Chromatin regulates origin activity in Drosophila follicle cells

It is widely believed that DNA replication in multicellular animals (metazoa) begins at specific origins to which a pre-replicative complex (pre-RC) binds. Nevertheless, a consensus sequence for origins has yet to be identified in metazoa. Origin identity can change during development, suggesting that there are epigenetic influences. A notable example of developmental specificity occurs in Drosophila, where somatic follicle cells of the ovary transition from genomic replication to exclusive re-replication at origins that control amplification of the eggshell (chorion) protein genes. This study shows that chromatin acetylation is critical for this developmental transition in origin specificity. Histones at the active origins are hyperacetylated, coincident with binding of the origin recognition complex (ORC). Mutation of the histone deacetylase (HDAC) Rpd3 induces genome-wide hyperacetylation, genomic replication and a redistribution of the origin-binding protein ORC2 in amplification-stage cells, independent of effects on transcription. Tethering Rpd3 or Polycomb proteins to the origin decreases its activity, whereas tethering the Chameau acetyltransferase increases origin activity. These results suggest that nucleosome acetylation and other epigenetic changes are important modulators of origin activity in metazoa (Aggarwal. 2004).

Chameau HAT and DRpd3 HDAC function as antagonistic cofactors of JNK/AP-1-dependent transcription during Drosophila metamorphosis
Animals homozygous for the amorphous allele chm14 lack zygotic chm function and die during late pupal stages. Pharate adults display morphological aberrations, especially a cleft of variable depth and width along the thorax midline. To quantitatively assess the strength of this phenotype, a system of four phenotypic classes, ranging from the absence to a mild, pronounced, or severe cleft, was adopted. chm is expressed in the two epithelia of wing discs, the columnar epithelium, and the peripodial membrane, and by 8 h after puparium formation (APF), when the two contralateral discs meet at the dorsal midline, transcription proceeds in fusion regions only. Together, the mutant phenotype and the expression pattern of chm support a function in migration and/or fusion of wing discs during metamorphosis (Miotto, 2006).

Since flies defective in JNK signaling display thorax defects similar to chm mutants, it was asked whether Chm might play a role in JNK signaling during thorax closure. Thus, genetic interactions were tested between chm and genes encoding components of the JNK pathway. Reducing the gene dosage of positive effectors, such as DJNKK/hemipterous (hep) and Dfos/kayak (kay), does not per se affect thorax closure but exacerbates the chm phenotype: Heterozygosity for hep raises the proportion of class II phenotypes from 14% to 39%; reducing Dfos activity has a more pronounced effect; class III rises from 6% to 97% in the case of the null allele kay1 and to 40% with the hypomorphic allele kay2. Conversely, heterozygosity for puckered (puc), a repressor of JNK activity, dominantly suppresses the thoracic cleft of chm mutants; no chm; puc/+ pharate adult presents a class III phenotype. Moreover, this genotype produces viable adult escapers, an effect already observed with lethal combinations of hep and Dfos alleles rescued by puc heterozygosity. These positive and negative genetic interactions indicate that Chm cooperates with JNK signaling during thorax closure. It was noticed, however, that heterozygosity for Djun does not exacerbate the chm phenotype. The observation that Dfos, but not Djun, alleles dominantly interact with chm suggests that DFos acts independently of DJun in the context of Chm-dependent thorax closure processes (Miotto, 2006).

AP-1 target gene transcription was examined. The puc gene is activated by JNK signaling in proximal peripodial cells. In chm mutant discs, puc expression is strongly reduced but can still be detected, consistent with a stimulatory rather than essential function of Chm for JNK signaling in these cells. To confirm such a function, transcription levels of puc and three additional bona fide JNK targets -- ance, chickadee (chic), and myospheroid (mys) -- were compared in wild-type and chm mutant wing discs. Results from quantitative RT-PCR show that the transcription of the four target genes is significantly reduced in a chm background. It is concluded that Chm supports JNK-dependent gene activation during wing disc development and thorax closure (Miotto, 2006).

