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. The expression pattern of chm supports the idea that it functions in migration and/or fusion of wing discs during metamorphosis (Miotto, 2006).
Reversible acetylation of histone tails plays an important role in chromatin remodelling and regulation of gene activity. While modification by histone acetyltransferase (HAT) is usually linked to transcriptional activation, evidence is provided for HAT function in several types of epigenetic repression. Chameau (Chm), a new Drosophila member of the MYST HAT family, dominantly suppresses position effect variegation (PEV), is required for the maintenance of Hox gene silencing by Polycomb group (PcG) proteins, and can partially substitute for the MYST Sas2 HAT in yeast telomeric position effect (TPE). Finally, in vivo evidence is provided that the acetyltransferase activity of Chm is required in these processes, since a variant protein mutated in the catalytic domain no longer rescues either PEV modification, telomeric silencing of SAS2-deficient yeast cells, or lethality of chm mutant flies. These findings emphasize the role of an acetyltransferase in gene silencing, which supports, according to the histone code hypothesis, the observation that transcription at a particular locus is determined by a precise combination of histone tail modifications rather than by overall acetylation levels (Grienenberger, 2002).
Mutations were generated by mobilization of a P element inserted 1.2 kb downstream of the 3′ end of chm. chm14 deletes the last three exons, breaking in the MYST domain; Df(2L)221 deletes most of the coding region. These deficiencies are recessive pupal lethal and genetically behave as null alleles of chm. They do not affect another essential gene, since the associated lethality is rescued by a heat shock construct of chm cDNA in transgenic animals raised at 25°C (Grienenberger, 2002).
To assay for a role in modulating chromatin structure and transcription, PEV, a phenomenon of epigenetic repression mediated by pericentric heterochromatin, was first analyzed. Genes that dominantly modify PEV are thought to encode products that affect chromatin structure, leading when mutated to an increased (suppressors of PEV) or a decreased (enhancers) transcription of neighboring genes. The wm4h inversion leads to a mosaic pattern of eye pigment mutation. Mutation of one chm copy gives significant increase of pigmented area, resulting in a 2.5-fold increase of pigment levels. chm mutation therefore dominantly suppresses variegation of white in wm4h. To assess whether chm also affects other variegating rearrangements, the γ238 minichromosome was used that contains close to centric heterochromatin the yellow (y) gene, responsible for the dark pigmentation of bristles. The number of dark bristles at the wing margin is significantly increased in animals heterozygous for chm, indicating a suppression of y variegation. To rule out a possible effect of the genetic background on PEV, whether Chm overexpression, provided by a HSchm transgene at 25°C, could reverse the y derepression seen in chm heterozygous flies was examined. This demonstrates that chm haploinsufficiency causes PEV suppression, consistent with a role of its product in heterochromatin-mediated gene silencing (Grienenberger, 2002).
Chromatin-mediated transcriptional control during development is best illustrated by the PcG and trxG groups of genes, whose function is required for appropriate maintenance of Hox gene expression. To address whether Chm plays a role in this process, the effect of chm mutation was tested on the activity of Fab-7, a fragment from the Bithorax complex that contains a Pc response element (PRE) and recapitulates many aspects of transcriptional repression mediated by PcG proteins. The strong PcG-dependent repression of mini-white by Fab-7 in 5F24 25.2 flies is impaired upon inactivation of one copy of chm. Heterozygosity for the amorphous PcXT109 allele produces comparable derepression of mini-white, showing that the effect of chm on Fab-7 is as strong as that of Pc. In a parallel control, reducing the dosage of mof fails to modify mini-white expression, indicating that Chm but not any MYST protein provides activity acting at Fab-7. Chm is thus required for transcriptional repression mediated by the Fab-7 PRE, suggesting a role in the formation and/or activity of silencing PcG complexes (Grienenberger, 2002).
To further examine whether Chm and PcG proteins act together to maintain Hox gene repression, the effect of a reduction of chm dosage was tested on homeotic transformations that result from mutations affecting either PcG transregulators or a PRE cis-regulatory element. The first PcG dominant phenotype examined was a T2 into T1 transformation. In the second leg disc of Pc male heterozygotes, derepression of Sex comb reduced leads to the formation on the second leg of a sex comb, a structure normally found on the first leg only. The mutation of one copy of chm significantly enhances this phenotype. chm and PcG gene interactions in the specification of adult abdomen identities were tested. In parasegment 9 (PS9) of males heterozygous for PcXT109 or for the ph410 allele of polyhomeotic (ph), inappropriate expression of Abdominal-B (Abd-B) produces a mild transformation of the fourth abdominal segment into the fifth (A4 into A5), as evidenced by patches of pigmentation in the anterior part of A4. This phenotype, which is never observed in a wild-type context, occurs at low frequency in males heterozygous for chm (less than 1%). Double heterozygotes for chm and for ph or Pc exhibit increased A4 into A5 transformation and/or increased number of transformed individuals, compared to single PcG mutants. Finally, the homeotic transformation induced by a PRE mutation was examined. McpB116 affects Abd-B silencing in PS9, giving rise to incomplete A4 into A5 transformations. This homeotic phenotype is stronger in double heterozygotes for chm14 and McpB116 and becomes further enhanced by the mutation of one copy of Pc. In the various genetic contexts reported here, chm was therefore found to genetically interact with PcG genes and the Mcp element in a positive manner. These synergistic effects strongly suggest that Chm collaborates with PcG proteins for PRE-mediated repression at Hox gene loci (Grienenberger, 2002).
