Interactive Fly, Drosophila

Fos-related antigen


Coactivators interfering with Fos functions

A short C-terminal sequence that is deleted in the v-ErbA oncoprotein (a C-terminally truncated form of the thyroid hormone receptor alpha [T3R alpha]) and conserved in members of the nuclear receptor superfamily, is required for normal biological function of its normal cellular counterpart, T3R alpha. An extensive mutational analysis was carried out in this region based on the crystal structure of the hormone-bound ligand binding domain of T3R alpha. Mutagenesis of either Leu398 or Glu401, which are surface exposed according to the crystal structure, completely blocks or significantly impairs T3-dependent transcriptional activation but does not affect or only partially diminishes interference with AP-1 activity. These are the first mutations that clearly dissociate these activities for T3R alpha. Substitution of Leu400, which is also surface exposed, does not affect interference with AP-1 activity and only partially diminishes T3-dependent transactivation. None of the mutations affect ligand-independent transactivation, consistent with previous findings that this activity is mediated by the N-terminal domain of T3R alpha. The loss of ligand-dependent transactivation for some mutants can largely be reversed in the presence of GRIP1 (a coactivator of T3Ralpha), which acts as a strong ligand-dependent coactivator for wild-type T3R alpha. There is excellent correlation between T3-dependent in vitro association of GRIP1 with T3R alpha mutants and their ability to support T3-dependent transcriptional activation. Therefore, GRIP1, previously found to interact with the glucocorticoid, estrogen, and androgen receptors, may also have a role in T3R alpha-mediated ligand-dependent transcriptional activation. When fused to a heterologous DNA binding domain, that of the yeast transactivator GAL4, the conserved C terminus of T3R alpha functions as a strong ligand-independent activator in both mammalian and yeast cells. However, point mutations within this region have drastically different effects on these activities compared to their effect on the full-length T3R alpha. It is concluded that the C-terminal conserved region contains a recognition surface for GRIP1 or a similar coactivator that facilitates its interaction with the basal transcriptional apparatus. While important for ligand-dependent transactivation, this interaction surface is not directly involved in transrepression of AP-1 activity. It is thought that transcripitonal interference between liganded nuclear receptors and AP-1 is due to competition for a cofactor that is required for efficient transactivation by either protein. Thus the C-terminal portion of T3R alpha has two functions: intertaction with Grip and interaction with a cofactor shared with AP-1 (Saatcioglu, 1997).

Down-regulation of c-Fos/c-Jun AP-1 dimer activity by sumoylation

The inducible transcriptional complex AP-1, composed of c-Fos and c-Jun proteins, is crucial for cell adaptation to many environmental changes. While its mechanisms of activation have been extensively studied, how its activity is restrained is poorly understood. Lysine 265 of c-Fos is shown to be conjugated by the peptidic posttranslational modifiers SUMO-1, SUMO-2, and SUMO-3 (see Drosophila SUMO), and c-Jun can be sumoylated on lysine 257 as well as on the previously described lysine 229. Sumoylation of c-Fos preferentially occurs in the context of c-Jun/c-Fos heterodimers. Using nonsumoylatable mutants of c-Fos and c-Jun as well as a chimeric protein mimicking sumoylated c-Fos, it has been shown that sumoylation entails lower AP-1 transactivation activity. Interestingly, single sumoylation at any of the three acceptor sites of the c-Fos/c-Jun dimer is sufficient to substantially reduce transcription activation. The lower activity of sumoylated c-Fos is not due to inhibition of protein entry into the nucleus, accelerated turnover, and intrinsic inability to dimerize or to bind to DNA. Instead, cell fractionation experiments suggest that decreased transcriptional activity of sumoylated c-Fos is associated with specific intranuclear distribution. Interestingly, the phosphorylation of threonine 232 observed upon expression of oncogenically activated Ha-Ras is known to superactivate c-Fos transcriptional activity. It also is shown to inhibit c-Fos sumoylation, revealing a functional antagonism between two posttranslational modifications, each occurring within a different moiety of a bipartite transactivation domain of c-Fos. Finally it is reported that the sumoylation of c-Fos is a dynamic process that can be reversed via multiple mechanisms. This supports the idea that this modification does not constitute a final inactivation step that necessarily precedes protein degradation (Bossis, 2005).