To determine whether chm contributes to JNK-dependent processes in addition to thorax closure, JNK-induced apoptotic cell death was examined. In the insertion trap line hepCA, JNK signaling is ectopically activated in the wing blade primordium; this causes cell death and induces notches of variable extent in the adult wing. Eliminating one copy of Djun, Dfos, or chm rescues the hepCA wing phenotype. Furthermore, loss of one copy of chm partially reverts the enhancement of the hepCA notching phenotype that is observed as a consequence of puc heterozygosity. Further supporting the notion of cooperation between JNK and Chm, cell death as detected by acridine orange staining in hepCA wing discs is abrogated in chm homozygous conditions. JNK-dependent apoptosis is also induced in response to morphogen gradient distortion caused by misexpression of scalloped (sd) or optomotor blind (omb). Heterozygosity for puc aggravates -- and for Djun, Dfos, or chm dominantly rescues -- the notched wing phenotype of flies heterozygous for sd or omb. Thus, Chm acts as a positive effector in the JNK-regulated apoptotic pathway. Interestingly, in this process chm cooperates with both Dfos and Djun genes (Miotto, 2006).

Based on the genetic interactions described above, the epistatic relationship between Chm and AP-1 factors was examined. To this end, UAS transgenes and the MZ980Gal4 driver, that promotes expression specifically in the proximal region of the wing disc and later along the prospective junction of the contralateral discs , were used. Driving UASchm in these cells fully rescues the thoracic cleft of chm mutants, while Dfos, Djun, or both Dfos and Djun expression in the MZ980 domain do not. Thus, chm is not epistatic to Dfos or Djun, which indicates that Chm is not upstream of AP-1 in the pathway and does not control the expression of Dfos and Djun. RT-PCR from RNA samples of wild-type and chm mutant wing discs confirmed that Dfos and Djun transcription is not affected by chm loss of function. This finding is consistent with Chm cooperating with JNK signaling at the level of the transcriptional effectors of the pathway (Miotto, 2006).

To further characterize the cooperation between DFos, DJun and Chm, reporter gene assays were performed in cultured cells. HEK293 cells were transiently transfected with an AP-1-dependent luciferase reporter in the presence or absence of Chm, DJun and DFos, DJNKK and DJNK. Chm either alone, or in the presence of DFos and DJun or of DJNKK and DJNK, had no significant effect. However, when DJNKK, DJNK, DFos, and DJun were supplied together, Chm stimulated AP-1-dependent transcription in a dose-dependent manner. Thus, Chm promotes the transactivation potential of AP-1 only when the JNK pathway is active. This effect seems to be specific for Chm, since expression of hTip60 (see Drosophila Tip60), another MYST HAT, does not change luciferase activity. These data identify Chm as a transcriptional coactivator of DFos and DJun (Miotto, 2006).

Focus was placed on the functional relevance of the Chm/DFos interaction. To investigate whether this interaction occurs on AP-1 target DNA, chromatin immunoprecipitation (ChIP) assays were performed in HEK293 cells transfected with an AP-1 reporter and constructs for HA-DFos and Flag-Chm. DFos efficiently and specifically binds the reporter promoter. Chm does so only when expressed together with DFos. No significant binding was observed on the vector DNA. To ascertain that Chm and DFos simultaneously associate with promoter sequences, a sequential ChIP experiment was performed. After a first ChIP with an antibody to HA (DFos) and dissociation of the antibodies from the immunoprecipitated complexes, a second ChIP was performed using anti-Flag (Chm) antibody. PCR detection then showed that the complex eventually pulled-down contains both DFos and Chm associated with the promoter. As a control, Chm but not hTip60 was coprecipitated by DFos from cells transfected with HA-DFos and either Flag-Chm or Flag-hTip60. It is concluded that Chm is recruited to chromatin in a sequence specific manner by DFos to AP-1-binding sites (Miotto, 2006).

