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Genetics, Vol. 174, 241-251, September 2006, Copyright © 2006
doi:10.1534/genetics.106.059980
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Department of Developmental Biology, Wenner-Gren Institute, Arrhenius Laboratories E3, Stockholm University, S-106 91 Stockholm, Sweden
2 Corresponding author: Department of Developmental Biology, Wenner-Gren Institute, Arrhenius Laboratories E3, Stockholm University, S-106 91 Stockholm, Sweden.
E-mail: mannervik{at}devbio.su.se
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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The Reptin protein, also known as TIP48, TIP49b, or RUVBL2, is related to bacterial RuvB, an ATP-dependent DNA helicase that promotes branch migration in Holliday junctions (KANEMAKI et al. 1999). Reptin, and the related Pontin (TIP49, TIP49a, or RUVBL1) protein, possess intrinsic ATPase and helicase activities and can heterodimerize (KANEMAKI et al. 1999; MAKINO et al. 1999). In yeast, both Reptin and Pontin are part of the INO80 chromatin-remodeling complex (SHEN et al. 2000), as well as the Swr1 complex that can exchange histone H2A with the variant histone H2A.Z (KROGAN et al. 2003; KOBOR et al. 2004; MIZUGUCHI et al. 2004). Reptin and Pontin appear to play antagonistic roles in development by regulating Wnt signaling (BAUER et al. 2000) and heart growth in zebrafish embryos (ROTTBAUER et al. 2002). Mammalian Reptin and Pontin are present in TIP60 HAT complexes, which are involved in induction of apoptosis in response to DNA damage and which interact with the c-Myc protein to promote its oncogenic activity (IKURA et al. 2000; WOOD et al. 2000; FUCHS et al. 2001; CAI et al. 2003; FRANK et al. 2003; DOYON et al. 2004).
TIP60 is a HAT of the MYST family (UTLEY and COTE 2003). The homologous yeast protein Esa1 is the catalytic subunit of the nucleosome acetyltransferase of H4 (NuA4) complex, which acetylates lysines in histone H4 and H2A (DOYON and COTE 2004). In Drosophila, the TIP60 complex acetylates the phosphorylated variant histone H2Av after DNA double-strand breaks and exchanges it with unmodified H2Av (KUSCH et al. 2004). The composition of TIP60 and NuA4 complexes has recently been determined (DOYON and COTE 2004). TIP60 (yeast Esa1), ING3 (Yng2), and Enhancer of Polycomb (EPC1, yeast Epl1) form a core complex that is sufficient for acetylation of histones in nucleosomes (BOUDREAULT et al. 2003; DOYON et al. 2004). Mammalian and Drosophila TIP60 complexes contain four subunits not present in yeast NuA4 (DOYON et al. 2004; KUSCH et al. 2004): Brd8, Reptin, Pontin, and Domino (also known as p400), the homolog of yeast Swr1.
Polycomb group (PcG) proteins are evolutionarily conserved chromatin regulators that maintain appropriate expression patterns of developmental control genes, such as the Hox genes (RINGROSE and PARO 2004). PcG proteins are generally repressors that maintain the off state of genes and exist in at least two distinct protein complexes. The Esc–E(z) complex is a histone methyltransferase that includes the catalytic subunit Enhancer of zeste [E(z)], as well as the extra sex combs (esc) and suppressor of zeste 12 [Su(z)12] subunits (BIRVE et al. 2001; CAO et al. 2002; CZERMIN et al. 2002; KUZMICHEV et al. 2002; MULLER et al. 2002). Another complex purified from Drosophila embryos, Polycomb repressive complex 1 (PRC1) has a mass of >1 MDa (SHAO et al. 1999). In addition to genetically identified PcG proteins, it includes TFIID subunits, the Reptin protein, and other polypeptides (SAURIN et al. 2001). The PRC1 complex can block chromatin remodeling by the SWI/SNF complex in vitro (LEVINE et al. 2004). A core PRC1 complex consisting of Polycomb (Pc), Posterior sex combs (Psc), Polyhomeotic (Ph), and dRING1/Sex combs extra (Sce) is sufficient for the in vitro activities of PRC1 (LEVINE et al. 2004). Recently, it was shown that dRing1/Sce as well as its mammalian orthologs are E3 ubiquitin ligases that monoubiquitylate histone H2A (DE NAPOLES et al. 2004; WANG et al. 2004).