Growth factor regulation of Fos DNA binding

Granulocyte-macrophage colony-stimulating factor (GM-CSF) provokes a proliferative response and induction of early-response genes (such as c-fos) in target cells. It also induces rapid tyrosine phosphorylation of cellular proteins, including the beta subunit (betac) of its functional receptor. However, locations and functions of phosphorylated tyrosine residues within the betac are unclear. To elucidate the mechanism of the human GM-CSF receptor signal transduction, mutational analyses were made of the cytoplasmic domain of the beta-c, using murine BA/F3 cells. Deletion of the conserved box 1 motif results in loss of tyrosine phosphorylation of the betac, thereby indicating an essential role for this motif in activating the tyrosine kinase that phosphorylates betac. A C-terminal truncated mutant at position 589 activates the c-fos promoter, and this activation is diminished by a substitution at tyrosine 577 (Tyr577). However, the same substitution in the full-length betac does not completely abrogate the c-fos promoter activation, hence, redundant signaling pathways probably exist. When signaling molecules functioning downstream of the beta-c were examined, it was found that Tyr577 is essential for Shc phosphorylation, while tyrosine phosphorylation of PTP1D is mediated through Tyr577 as well as through other site(s). It is suggested that GM-CSF stimulates at least two modes of signals leading to Ras activation, an event which ultimately gives rise to promoter activation of c-fos (Itoh, 1996).

Interleukin-3 (IL-3) or granulocyte-macrophage colony-stimulating factor (GM-CSF) is known to activate JAK2 in various cells, but the role of JAK2 in IL-3 or GM-CSF receptor signal transduction is largely unknown. In BA/F3 cells expressing the human granulocyte-macrophage colony-stimulating factor receptor (hGMR), activation of JAK2 by hGM-CSF requires the box1 region of hGMR beta. Dominant negative JAK2 (delta JAK2), which lacks the kinase domain, suppresses mIL-3 or hGM-CSF-induced c-fos promoter activation as well as c-myc promoter activation/cell proliferation, thereby suggesting that JAK2 is involved in the signaling of both pathways. Further analyses of the role of JAK2 in c-fos gene activation in BA/F3 cells expressing hGMR reveals that delta JAK2 inhibits hGM-CSF-induced phosphorylation of Shc and protein tyrosine phosphatase 1D. Within hGMR beta, the several existing tyrosine residues are related either to activation of Shc or protein tyrosine phosphate 1D, and are phosphorylated in response to hGM-CSF stimulation. Delta JAK2 inhibits hGM-CSF-induced phosphorylation of hGMR beta. Taken together, these results suggest that JAK2 activated by the box1 region of hGMR mediates hGM-CSF-induced c-fos promoter activation through phosphorylation of hGMR (Watanabe, 1996).

Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, regulates survival and apoptosis of several neuronal populations. These effects are initiated by high-affinity membrane receptors displaying tyrosine kinase activity (trk). BDNF stimulates AP1 binding activity in primary cerebellar neurons. This binding corresponds to a functional complex, since it is associated with the induction of AP1-dependent transactivation. Application of AP1 partner mRNAs shows an increase in levels of c-fos and c-jun mRNAs after BDNF treatment, resulting from an induction of their promoters. The cis-acting elements by which BDNF stimulates c-fos transcription were further studied. BDNF impinges on multiple regulatory elements, including the serum-responsive element, Fos AP1-like element, and cyclic AMP (cAMP)-responsive element (CRE) sequences. The latter were stimulated without any detectable increase in cAMP or Ca2+ levels. To confirm that BDNF induces c-fos transcription independently of the protein kinase A/cAMP pathway, a dominant inhibitory mutant of the regulatory subunit of protein kinase A was transfected. The overexpression of this mutant does not affect the c-fos promoter transactivation by BDNF. Thus BDNF stimulates AP1- and CRE-dependent transcription through a mechanism that is distinct from the cAMP- and Ca(2+)-dependent pathways in CNS neurons (Gaiddon, 1996).

