jun dimers but are unaffected in DNA binding or transcriptional activity, it has been shown that expression of interleukin (IL)-2, granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-3, IL-4, MIP1alpha and Fas ligand mRNAs are all absolutely dependent on cooperation between NFAT and Fos-Jun; in contrast, NFAT induces tumor necrosis factor alpha mRNA and IL-13 promoter activity without any necessity to recruit Fos and Jun. Furthermore, NFAT-Fos-Jun cooperation is also essential to elicit the NFAT-dependent program of activation-induced cell death. These results support the hypothesis that even in a single cell type, NFAT activation can evoke two distinct biological programs of gene expression, dependent or independent of NFAT-AP-1 cooperation (Macian, 2000).

Zygotic expression of the BMP-4 gene in Xenopus embryos is regulated by an auto-regulatory loop. Since AP-1 is known as a mediator of auto-regulatory loops both in the case of the Drosophila dpp and the mammalian TGF-beta genes, the potential of Xenopus c-Jun (AP-1) as a mediator of BMP-4 expression during Xenopus development was analyzed. RNA injection experiments reveal that both heteromeric c-Fos/c-Jun and homodimeric c-Jun/c-Jun strongly activate BMP-4 transcription, whereas BMP signaling activates the Xenopus c-Jun gene only to a rather low extent. In addition, the lack of zygotic c-Jun transcripts until the end of gastrulation should exclude a role of AP-1 in the activation and the early expression of BMP-4 during gastrulation in vivo. However, at later stages of Xenopus development, a spatial overlap of c-Jun and BMP-4 transcripts was found which suggests that AP-1 might serve as an additional activatory component for the auto-regulation of BMP-4. Promoter/reporter and gel mobility shift assays demonstrate multiple responsive sites for AP-1 in the 5' flanking region and two in the second intron of the BMP-4 gene. AP-1 acts independently of Xvent-2, which mediates the early expression of BMP-4 in gastrula stage embryos. In summary, it has been shown both for the wild type gene and for promoter/reporter constructs that the Xenopus BMP-4 gene is activated by c-Jun (AP-1). Corresponding target sites are localized in the 5' flanking region and in the second intron. Results obtained from biological and molecular investigations lead to the conclusion that c-Jun (AP-1) is a transcriptional activator of the BMP-4 gene and, since the c-Jun gene is weakly activated by BMP signaling, c-Jun is another potential mediator of the BMP-4 auto-regulatory loop (Knochel, 2000).

Dickkopf-1 (Dkk-1) is a potent inhibitor of Wnt/ß-catenin signaling. Expression of Dkk-1 overlaps significantly with the sites of programmed cell death in normal as well as mutant vertebrate limb development. Several of Dkk-1's upstream regulators, one of which is Bmp-4, have been identified. Interestingly, Bmp-4 activates Dkk-1 only when it concomitantly induces apoptosis. Moreover, Dkk-1 is heavily up-regulated by UV irradiation and several other genotoxic stimuli. Normal expression of Dkk-1 is dependent on the Ap-1 family member c-Jun and overexpression of Dkk-1 enhances Bmp-triggered apoptosis in the vertebrate limb. Taken together, these results provide evidence for an important role of Dkk-1-mediated inhibition of Wnt/ß-catenin signaling in response to different stress signals that all converge on the activation of c-Jun in vivo (Gotewold, 2002).