Here, we investigate the role of Drosophila Reptin in chromatin regulation. We show that it interacts genetically with PcG gene products and suppresses position-effect variegation (PEV), properties shared by other Drosophila TIP60 complex components. We suggest that the fly TIP60 complex regulates epigenetic processes leading to a repressive chromatin state. This is a novel activity of a HAT complex that has previously been implicated in transcription activation and DNA repair.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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The P element in rept was mobilized using a transposase source, and progeny were scored for loss of the rosy marker gene. reptl(3)06945 [ry+] ry/
2-3 Dr ry males were crossed to Ki ry/TM3 ry virgin females and rosy-eyed P* ry/Ki ry male progeny were crossed to CxD/TM3, Sb females to balance the excision chromosome over CxD. Both viable and lethal strains were obtained. Genomic DNA from two of the viable strains was sequenced to confirm that the excisions were precise. One of these, reptex1, was used as a control in the genetic interaction tests.
To make a precise excision of the P[lacW] insertion in dMRG15, we first crossed the dMRG15j6A3 allele with a transposase source and selected progeny that had lost the mini-white marker gene. No homozygous viable w minus strain was obtained, indicating the presence of a second-site lethal mutation on the chromosome. We crossed the w strains to Df(3R)ea, which uncovers the dMRG15 locus, and found one viable precise excision strain. Next, the dMRG15j6A3 [w+] allele was outcrossed to w1118, and recombinants that had lost the second-site lethal mutation were selected by crossing to the dMRG15 precise excision [w–] strain (that still contained the second-site lethal mutation). Viable dMRG15j6A3 [w+]/dMRG15 precise excision [w–] males were selected and crossed to w; CxD/TM3, Sb to establish a clean dMRG15j6A3 stock, designated dMRG15P. To remove the lethal mutation from the dMRG15 precise excision [w–] chromosome, it was recombined with dMRG15P [w+]. Putative w- recombinants balanced over CxD were crossed with the original dMRG15 precise excision/TM3, Sb stock, and viable nonbalancer flies selected. Stocks were established from their Sb siblings, and one of the strains, dMRG15ex1, was used in the genetic interaction experiments.
Genetic interactions with PcG genes were scored by crossing PcG mutant stocks to wild-type, reptex1, reptl(3)06945/TM3, Sb, dMRG15P/TM3, Sb, or dMRG15ex1 flies at 25°. Male progeny were examined for the presence of sex combs on the second and third pair of legs. During the course of these experiments, the penetrance of the extra sex combs phenotype in PcG alleles increased, perhaps because propionic acid was added to the fly food during later stages of this work. However, enhancement of the phenotype by rept or dMRG15 alleles was always compared to control crosses performed in parallel.
Rescue of the genetic interaction was obtained with a reptin cDNA cloned into the pUASp vector (RORTH 1998). The expressed sequence tag (EST) clone LD12420 was PCR amplified with Pfu polymerase and ligated into the XbaI and blunted KpnI sites of the pUASp vector. The construct was sequenced to ensure that no mutations were introduced during PCR and then injected into w1118 flies following standard procedures (RUBIN and SPRADLING 1982). An insertion on the second chromosome was used to establish a w; UASp-reptin; reptl(3)06945/TM3, Sb stock, which was crossed to a w; actin5C-Gal4/+; Pc11/TM3, Sb stock. w; UASp-reptin/actin5C-Gal4; Pc11/reptl(3)06945 males could be distinguished from w; UASp-reptin/+; Pc11/reptl(3)06945 males by their stronger eye color. As a control, we crossed w; UASp-reptin; reptl(3)06945/TM3, Sb flies to a Pc11/TM3, Sb Ser stock. w; UASp-reptin/+; Pc11/reptl(3)06945 males were compared to their w; UASp-reptin/+; Pc11/TM3, Sb brothers.
Pc and rept mutant stocks were crossed to transgenic flies containing Polycomb response elements (PRE) linked to the mini-white reporter gene and the eye color observed in progeny. Different insertions of 8.2 XbaI derived from the Scr gene (GINDHART and KAUFMAN 1995), two constructs from the Mcp element (44.16.1 and 43.36.B11, construct nos. 19 and 12 described in MULLER et al. 1999), and 5F24 from the Fab7 element of the bithorax complex (CAVALLI and PARO 1999) were tested.
Effects on position-effect variegation was determined by crossing wm4h females or females carrying P elements with variegating mini-white expression (kindly provided by Lori Wallrath and Steve Henikoff) to wild-type, Su(var)3-91/TM3 Sb Ser, reptex1, or reptl(3)06945/TM3 Sb males at room temperature. The eye color of male progeny was examined, and representative eyes for each genotype were photographed. Modification of Notch variegation was examined by crossing T(1;4)wm258-21 y1 wa/FM4 females to wild-type, Su(var)3-91/TM3 Sb Ser, reptex1, reptl(3)06945/TM3 Sb, dMRG15ex1, or dMRG15P/TM3, Sb males. The number of notches per wing was scored in female progeny without the FM4 balancer chromosome.