The immediate early genes are regulated by a variety of extracellular signals, including pleiotropic cytokines. The effects of the testicular cytokines [interleukin-6 (IL-6) and interferon-gamma (IFN-gamma)] on signal transducers and activators of transcription 3 and 1 (STAT-3 and STAT-1) and on c-fos gene expression in primary Sertoli cells are suggestive of their roles in differential function. Using the tyrosine phosphorylation inhibitor, genistein, and electrophoretic mobility shift assay, it has been shown that IL-6 and IFN-gamma induce nuclear factor STAT-3 and STAT-1 DNA-binding activity to the sis-inducible element of c-fos in a genistein-dependent pathway. Quantitative solution hybridization, Northern blot, and nuclear run-on analysis show that differential induction of c-fos, junB, and c-myc messenger RNA (mRNA) by these cytokines occur at transcriptional levels. IL-6 stimulates c-fos mRNA levels 6-fold while increasing junB levels 2-fold. IFN-gamma increases c-fos message 2-fold, but has no effect on junB mRNA levels. Furthermore, genistein treatment blocks the induction of c-fos and junB gene expression, demonstrating that tyrosine phosphorylation of STAT proteins is involved in the cytokine regulation of the Sertoli immediate early genes. H7, a serine/threonine phosphorylation inhibitor, also blocks c-fos gene induction by IL-6 and IFN-gamma, but does not affect the DNA-binding activities of STAT-3 and STAT-1. IL-6 treatment of Sertoli cells (3-6 h) increases the amounts of activating protein-1 binding to activating protein-1 element and c-myc transcription (Jenab, 1997).

Fos binding to DNA: DNA bending and chromatin restructuring

The transcription activation domains of both Fos and Jun induce DNA bending. In chimeric proteins, these same domains induce DNA bending independent of the DNA-binding domains. DNA bending by the chimeric proteins is directed diametrically away from the transcription activation domains. Therefore, the opposite directions in which the DNAs bend are caused, in part, by the opposite locations of the Fos and Jun transcription activation domains, relative to the DNA-binding domains in these proteins. DNA bending is reduced in the presence of multivalent cations, indicating that electrostatic interactions contribute to DNA bending by Fos and Jun (Kerppola, 1997).

Interactions among transcription factors that bind to separate promoter elements depend on the distortion of DNA structure and the appropriate orientation of transcription factor binding to allow juxtaposition of complementary structural motifs. Fos and Jun induce distinct DNA bends at different binding sites; heterodimers bind to AP-1 sites in a preferred orientation. Sequences on each side of the consensus AP-1 recognition element have independent effects on DNA bending. A single base pair substitution outside the sequences contacted in the X-ray crystal structure alters DNA bending. Substitution of sequences flanking the AP-1 site has converse effects on DNA bending in the opposite direction, suggesting that the extent of DNA bending by Fos and Jun is determined in part by the anisotropic bendability of sequences flanking the AP-1 site. DNA bending by Fos and Jun, and the orientation of heterodimer binding are interrelated. Reversal of the orientation of heterodimer binding causes a shift in the direction of DNA bending. The preferred orientation of heterodimer binding is determined both by contacts between a conserved arginine in the basic region of Fos and the central asymmetric guanine, as well as the structure of sequences flanking the AP-1 site. Consequently, the structural adaptability of the Fos-Jun-AP1 complex may contribute to its functional versatility at different promoters (Rajaram, 1997).

An important first step in the chromatin remodeling 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).