It is propose that the c-Jun-mediated activation of Dkk-1 is fundamental for Bmp-induced apoptosis. The cytoplasmic kinase TAK-1 has been reported to be essential for Bmp-2-induced apoptosis, and Bmp-4 can also directly activate this kinase. There are several further implications for TAK-1 in apoptosis. Importantly, overexpression of TAK-1 in the Drosophila visual system leads to ectopically induced apoptosis mediated by JNK. Enhanced apoptosis has also been observed in transgenic frogs and mice overexpressing TAK-1. TAK-1 activates Jnk signaling, which in turn activates c-Jun. Transcription of c-Jun is then autoregulated by the c-Jun protein, the overexpression of which is sufficient to induce apoptosis. This cascade might provide the link between Bmp and the induction of c-Jun reported in this study. It is suggested that the predominant activation of a particular intracellular signaling cascade downstream of the Bmp receptor also contributes to the different effects that Bmps have on limb mesodermal cells. According to this model, Bmp would induce apoptosis when the Bmp/Jnk pathway dominates the Bmp/Smad pathway to activate certain genes, as is the case for Dkk-1. Further support for this model comes from a study showing that the distortion of positional information determined by dpp and wg signaling gradients leads to the activation of the Drosophila JNK apoptotic pathway, which subsequently induces cell death in the Drosophila wing. This pathway is likely to be latent in normal wing development, but is activated upon abnormal dpp signaling to maintain proper development. Thus, the possibility that this pathway might only be used upon inappropriate signaling cannot be ruled out (Gotewold, 2002).

Ligand-dependent transcription by the nuclear receptor glucocorticoid receptor (GR) is mediated by interactions with coregulators. The role of these interactions in determining selective binding of GR to regulatory elements remains unclear. Recent findings indicate that a large fraction of genomic GR binding coincides with chromatin that is accessible prior to hormone treatment, suggesting that receptor binding is dictated by proteins that maintain chromatin in an open state. Combining DNaseI accessibility and chromatin immunoprecipitation with high-throughput sequencing, the activator protein 1 (AP1) was identified as a major partner for productive GR-chromatin interactions. AP1 is critical for GR-regulated transcription and recruitment to co-occupied regulatory elements, illustrating an extensive AP1-GR interaction network. Importantly, the maintenance of baseline chromatin accessibility facilitates GR recruitment and is dependent on AP1 binding. A model is proposed in which the basal occupancy of transcription factors acts to prime chromatin and direct inducible transcription factors to select regions in the genome (Biddie, 2011).

Activator protein-1 (AP-1) is a mediator of BMP or FGF signaling during Xenopus embryogenesis. However, specific role of AP-1 in activin signaling has not been elucidated during vertebrate development. This study provides new evidence showing that overexpression of heterodimeric AP-1 comprised of c-jun and c-fos [AP-1(c-Jun/c-Fos)] induces the expression of BMP-antagonizing organizer genes (noggin, chordin and goosecoid) that were normally expressed by high dose of activin. AP-1(c-Jun/c-Fos) enhanced the promoter activities of organizer genes but reduced that of PV.1, a BMP4-response gene. A loss of function study clearly demonstrated that AP-1(c-Jun/c-Fos) is required for the activin-induced organizer and neural gene expression. Moreover, physical interaction of AP-1(c-Jun/c-Fos) and Smad3 cooperatively enhanced the transcriptional activity of goosecoid via direct binding on this promoter. Interestingly, Smad3 mutants at c-Jun binding site failed in regulation of organizer genes, indicating that these physical interactions are specifically necessary for the expression of organizer genes. In is concluded that AP-1(c-Jun/c-Fos) plays a specific role in organizer gene expression downstream of activin signal during early Xenopus embryogenesis (Lee, 2011).

Jun interaction with TFIID

Sequence-specific DNA-binding activators, key regulators of gene expression, stimulate transcription in part by targeting the core promoter recognition TFIID complex and aiding in its recruitment to promoter DNA. Although it has been established that activators can interact with multiple components of TFIID, it is unknown whether common or distinct surfaces within TFIID are targeted by activators and what changes if any in the structure of TFIID may occur upon binding activators. As a first step toward structurally dissecting activator/TFIID interactions, the three-dimensional structures of TFIID bound to three distinct activators (i.e., the tumor suppressor p53 protein, glutamine-rich Sp1 and the oncoprotein c-Jun) was determined and their structures were compared as determined by electron microscopy and single-particle reconstruction. By a combination of EM and biochemical mapping analysis, these results uncover distinct contact regions within TFIID bound by each activator. Unlike the coactivator CRSP/Mediator complex that undergoes drastic and global structural changes upon activator binding, instead, a rather confined set of local conserved structural changes were observed when each activator binds holo-TFIID. These results suggest that activator contact may induce unique structural features of TFIID, thus providing nanoscale information on activator-dependent TFIID assembly and transcription initiation (Liu, 2009).

