BIOLOGICAL OVERVIEW

Gene regulation by AP-1 transcription factors in response to Jun N-terminal kinase (JNK) signaling controls essential cellular processes during development and in pathological situations. The histone acetyltransferase (HAT) Chameau and the histone deacetylase DRpd3 act as antagonistic cofactors of DJun and DFos to modulate JNK-dependent transcription during pupal thorax metamorphosis and JNK-induced apoptosis in Drosophila. It has been demonstrated, in cultured cells, that DFos phosphorylation mediated by JNK signaling plays a central role in coordinating the dynamics of Chameau and DRpd3 recruitment and function at AP-1-responsive promoters. Activating the pathway stimulates the HAT function of Chameau, promoting histone H4 acetylation and target gene transcription. Conversely, in response to JNK signaling inactivation, DRpd3 is recruited and suppresses histone acetylation and transcription. This study establishes a direct link among JNK signaling, DFos phosphorylation, chromatin modification, and AP-1-dependent transcription and its importance in a developing organism (Miotto, 2006).

The transcriptional response to signal transduction via the evolutionary conserved Jun N-terminal kinase (JNK) pathway controls essential cellular processes, including morphogenesis, differentiation, and apoptosis during development and in physiopathological situations. A large body of research supports the model that in response to extracellular stimulation, JNK activates the transcriptional effector AP-1 by phosphorylation, and thereby reprograms target gene expression. AP-1 mainly consists of Fos and Jun family proteins that can form homodimers or heterodimers and bind DNA through conserved bZIP domains. Given the importance of chromatin dynamics in the control of gene expression, recent work has focused on AP-1 interaction partners capable of chromatin modification and remodeling, notably enzymes able to reversibly modify histone tails by acetylation. The nuclear receptor corepressor (NCoR)/histone deacetylase 3 (HDAC3) complex was thus found to inhibit the JNK pathway. The checkpoint function of the NCoR complex is relieved by c-Jun phosphorylation, which directs its removal from the promoter and promotes the recruitment of the TAF7 RNA polymerase II (RNA pol II) subunit. AP-1 recruits DRpd3/HDAC1 to reverse histone acetylation at the promoter of the attacin-A gene and its activation by the NF-kappaB transcription factor Relish associated with the histone acetyltransferase (HAT) Pcaf. Other reports on stimulation of Fos or Jun activities by CBP suggest that HAT coactivator complexes, recruited at target promoters, mediate nucleosome acetylation and stimulate transcription. These findings, gained from experiments performed in vitro and in cultured cells, indicate that chromatin dynamics play a central role in the cellular response to JNK signaling. However, the epigenetic mechanisms that control the transcriptional response in intact organisms remain unclear (Miotto, 2006 and references therein).

JNK signaling regulates a number of different processes during Drosophila development, including pupal thorax closure and apoptotic cell death in imaginal discs. The role of JNK signaling is well established in these two morphogenetic events. In the former, it drives the migration of wing discs toward the midline and their fusion into a continuous epidermal structure; in the latter, it apoptotically eliminates cells exposed to inappropriate proximodistal patterning cues. Genetic and molecular evidence is provided that Chameau (Chm), a MYST domain HAT previously reported to act in epigenetic mechanisms of transcriptional control (Grienenberger, 2002) and to support histone acetylation at replication origins (Aggarwal, 2004), and the HDAC DRpd3, modulate the transactivation potential of AP-1 during thorax closure and JNK-induced apoptosis in an antagonistic manner. Furthermore, by deciphering the mode of action of Chm and DRpd3 in vitro and in cultured cells, mechanistic insights are provided into the regulation of AP-1 function by chromatin (Miotto, 2006).

A direct link exists between JNK signaling and chromatin modification via AP-1 transcription factors in an intact developing organism, an effect that rests on the HAT coactivator Chm and the HDAC corepressor DRpd3. This conclusion holds for thoracic closure and JNK-induced apoptosis but not for another JNK-dependent morphogenetic event, the embryonic dorsal closure. Although this process shares many similarities with thorax closure, it is not affected in embryos deprived of maternal and zygotic chm contribution. Thus, Chm is not required for all JNK signaling-dependent mechanisms. Furthermore, when it is involved, Chm is not essential for JNK signaling: The incomplete penetrance of chm phenotypes and the decreased but remaining expression of AP-1 target genes in mutant discs illustrate that chm loss-of-function compromises but does not block JNK signaling. Thus, Chm is not an obligatory component in the pathway but its recruitment supports the transcriptional efficiency of AP-1 during imaginal development. DRpd3 counteracts this function, indicating that the balanced activity of the two antagonistic factors constitutes a new regulatory mechanism for JNK-dependent transcription (Miotto, 2006).

The functional partnership between Chm and DRpd3 in the control of JNK signaling seems specific to this HAT/HDAC pair, since mutations in other HDAC or HAT genes do not modify the chm thorax closure phenotype. Particularly illustrative is the absence of genetic interactions with mutant alleles of mof, since Mof is a MYST HAT of the same substrate specificity as Chm (Histone4 K16) and DFos interacts with Chm through the conserved MYST domain. Interestingly, a recent report has connected Chm and DRpd3 functions during Drosophila oogenesis (Aggarwal, 2004). The two proteins exhibit opposite effects on H4 acetylation and activity of replication origins, indicating that the functional antagonism of Chm and DRpd3 regulates several chromatin-dependent processes, including transcription and replication (Miotto, 2006).