Histone acetylation and phosphorylation have separately been suggested to affect chromatin structure and gene expression. These two modifications are synergistic. Stimulation of mammalian cells by epidermal growth factor (EGF) results in rapid and sequential phosphorylation and acetylation of H3, and these modified H3 molecules are preferentially associated with the EGF-activated c-fos promoter in a MAP kinase-dependent manner. In addition, the prototypical histone acetyltransferase Gcn5 displays an up to 10-fold preference for phosphorylated (Ser-10) H3 over nonphosphorylated H3 as substrate in vitro, suggesting that H3 phosphorylation can affect the efficiency of subsequent acetylation reactions. Together, these results illustrate how the addition of multiple histone modifications may be coupled during the process of gene expression (Cheung, 2000).

Acylation of histones upon Fos activation

p300 and CREB-binding protein are functional homologs and global transcriptional coactivators that are involved in the regulation of various DNA-binding transcription factors. p300/CBP interacts with nuclear receptors CREB, c-Jun, C-Myb, c-Fos, and MyoD. DNA-binding factors recruit p300/CBP by both direct and indirect interactions through cofactors. p300/CBP is both a transcriptional adaptor and a histone acetyltransferase. The p300/CBP-histone acetyltransferase domain has no obvious sequence similarity to GCN5, another protein with known histone acetyltransferase activity, or to other previously described acetyltransferases. P300 acetylates all core histones in mononucleosomes and the four lysines in the Histone H4 N-terminal tail. These observations suggest that p300/CBP is not a simple adaptor between DNA binding factors and cellular p300/CBP associated factor (PCAF) or transcription factors; rather, p300/CBP per se may contribute directly to transcriptional regulation via targeted acetylation of chromatin (Ogryzko, 1996 and references).

The induction of immediate-early (IE) genes, including proto-oncogenes c-fos and c-jun, correlates well with a nucleosomal response, with the phosphorylation of histone H3 and is HMG-14 mediated via extracellular signal regulated kinase or p38 MAP kinase cascades. Phosphorylation is targeted to a minute fraction of histone H3, which is also especially susceptible to hyperacetylation. Direct evidence is provided that phosphorylation and acetylation of histone H3 occur on the same histone H3 tail on nucleosomes associated with active IE gene chromatin. Chromatin immunoprecipitation (ChIP) assays were performed using antibodies that specifically recognize the doubly-modified phosphoacetylated form of histone H3. Analysis of the associated DNA shows that histone H3 on c-fos- and c-jun-associated nucleosomes become doubly-modified, the same H3 tails becoming both phosphorylated and acetylated, only upon gene activation. This study reveals potential complications of occlusion when using site-specific antibodies against modified histones, and shows also that phosphorylated H3 is more sensitive to trichostatin A (TSA)-induced hyperacetylation than non-phosphorylated H3. Because MAP kinase-mediated gene induction is implicated in controlling diverse biological processes, histone H3 phosphoacetylation is likely to be of widespread significance (Clayton, 2000).

Fos and photoperiod response

Light-induced phase shifts of circadian rhythmic locomotor activity are associated with the expression of c-Jun, JunB, c-Fos and FosB transcription factors in the rat suprachiasmatic nucleus, as shown in the present study. In order to explore the importance of c-Fos and JunB, the predominantly expressed AP-1 proteins for the phase-shifting effects of light, the expression of c-Fos and JunB were blocked in the suprachiasmatic nucleus of male rats, housed under constant darkness, by intracerebroventricular application of 2 microliters of 1 mM antisense phosphorothioate oligodeoxynucleotides (ASO) specifically directed against c-fos and junB mRNA. A light pulse (300 lux for 1 h) at circadian time 15 induces a significant phase shift (by 125 +/- 15 min) of the circadian locomotor activity rhythm, whereas application of ASO 6 h before the light pulse completely prevents this phase shift. Application of control nonsense oligodeoxynucleotides has no effect. ASO strongly reduces the light-induced expression of c-Fos and JunB proteins. In contrast, light pulses with or without the control nonsense oligodeoxynucleotides evokes strong nuclear c-Fos and JunB immunoreactivity in the rat suprachiasmatic nucleus. These results demonstrate for the first time that inducible transcription factors such as c-Fos and JunB are an essential part of fundamental biological processes in the adult mammalian nervous system, e.g. of light-induced phase shifts of the circadian pacemaker (Wollnik, 1995).