Direct evidence for a role of Chm in Hox gene silencing was obtained from the examination of Ubx expression in imaginal discs. Whereas Ubx is not detected in the columnar epithelium of a wild-type wing disc, derepression is observed in few cells from discs heterozygous for Pc, and a more extended activation occurs in discs heterozygous for both chm and Pc. These results confirm that Chm and PcG proteins act together to repress Hox genes. No misexpression of Ubx could be detected, however, in discs from chm homozygous larvae. Thus, chm can be classified as an enhancer of PcG mutations instead of a novel PcG gene (Grienenberger, 2002).
Recombinant Chm turned out unable to acetylate histones in vitro, as its human homolog HBO1 (Iizuka, 1999). Thus, assays of acetyltransferase activity in vivo were carried out. The heterologous yeast system was used. The rationale was to test, using a three step process, whether the ability of Chm to substitute for a MYST yeast protein is lost upon enzymatic inactivation. In a first step, it was established that chm can replace SAS2 in TPE. As already described, disruption of SAS2 derepresses telomeric silencing and impairs cells carrying a subtelomeric URA3 gene to grow on 5-FOA. Chm overexpression in sas2Δ cells partially restores TPE as shown by the increased colony-forming ability on 5-FOA. As a control, Chm is unable to rescue telomeric silencing defect caused by Set1 deficiency, a SET domain protein devoid of MYST domain, and, reciprocally, Set1 cannot restore defect in silencing associated with loss of Sas2. These experiments indicate that Chm does not act as a general signal in telomere silencing but rather specifically replaces Sas2 in TPE. A Chm variant was generated where the glycine at position 680 was mutated into glutamate. This glycine lies at a central position in the Q/RxxGxG motif and is essential for enzymatic activity. The variant protein fails to restore TPE, indicating that Chm acetyltransferase activity is needed for the assembly of repressive telomeric chromatin in sas2Δ cells (Grienenberger, 2002).
In a second step, PEV modification was examined. As reported above, Chm overexpression can reverse the defect of y repression caused by chm heterozygosity in γ238 flies. Providing ChmG680 instead of wild-type Chm in an otherwise similar background has no effect on y expression. ChmG680 mutation does not impair protein stability, since extracts from HSchm and HSchmG680 animals raised at 25°C react similarly on Western blot. Thus, ChmG680 cannot modify PEV, indicating that heterochromatin-mediated silencing requires Chm acetyltransferase activity (Grienenberger, 2002).
Third, whether ChmG680 could rescue the lethality of chm14 animals was examined. If the acetyltransferase activity is essential for development, then either no or a far less efficient rescue is expected from heat shock constructs providing ChmG680 instead of wild-type Chm. Two lines transgenic for the wild-type and four for the variant were generated. Efficient rescue by HSchm was obtained, raising one line at 25°C and providing the other with a larval heat pulse. In contrast, HSchmG680 does not allow any rescue at 25°C. For two of the four transgenic lines, however, rare escapers were eventually obtained after larval induction. These results indicate that the acetyltransferase activity of Chm is required for normal development. The fact that ChmG680 can rarely rescue lethality suggests either that the variant still possesses residual acetyltransferase activity and/or that other regions of the protein encode distinct essential functions (Grienenberger, 2002).
These results extend previous observations that MYST proteins are important for the definition of silent heterochromatin. Thus, H4 lysine 16 is a likely in vivo target of Sas2, and this HAT activity is required for telomere silencing mediated by a complex formed by Sas2 and the chromatin assembly factors CAF-1 and Asf1. The ability of Chm but not of ChmG680 to partially rescue the sas2Δ silencing defect suggests it may have a similar HAT activity. Other findings have connected MYST acetyltransferases, ORC, and heterochromatin-mediated silencing. HBO1, the human homolog of Chm, interacts with ORC1, and DmOrc2 is, as chm, a dominant suppressor of PEV. Furthermore, ORC subunits and the Su(var) proteins HP1 and HOAP form a complex required for heterochromatin assembly. In this context, the recruitment of HBO1/Chm might provide specific activity needed for the reestablishment of acetylation patterns after DNA replication and for the ORC/HP1 function in the process of heterochromatin formation (Grienenberger, 2002).