Three D density difference maps generated from reconstructions of the three independent activator/TFIID assemblies (i.e., p53-IID, Sp1-IID, and c-Jun-IID) and free holo-TFIID have served as a method to map the most likely contact sites of these activators within the native TBP-TAF complex. Remarkably, each activator contacts TFIID via select TAF interfaces within TFIID. The unique and localized arrangements of these three activators contacting different surfaces of TFIID could be indicative of the wide diversity of potential activator contact points within TFIID that would be dependent on both the specificity of activation domains as well as core promoter DNA sequences appended to target gene promoters. It is also possible, however, that these distinct activator-TFIID contacts can form a common scaffold when TFIID binds to the core promoter DNA (Liu, 2009).

It is well established that activators including p53, Sp1, and c-Jun frequently work synergistically with each other or other activators to potentiate selective gene expression programs in response to a variety of stimuli in vivo. Therefore, combinatorial mechanisms of promoter activation might favor distinct nonoverlapping activator-binding sites within TFIID, which can be achieved by specific interactions between selective TAF subunits and activators. Indeed, it was established that TAF1 and TAF4 serve as coactivators for Sp1, while TAF1, TAF6, and TAF 9 mediate p53-dependent transactivation and TAF1 and TAF7 subunits are thought to be coactivators for c-Jun. Since activators make sequence-specific contacts with the DNA template at various positions upstream of the core promoter, it is also plausible that activators bound to unique surfaces of TFIID can influence specific structures of a promoter as the DNA traverses along TFIID resulting in distinct activator/promoter DNA structures (Liu, 2009).

Activator mapping results also complement and structurally extend the functional relevance of previous biochemical and immunomapping studies of TFIID. For example, label transfer studies show that the N-terminal activation domain of p53 contacts TAF6, confirming previous biochemical evidence showing that amino acids 1-42 of p53 contact TAF6/9. In support of this observation, the p53-IID 3D structure indicates that p53 contacts TFIID at lobes A and C where TAF6/9 are located as determined by EM immunomapping. In addition, previous studies have shown that both TBP and TAF1 can directly contact p53 in the absence of additional TFIID subunits. Interestingly, body-labeled p53 cross-linked to TAF1, TAF5, and weakly to TBP, thus extending the immunomapping studies that determined the locations of TBP and the N terminus of TAF1 at lobe C. Thus, EM activator mapping studies show a significant interface between p53 and specific TAFs located at lobes A and C of TFIID. Likewise, Sp1 label transfer results confirmed previous biochemical data showing a direct interaction between TAF4 and the N-terminal glutamine-rich domains of Sp1. In addition to TAF4, TAF6 was identified as weakly cross-linked to Sp1, suggesting that TAF6 may also be in the vicinity but perhaps more distal to the N terminus of Sp1. The largest TFIID subunit, TAF1, was cross-linked when body-labeled Sp1 was used. This result was not entirely unexpected, since previous studies found that TAF1 is required for Sp1-dependent transactivation, possibly through a direct interaction between TAF1 and Sp1 (Liu, 2009).

In comparison with p53 and Sp1, body-labeled c-Jun was shown to contact TAF1 and TAF6 in label transfer studies with no subunits contacting the N-terminal activation domain of c-Jun. This N-terminal activation domain of c-Jun may be structurally flexible or predominantly unstructured and is apparently positioned away from TFIID contacts. Indeed, successful structural studies of c-Jun thus far have been limited to the C-terminal leucine zipper DNA-binding region when bound to DNA. Previous biochemical assays have shown that the C-terminal basic leucine zipper DNA-binding region also contacts the N terminus of TAF1 (Liu, 2009).