Although genetic experiments emphasize the specificity of Chm/DRpd3 partnership, the data do not imply that DRpd3's only role in JNK signaling is in balancing Chm function. Neither do they exclude the possibility that other HDACs or HATs participate in the JNK response. For example, in cultured cells DRpd3 recruitment by AP-1 changes histone acetylation levels established earlier by the HAT Pcaf at the attacin promoter from a permissive to a repressive status, and a HDAC3 repressor complex maintains c-Jun transcriptionally inactive. It is therefore likely that different HATs and HDACs control AP-1-regulated transcription, depending on the physiological context. What specifies HAT and HDAC commitment in a given process remains to be explored (Miotto, 2006).

Insights into the mechanism by which Chm supports JNK target gene transcription were initially obtained from observations made in the developing animal. (1) Chm improves the activation of a lexA-acZ reporter by the LexA-DFos fusion protein, revealing that Chm stimulates the transactivating potential of DFos when it is tethered to a promoter. (2) The thorax cleft phenotype caused by DFos^NAla, a protein deficient for the JNK phosophorylation sites, is not rescued upon simultaneous overexpression of Chm. Since the DFos^NAla variant recruits Chm as efficiently as the wild-type protein, this suggests that transcriptional improvement by Chm requires DFos phosphorylation, providing an attractive link between Chm function and JNK signaling (Miotto, 2006).

Study of the mode of action of the antagonistic cofactors Chm and DRpd3 in cultured cells has provided further insights into a chromatin-based mechanism that executes a modulation of the transcriptional response to JNK signaling. The following model is proposed, based on the dynamics of cofactor recruitment and activity, chromatin modification and transcriptional status related to reversible activation of the pathway. In response to JNK signaling, Chm HAT activity sets up a histone modification pattern that is instructive for transcriptional enhancement. Consistent with this notion Chm acetylates H4, with a marked preference for K16, and facilitates H3K4 trimethylation. However, AP-1 likely also engages HATs of different substrate specificity, since H4K8 acetylation, a modification required for the recruitment of the SWI/SNF-activating complex, is directed by DFos in the absence of Chm. After JNK signaling has ceased, DRpd3 gets recruited to promoters occupied by unphosphorylated DFos and counteracts the effects of Chm by reversing histone modifications, which results in transcriptional down-regulation. Strikingly, the recruitment of DRpd3 seems not to result from the displacement of Chm from the promoter, since invariant levels of Chm are associated with the promoter in sorbitol experiments, whereas DRpd3 starts to be recruited only once the signal has been eliminated. Thus, as opposed to an exchange of a HAT coactivator complex for an HDAC corepressor complex, which occurs for instance between Pcaf/NF-kappab and DRpd3/AP-1 complexes at the attacin promoter (Kim, 2005), a complex containing both Chm and DRpd3 could then form at the target promoter whose activity changes the histone modification pattern back to a pattern less permissive to transcription. Thus, DRpd3 most likely functions during a transient phase from a transcriptionally active to silent status. Its absence from the promoter at the inactive steady state in nonstimulated cells, suggests that unphosphorylated DFos then lies in a conformational environment that prevents DRpd3 recruitment by the ZIP domain (Miotto, 2006).

AP-1 phosphorylation plays a pivotal role in regulating the epigenetic response to JNK signaling. An 'activation by derepression' model has been recently proposed following which JNK-dependent phosphorylation induces the release of an HDAC3 repressor complex from the N terminus of c-Jun (Weiss, 2003; Ogawa, 2004). DFos^bZIP, which lacks JNK phosphorylation sites and domains other than the bZIP that could recruit HDAC complexes, can promote transcription in nonstimulated cells. This suggests that a phosphorylation-dependent process similar to the activation by derepression model mediates transactivation by full-length DFos when JNK signaling is active. The work furthermore supports additional regulatory roles for AP-1 phosphorylation as indicated by the dependence of Chm HAT activity and DRpd3 recruitment on the DFos phosphorylation state. It is proposed that phosphorylation not only releases an HDAC corepressor complex (other than DRpd3) and leads to activation by derepression but also unmasks Chm HAT function and results in increased transcriptional efficiency, whereas dephosphorylation promotes the recruitment of DRpd3 to reverse transcriptional activation. Considering the opposite functions of chm and Drpd3 in JNK-dependent thorax closure and apoptosis during fly development, it is tempting to speculate that the balanced activities of the Chm HAT and the DRpd3 HDAC allow transient JNK target gene activation as well as fine-tuning of JNK transcriptional output in vivo (Miotto, 2006).

GENE STRUCTURE

cDNA clone length - 2919

Bases in 5' UTR - 54

Exons - 8

Bases in 3' UTR - 429

PROTEIN STRUCTURE

Amino Acids - 811

Structural Domains

MYST proteins have been identified from yeast to human (Sterner, 2000). The family includes, in yeast, Sas2 and Sas3, first identified for their roles in silencing at HML and telomeres, and Esa1; in Drosophila, Mof, involved in gene dosage compensation; in human, Moz, Morf, Tip60, and HBO1. Sas3, Esa1, Mof, Morf, Moz, and Tip60 possess intrinsic HAT activity; HBO1 transacetylates histones only as part of a multiprotein complex (Iizuka, 1999). Genetic evidence indicates that Sas2 is required for H4 lysine 16 acetylation in vivo. Sequence comparison indicates that Chm corresponds to the fly counterpart of HBO1. HBO1 was shown to interact with the ORC largest subunit, providing the complex with HAT activity (Iizuka, 1999) with the androgen receptor (Sharma, 2000), and with the replication factor MCM2 (Burke, 2001), suggesting chromatin-mediated roles in both DNA replication and transcription regulation (Grienenberger, 2002).

date revised: 2 July 2006

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