Next, it was asked whether Chm acts by promoting DFos DNA-binding or transactivating potential. This was addressed in an in vivo assay, using a fly line that expresses a chimera between the DNA-binding LexA protein and DFos, and permits monitoring transactivation by DFos independent of its DNA-binding activity. It was observed that a lacZ reporter cloned downstream of LexA-binding sites is strongly activated in the larval epidermis by DFos-LexA and that chm mutation reduces lacZ transcription. This result suggests that Chm supports gene activation in the animal by a mechanism improving DFos transactivation capacity (Miotto, 2006).

So far, it has been shown that DFos and Chm physically interact and functionally cooperate in the execution of JNK signals to mediate apoptosis, morphogenesis, and gene activation. Next, how JNK influences the function of DFos and Chm at the molecular level was addressed. DFos is a substrate for JNK and several phosphorylation sites have been reported. The relevance of JNK-mediated phosphorylation of DFos in thorax closure is illustrated by the strong cleft phenotype induced by the expression in the MZ980 domain of a variant, DFosNAla, in which the two DJNK threonine residues that represent DJNK targets were mutated into alanine. Interestingly, this effect cannot be reversed by Chm overexpression, indicating that the protein cannot promote gene activation by DFosNAla. To further validate this interpretation, reporter gene assays were performed in HEK293 cells. The luciferase reporter is activated by DFos, DJNKK and DJNK, and in this context Chm expression boosts transcription in a concentration-dependent manner whereas no such effect is observed using DFosNAla instead of the wild-type protein. These data strongly suggest that the cooperation between DFos and Chm in gene activation is regulated by a mechanism that involves DFos phosphorylation. Interestingly, phosphorylation by DJNK does not seem to be required for binding of Chm to DFos since DFosNala recruits Chm to chromatin as efficiently as the wild-type protein (Miotto, 2006).

The recruitment of Chm to AP-1-binding sites by DFos suggests a mechanism for gene activation that involves histone modification by the HAT activity of Chm. Since the bZIP domain of DFos is sufficient to recruit Chm, and to avoid the additional effects of the transactivation and phosphorylation domains of DFos, a truncated version of DFos was used that essentially comprises the bZIP domain. Thus, a simple experimental system relying on DFosbZIP expression in HEK293 cells was devised to explore the mode of action of Chm (Miotto, 2006).

The requirement of Chm HAT activity for DFos-mediated transcription was tested. Coexpression of DFosbZIP with Chm results in a significant stimulation of reporter activity. This is dependent on the enzymatic activity of Chm, because a point mutation in the acetyl-CoA-binding site (G680E) that abolishes HAT activity abrogates reporter activation. The effect of ChmG680E is dominant as indicated by luciferase inhibition when the level of the mutant protein is raised leaving that of the wild-type constant. In these experiments, similar levels of mutant and wild-type Chm proteins are expressed and bind chromatin in transfected HEK293 cells. The dominant negative effect of ChmG680E was also observed using the full-length DFos protein (Miotto, 2006).

To assess whether HAT-dependent AP-1 reporter activation correlates with changes in histone modification, the acetylation pattern at the reporter gene promoter and its dependence on Chm enzymatic activity was examined by performing ChIP experiments with acetylation site-specific antibodies. Chm recruitment by DFosbZIP results in an increase of H4 tetra-acetylation but does not change K9/K14 acetylation of H3, while ChmG680E has no significant effect. Thus, H4 is a likely target of DFos-directed acetylation by Chm. In ChIP assays with antibodies to mono-acetylated H4 isoforms, Chm enhances the H4K16 acetylation over levels observed in the presence of DFosbZIP alone, an effect that is not supported by ChmG680E. Thus, H4K16 is a target lysine of Chm HAT activity. Interestingly, DFosbZIP promotes H4K8 acetylation but Chm addition does not raise the level further. This indicates that H4K8 is not a Chm target, and therefore suggests that DFosbZIP recruits another HAT to modify this lysine (Miotto, 2006).