It is worth noting that the extra density representing c-Jun and the other activator polypeptides in EM studies may not reflect the full-expected size of the activators. This is due to the presence of large unstructured regions in these proteins that are averaged out during structural analysis. As activators contain multiple molten globular domains that likely interact with different partners, one would expect a high degree of structural disorder in the domains that are not in direct contact with TFIID. Thus, the extra density associated with each activator determined from the single-particle reconstructions likely only represents minimally the most stably associated portion of activators bound to TFIID. This common situation would invariably lead to underrepresenting the actual size of the activator in a manner not unlike crystal structures of domains with flexible loops that become 'invisible' in the crystal structure (Liu, 2009).

Based on EM immunomapping, there are two copies of TAF6 within TFIID, wherein one copy resides in lobe A and another in lobe B. Collectively, the current studies suggest that two distinct activators (p53 and c-Jun) strongly contact the two different TAF6 subunits that are each located in different lobes of TFIID. It is unknown how p53 or c-Jun discriminates between TAF6 on lobe A versus B when binding to TFIID. In the future, it will be interesting to investigate if these two activators can bind to a single TFIID molecule simultaneously and decipher 3D structures of TFIID assemblies bound to select endogenous promoter DNA sequences in the presence and absence of distinct activators that are engaged in synergistic transcriptional activation (Liu, 2009).

It is of note that unlike the radical, diverse, and global structural changes observed with CRSP/Mediator complexes upon activator binding, TFIID largely retains its overall architecture when bound by three different activators. Interestingly, this study found that two of the activator/IID structures, p53-IID and Sp1-IID assemblies appear to be more constricted around the central cavity with narrower ChB-D and ChA-B channels, while the third structure, c-Jun-IID, remains most similar to free holo-TFIID. In particular, the p53-IID structure more closely resembles the closed conformational state of the previous cryo-TFIID structure. To test if p53-bound TFIID mimics the most closed conformational form of holo-TFIID, 3D reconstructions were performed using either the most closed or 'open' cryo-TFIID structures as an initial reference volume for refinement. Interestingly, it was found that both newly refined 3D structures generated from either the closed or open reference volume are fairly similar, with possibly a partial occupancy of p53 on lobe A. These findings suggest that the overall p53-TFIID structure tends to move toward the closed conformation with moderate movement at the outer tips of lobes A and B, even though p53-IID is predominantly observed in an intermediate average conformational form between the most closed and open forms. Perhaps factors contacting lobe A or C can induce certain coordinated movements within lobes that lead to a closed conformation of TFIID (Liu, 2009).

Although TFIID largely retains its prototypic global architecture upon activator binding, several common localized structural changes induced upon activator binding were observed in the 3D reconstruction. For example, a prominent and consistent induced extra density protrusion located in lobe D was observed when each of the three different activators binds TFIID. Given that all these activators are represented by distinct densities with unique sizes and shapes within the bound TFIID structure, and the fact that it has been demonstrated that they each can target different subunits within TFIID by a number of independent biochemical assays, it seems reasonable to assign 'unique and significant' extra densities located at distinct sites as representing the different bound activators. In contrast, the common similarly sized extra density seen at lobe D of each activator-IID structure most likely represents a conserved conformational change induced by these three different activators. Interestingly, this protrusion in lobe D resides distal to each of the activator-binding sites, suggesting that these three activators may potentially induce a long-range internal conformational change within TFIID. It would be intriguing to identify which TAF subunits are located at the tip of lobe D and eventually determine the function, if any, of this extended lobe in activator-induced transcription initiation. However, despite the potential significance of these structural changes induced by activators, it is premature to speculate regarding their functional importance (Liu, 2009).

Table of contents

DJun: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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