The role of c-fos was examined in the synchronization of circadian rhythms to environmental light cycles using a line of gene-targeted mice carrying a null mutation at this locus. Circadian locomotor rhythms in mutants have similar periods as wild-type controls but take significantly longer than controls to entrain to 12:12 light-dark cycles. Light-induced phase shifts of rhythms in constant dark are attenuated in mutants although the circadian timing of phase delays and advances is not changed. A functional retinohypothalamic projection is indicated from behavioral results and light-induced jun-B expression in the SCN. The results indicate that while c-fos activation is not an absolute requirement for either rhythm generation or photic responses, it is required for normal entrainment of the mammalian biological clock (Honrado, 1996).

The suprachiasmatic nuclei (SCN) contain the principal circadian clock governing overt daily rhythms of physiology and behavior. The endogenous circadian cycle is entrained to the light/dark via direct glutamatergic retinal afferents to the SCN. To understand the molecular basis of entrainment, it is first necessary to define how rapidly the clock is reset by a light pulse. A two-pulse paradigm was used, in combination with cellular and behavioral analyses of SCN function, to explore the speed of resetting of the circadian oscillator in Syrian hamster and mouse. Analysis of c-fos induction and cAMP response element-binding protein phosphorylation in the retinorecipient SCN demonstrates that the SCN are able to resolve and respond to light pulses presented 1 or 2 hr apart. Analysis of the phase shifts of the circadian wheel-running activity rhythm of hamsters presented with single or double pulses demonstrates that resetting of the oscillator occurs within 2 hr. This is the case for both delaying and advancing phase shifts. Examination of delaying shifts in the mouse shows resetting within 2 hr and in addition shows that resetting is not completed within 1 hr of a light pulse. These results establish the temporal window within which to define the primary molecular mechanisms of circadian resetting in the mammal (Best, 1999).

The molecular basis of the mammalian clock is unknown, and so the recent cloning of mammalian homologs of the insect period gene is an important development. Whatever their nature, it is clear that resetting mechanisms are engaged very quickly, between 1 and 2 hr after a pulse. Thus, the rapid photic induction of mPer1 and mPer2 expression, which peaks after 1 hr and 2 hr, respectively, provides a molecular correlate of the overt resetting reported here. Increases in their expression and subsequent abundance of their putative protein products may be the cause of resetting to a new phase. Such a model is based on the Neurospora clock in which rapid resetting is associated with equally rapid induction of the gene frq, which has been shown to encode a state variable of the oscillation, i.e., the relative abundance of this transcript, and its protein products actually define circadian phase. The report that light-induced advances remain sensitive to blockade by inhibitors of protein synthesis for the 2-4 hr after a pulse supports the possibility that the mammalian clock is based on such an autoregulatory transcriptional cycle. The rapid response of the SCN to light is dependent on glutamatergic signaling by retinal ganglion cell afferents (Best, 1999 and references).

Light also causes rapid phosphorylation of CREB (see Drosophila CREB) in retinorecipient SCN neurons, probably via NMDA-mediated calcium influx. Phosphorylated CREB is a positive regulator of the c-fos gene via the calcium response element, although its potential role in the induction of mPer1 and mPer2 (see Drosophila Period) awaits clarification. The changes in phospho-CREB expression are extremely rapid, occurring significantly before increases in c-fos or mPer mRNA, although the magnitude of the response is not related to the size or direction of a phase shift. Moreover, in both hamsters and mice, it occurs after presentation of a light pulse at all phases of circadian night but is not induced by light during the subjective day, closely matching the temporal induction of mPer1 regardless of whether light advances or delays the clock. If mPer1 does encode a state variable of the oscillator, it should be induced repeatedly by serial resetting light pulses. Moreover, the observation that photic induction of mPer2 is a marker for delaying pulses (early subjective night) but not advancing pulses (late subjective night) suggests a novel means for mapping the phase of the oscillator during serial resetting by using a molecular index. If the inducibility of mPer2 is a marker for early subjective night, it should be possible to show that serial pulses delivered in the delay zone hold the oscillator at early subjective night, i.e., mPer2 induction is sustained. This approach is comparable to using the onset and offset of photic induction of c-fos to define the total limits of subjective night (Best, 1999 and references).