Then, Chm function was examined in transcription activation. H3K4 trimethylation constitutes a chromatin mark for transcriptionally active loci. Consistent with the coactivator function of Chm, the level of H3K4 trimethylation at the promoter is increased in the presence of both Chm and DFosbZIP. Again, Chm HAT activity is required in this process; using the ChmG680E variant has no effect. This suggests that H4 acetylation by Chm is a prerequisite for the definition of a histone modification pattern instructive for improved transcription (Miotto, 2006).

The identification of HAT activities as mediators of DFos-dependent histone acetylation and gene activation raises the question of which HDACs might function to counter these processes. A hypomorphic mutation of the gene encoding the Drosophila homolog of HDAC1, Drpd3, dominantly suppresses the thoracic cleft of chm mutants, whereas available mutations in other HDAC genes, including DHDAC4 and Dsir2, have no effect. Furthermore, heterozygosity for Drpd3 enhances the hepCA notched wing phenotype and counteracts the phenotypic rescue caused by the mutation of one copy of chm. Thus, Drpd3 and chm antagonistically control JNK signaling during thorax closure and the JNK-dependent apoptotic pathway. Since H4K8 is modified by HAT, other than Chm, genetic interactions were tested with available mutations in HAT genes. Reducing the gene dosage of nejire/CBP, mof, enok, or deco neither aggravated nor rescued the chm thorax phenotype (Miotto, 2006).

To examine the effect of DRpd3 on AP-1-dependent transcription at the molecular level, it was asked whether DRpd3 could directly interact with DFos, as does Chm. DRpd3 coprecipitates with TAP-DFos from nuclear extracts of larvae expressing UASTAP-Dfos under the control of armGal4. GST pull-down assays reveal that DFos physically interacts with DRpd3 and that the only DFos fragments that precipitate DRpd3 share the ZIP domain. Thus, DRpd3 and Chm physically interact with the ZIP and the basic domains of DFos, respectively. Finally, to assess the functional relevance of these interactions, the effect of DRpd3 expression on DFosbZIP/Chm-induced transcription and histone modifications was tested in HEK293 cells. Increasing DRpd3 levels while leaving Chm constant results in reporter repression. Consistent with this inhibitory effect, DRpd3 reduces H4 tetra-acetylation and H3K4 trimethylation mediated by DFosbZIP/Chm at the promoter, indicating that DRpd3 restores a repressive transcriptional environment. Thus, Chm and DRpd3 behave as antagonistic cofactors of DFosbZIP in HEK293 cells. Considered together, these data strongly suggest that the nuclear response to JNK signaling is determined by the opposing activities of this HAT/HDAC pair (Miotto, 2006).

To investigate the roles of Chm and DRpd3 in JNK phosphorylation-dependent transactivation, experiments were performed with the full-length version of DFos. To modulate JNK activity, sorbitol treatment (which can reversibly activate the pathway in cultured cells) was employed. In HEK293 cells transfected with Chm, DRpd3 and DFos vectors, luciferase transcription rapidly increases after sorbitol addition and is progressively shut-down after sorbitol removal. Therefore, reversible stimulation by sorbitol constitutes an appropriate system to correlate molecular changes at AP-1-responsive promoter with JNK-dependent transcription (Miotto, 2006).

ChIP assays were performed to assess changes in reporter promoter occupancy throughout the experiment. It was found that Chm is always and at invariant levels associated with the promoter, indicating that its recruitment by DFos does not depend on JNK signaling. H4 acetylation levels at the promoter, as monitored by the relative abundance of H4K16 and tetra-acetylated H4, dynamically changes throughout the time course of induction and correlates with transcriptional activity. Because DFos-directed acetylation by Chm is responsible for these modifications, this result indicates that the protein, while present at the promoter, is unable to acetylate H4 in nonstimulated cells, and therefore that JNK signaling stimulates the latent HAT activity of Chm and permits subsequent transcriptional activation (Miotto, 2006).