The results provide evidence that Chm is required for PcG-mediated silencing during larval development. This is presumably not the case during embryogenesis, since chm mutation has no effect on cuticular identity. Two PcG protein complexes have been isolated, only from embryos so far, and both contain histone modifying activities. The E(z)/Esc complex, partially characterized, contains the RPD3 HDAC. The 30 proteins from PRC1 have been identified, among which are RPD3 and the TAFII250 HAT. This indicates that PRC1 needs some HAT activity to mediate repression and that the silent chromatin state likely results from a steady-state acetylation level defined by the combination of TAFII250 acetylating and RPD3 deacetylating activities. Chm has not been found in PRC1, consistent with the conclusion that it is dispensable for PcG silencing during embryogenesis. It will be interesting to check for the presence of Chm in PcG repressive complexes acting during imaginal development (Grienenberger, 2002).
Further work is needed to elucidate how Chm conveys chromatin-mediated silencing. One possibility that would obey the general correlation between histone acetylation and gene activation is that Chm promotes acetylation and allows transcription at loci required for repression, including PcG and Su(var) genes. The ability of Chm to replace Sas2 in TPE in a process dependent of its acetyltransferase activity rather suggests a direct involvement in silencing. Considering the novel conceptual frame of the histone code, Chm may thus provide acetylation marks required for the definition of histone tail modification patterns allowing the recruitment of silencing complexes, such as ORC/HP1 at heterochromatin or PcG complexes at PREs. Alternatively, Chm might contribute to epigenetic silencing by modifying chromatin proteins other than histones, such as PcG and heterochromatic proteins (Grienenberger, 2002).
Reference names in red indicate recommended papers.
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Burke, T. W., Cook, J. G., Asano, M. and Nevins, J.R. (2001). Replication factors MCM2 and ORC1 interact with the histone acetyltransferase HBO1. J. Biol. Chem. 276: 15397-15408. 11278932
Doyon, Y., et al. (2006). ING tumor suppressor proteins are critical regulators of chromatin acetylation required for genome expression and perpetuation. Mol. Cell. 21(1): 51-64. 16387653
Georgiakaki, M., et al. (2006). Ligand-controlled interaction of HBO1 with the N-terminal transactivating domain of progesterone receptor induces SRC-1-dependent co-activation of transcription. Mol. Endocrinol. [Epub ahead of print]. 16645042
Grienenberger. A., et al. (2002). The MYST domain acetyltransferase Chameau functions in epigenetic mechanisms of transcriptional repression. Curr. Biol. 12(9): 762-6. 12007422
Iizuka, M. and Stillman, B. (1999). Histone acetyltransferase HBO1 interacts with the ORC1 subunit of the human initiator protein. J. Biol. Chem. 274: 23027-23034. 10438470
Iizuka, M., Matsui, T., Takisawa, H. and Smith, M. M. (2006). Regulation of replication licensing by acetyltransferase Hbo1. Mol. Cell. Biol. 26(3): 1098-108. 16428461
Kim, T., Yoon, Y., Cho, H., Lee, W-B., Kim, J., Song, Y-H., Kim, S.N., Yoon, J.H., Kim-Ha, J. and Kim, Y-J. (2005). Downregulation of lipopolysaccharide response in Drosophila by negative crosstalk between the AP-1 and the NF-kappaB signaling modules. Nat. Immunol. 6: 211-218. 15640802
Miotto, B., Sagnier, T., Berenger, H., Bohmann, D., Pradel, J. and Graba, Y. (2005). Chameau HAT and DRpd3 HDAC function as antagonistic cofactors of JNK/AP-1-dependent transcription during Drosophila metamorphosis. Genes Dev. 20(1): 101-12. 16391236
Ogawa, S., Lozach, J., Jepsen, K., Sawka-Verhelle, D., Perissi, V., Sasik, R., Rose, D. W., Johnson, R. S., Rosenfeld, M. G., and Glass, C. K. (2004). A nuclear receptor corepressor transcriptional checkpoint controlling activator protein 1-dependent gene networks required for macrophage activation. Proc. Natl. Acad. Sci. 101: 14461-14466. 15452344
Sharma, M. Zarnegar, M., Li, X., Lim, B. and Sun, J. (2000). Androgen receptor interacts with a novel MYST protein, HBO1. J. Biol. Chem. 275: 35200-35208. 10930412
Stedman, W., Deng, Z., Lu, F. and Lieberman, P. M. (2004). ORC, MCM, and histone hyperacetylation at the Kaposi's sarcoma-associated herpesvirus latent replication origin. J. Virol. 78(22): 12566-75. 15507644
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Weiss, C., Schneider, S., Wagner, E.F., Zhang, X., Seto, E., and Bohmann, D. (2003). JNK phosphorylation relieves HDAC3-dependent suppression of the transcriptional activity of c-Jun. EMBO J. 22: 3686-3695. 12853483
Zong, H., et al. (2005). Cyclin-dependent kinase 11(p58) interacts with HBO1 and enhances its histone acetyltransferase activity. FEBS Lett. 579(17): 3579-88. 15963510
date revised: 2 July 2006
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