The immediate early gene c-fos is also a potential component of the photic resetting pathway. Phase shifts are impaired in c-fos knock-out mice, and central infusion of antisense oligonucleotides to c-fos and jun-B are reported to block light-induced resetting in the rat. Induction of c-fos mRNA by a light pulse peaks after 30 min and is undetectable after 60 min. Moreover, the induction of c-fos protein, which is detectable after 7.5 min and peaks after 1 to 2 hr, is simultaneous with the induction of mPer1 mRNA, raising the possibility that c-fos contributes to resetting by modulating expression of the mPer1 gene via changes in AP-1 activity, which is known to occur after a light pulse in subjective night. In situ hybridization and Western blots reveal that the second resetting pulse causes de novo induction of c-fos, although the second pulse was presented when c-fos protein levels were high. In other contexts, it has been shown that c-fos protein can downregulate expression of the c-fos gene via upstream promoters, although it is clear that autoregulation of c-fos does not occur in the SCN (Best, 1999 and references).

Nonphotic stimuli can reset and entrain circadian activity rhythms in hamsters and mice; serotonin is thought to be involved in the phase-resetting effects of these stimuli. The present study examines the effect of the serotonin agonist quipazine on circadian activity rhythms in three inbred strains of rats (ACI, BH, and LEW). Also studied was the effect of quipazine on the expression of c-Fos in the mammalian circadian pacemaker, the suprachiasmatic nucleus (SCN). Quipazine reduces the amount of running wheel activity for 3 h after treatment, however, no long-term changes in the activity level are observed. More important, quipazine induces significant phase advances of the activity rhythm and c-Fos production in the SCN at the end of the subjective night (Circadian Time [CT] 22), whereas neither phase shifts nor c-Fos induction are observed during the subjective day. Quipazine injections also result in moderate phase delays at the beginning of the subjective night (CT 14). A similar phase-response characteristic typically can be observed for photic stimuli. By contrast, nonphotic stimuli normally produce phase advances during the subjective day. The present results suggest species differences between the hamster and the rat with respect to the serotonergic action on circadian timekeeping and indicate that serotonergic pathways play a role in the transmission of photic information to the SCN of rats (Kohler, 1999).

AP-1 family members act with Sox9 to promote chondrocyte hypertrophy

An analysis of mammalian Sox9 binding profiles in developing chondrocytes identified marked enrichment of an AP-1-like motif. This study has explored the functional interplay between Sox9 and AP-1 (see Drosophila Fos and Jun) in mammalian chondrocyte development. Among AP-1 family members, Jun and Fosl2 were highly expressed within prehypertrophic and early hypertrophic chondrocytes. Chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq) showed a striking overlap in Jun- and Sox9-bound regions throughout the chondrocyte genome. The similar profiles reflect direct binding of each factor to the same enhancers and a potential for protein-protein interactions within AP-1 and Sox9 containing complexes. In vitro reporter analysis indicated that direct co-binding of Sox9 and AP-1 at target motifs promoted gene activity. In contrast, where only one factor can engage its DNA target, the presence of the other factor suppresses target activation consistent with protein-protein interactions attenuating transcription. Analysis of prehypertrophic chondrocyte removal of Sox9 confirmed the requirement of Sox9 for hypertrophic chondrocyte development, while in vitro and ex vivo analyses showed AP-1 promotes chondrocyte hypertrophy. Sox9 and Jun co-bound and co-activated a Col10a1 enhancer in Sox9 and AP-1 motif-dependent manners consistent with their combined action promoting hypertrophic gene expression. Together, the data support a model where AP-1-family members contribute to Sox9-action in the transition of chondrocytes to the hypertrophic program (He, 2016).

Fos transcriptional targets

Continued: Evolutionary homologs part 4/4 | back to part 1/4 | part 2/4 |

Fos-related antigen: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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