Whereas Chm is found constitutively associated, DRpd3 recruitment to the promoter depends on JNK signaling. Before sorbitol addition, which is considered as the inactive steady state, DRpd3 does not associate the promoter (1.35-fold over background). DRpd3 thus appears dispensable for maintenance of the inactive steady state. While activating the pathway upon sorbitol addition has no significant effect on DRpd3 tethering, the HDAC starts to be recruited to the promoter after sorbitol elimination (14-fold over background 1 h after sorbitol removal) and the levels of H4 acetylation and transcription then decrease simultaneously and progressively. Thus, the down-regulation of the transcriptional response to JNK activation after dissipation of the signal is at least in part mediated by recruitment of DRpd3 to the promoter already occupied by Chm. Supporting this notion, DRpd3 is found on chromatin fragments immunoprecipitated by Chm and conversely Chm is associated with fragments containing DRpd3. Together, these results suggest that DRpd3 assumes a transient function to reduce histone modification and transcription levels previously stimulated by Chm (Miotto, 2006).

The dynamics of Chm activation and DRpd3 recruitment to the promoter were next considered with regard with DFos phosphorylation. Sorbitol addition increases the ratio of phosphorylated DFos (P-DFos) to nonphosphorylated DFos in the chromatin-bound fraction, and sorbitol removal decreases the ratio. Thus, DFos phosphorylation correlates with Chm-mediated transcriptional activation and DFos dephosphorylation with down-regulation by DRpd3. Furthermore, Western analysis of immunoprecipitated chromatin revealed that 1 h after sorbitol removal Chm, DFos and P-DFos are present on the same chromatin fragments, whereas DRpd3 coprecipitates predominantly with fragments containing unphosphorylated DFos. Pull-down assays indicate that DFos interacts in vitro with both Chm and DRpd3, and P-DFos only with Chm. Together, these data suggest that the DFos phosphorylation state is the likely connection between sorbitol stimulus, Chm HAT activity unmasking and DRpd3 recruitment. To investigate this point further, sorbitol experiments were performed using the DFosNAla variant, which cannot be phosphorylated, instead of the wild-type protein. Consistent with an earlier finding, Chm still is tethered to the promoter. However, sorbitol addition no longer stimulates H4 acetylation and target gene transcription. This lack of JNK responsiveness correlates with a significantly reduced change in promoter occupancy by DRpd3 in response to JNK signaling. Thus, the DFos phosphorylation status controls Chm HAT activity and DRpd3 recruitment to the promoter (Miotto, 2006).

Protein Interactions

Whether Chm directly binds to DFos and/or DJun was investigated using GST pull-down assays. The C-terminal half of Chm (amino acids 494-812), which contains the MYST domain, displays strong in vitro affinity for an N-terminal fragment of DFos (including the N terminus and the basic DNA-binding domain), and binds DJun as well, although less efficiently. Conversely, the cytoplasmic kinase Basket (Bsk)/DJNK does not bind to the His-Chm fusion protein. The Chm N terminus (amino acids 20-400) does not associate with DFos or DJun. Similar experiments with GST-fused DFos deletion mutants identified the basic region of DFos as the predominant Chm-interacting domain, although significant association with the C-terminal part of DFos was also observed. Immunoprecipation assays followed by Western analyses confirmed that these interactions occur in vivo. Both DFos and DJun coprecipitate with Myc-Chm from nuclear extracts of larvae expressing a Myc-tagged version of the protein. DFos is eluted from the immunoprecipitate at higher salt concentrations than DJun; this indicates a more stable association with Chm and that Chm to DFos interaction can occur in the absence of DJun. Confirming the specificity of the assay, nuclear proteins unrelated to JNK signaling, the chromatin-associated protein Modulo (Mod), the homeodomain transcription factors Ultrabithorax (Ubx) and Engrailed (En), as well as the basic helix-loop-helix (bHLH) factor DMyc are not precipitated by Myc-Chm. In reciprocal experiments, an anti-TAP antibody coprecipitates Myc-Chm from nuclear extracts of larvae ubiquitously expressing Myc-Chm and TAP-DFos or TAP-DJun. The results of the in vitro and the in vivo experiments, taken together, show that Chm, DFos and DJun can directly interact and form multimeric protein complexes in larvae (Miotto, 2006).

chameau: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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