In situ hybridization and immunohistochemistry:
Embryos were collected from the reptl(3)06945 stock balanced over TM3, Ubx-lacZ, and from Oregon-R flies (wild-type controls) on apple juice plates and aged appropriately. RNA in situ hybridization using a digoxigenin-labeled antisense rept RNA probe was performed as previously described (TAUTZ and PFEIFLE 1989; JIANG et al. 1991).
Immunohistochemistry was performed essentially as described previously. In brief, reptl(3)06945/TM6 Tb, Pc11/TM3 Sb Ser, and Psc1/CyO mutant stocks were used to generate rept, Pc, and Psc heterozygous as well as rept/Pc and Psc/+; rept/+ mutant third instar larvae. The larvae were dissected in phosphate-buffered saline (PBS), transferred to ice-cold 4% paraformaldehyde in PBS, and fixed for 20 min at room temperature, followed by washes in PBS and PBT (PBS + 0.1% Tween 20). They were blocked in PBSBT (PBS + 0.1% Triton X-100 + 0.5% BSA) and incubated with anti-Scr monoclonal antibody 6H4.1 (1:10 dilution, Develpmental Studies Hybridoma Bank) at 4° overnight. After washing with PBSBT, they were developed with the Vectastain ABC Elite kit. After being washed in PBT solution, embryos were rinsed in PBS/50% glycerol, followed by mounting and dissection of leg imaginal discs in 80% glycerol.
Induction of clones and immunofluorescence:
Since reptin mutant clones are very small (data not shown), we used a Minute background. reptl(3)06945 FRT2A/TM6b Tb flies were crossed to yw hsFLP; M(3)i55 hs-nGFP FRT2A/TM6b Tb and F1 larvae were heat-shocked for 1 hr at 37° as first and second instar larvae. Prior to dissection of non-Tb larvae, they were subjected to one more 1-hr heat shock followed by a 1-hr recovery to induce expression of the GFP protein. For detection of GFP mRNA, wing imaginal discs of wandering third instar larvae were dissected immediately after a 30-min heat shock. As a control, we used Su(z)124 FRT2A/TM3 Sb flies crossed to hsFLP; ubi-GFP FRT2A to generate hsFLP; Su(z)124 FRT2A/ubi-GFP FRT2A larvae that were heat-shocked for 1 hr at 37° as first and second instar larvae.
A Ubx 1.6-kb PRE was recombined onto the reptl(3)06945 FRT2A chromosome. A yw; >PBX>-PRE1.6-IDE-Ubx-nlacZ [y+] strain (FRITSCH et al. 1999) was crossed with reptl(3)06945 FRT2A flies, and y+ w+ recombinants were selected. These were crossed to a reptl(3)06945/TM6b Tb stock to test for the presence of the reptin mutation. The resulting PRE1.6-lacZ reptl(3)06945 FRT2A/TM6b Tb stock was crossed to yw hsFLP; M(3)i55 hs-nGFP FRT2A/TM6b Tb flies and larval progeny were heat-shocked as described above.
Wing imaginal discs of wandering third instar larvae were dissected, fixed in 4% paraformaldehyde, blocked in block solution (1% BSA in PBT), and incubated with a rabbit ß-gal antibody (Cappel) diluted 1:150 or with a monoclonal anti-Ubx antibody (WHITE and WILCOX 1984) diluted 1:75 at 4° overnight. To identify homozygous mutant clones, a rabbit anti-GFP (1:1000, Promega, Madison, WI) or a mouse anti-GFP (1:500, Sigma, St. Louis) antibody was mixed with the primary antibodies. Discs were washed in block solution and incubated with Cy3-conjugated anti-rabbit (1:500), and Cy2-conjugated anti-mouse (1:200) secondary antibodies (Jackson Laboratories) at room temperature for 2 hr. After being washed in PBT solution, wing discs were rinsed in PBS and in PBS/50% glycerol and then mounted in PBS/80% glycerol. A laser-scanning microscope (Zeiss) was used for confocal imaging. Acquired images were processed with LSM510 software (Zeiss).
Reptin mRNA expression in wing discs was determined by fluorescent in situ hybridization using tyramide signal amplification (TSA; Perkin-Elmer, Norwalk, CT). Larvae at the late third instar stage containing reptin mutant clones were dissected, fixed in 1 ml 4% paraformaldehyde (PFA) in PBS at room temperature for 20 min. Samples were then washed with PBS, 50% methanol, and methanol and stored at –20° overnight. After five washes in ethanol, samples were incubated in a mixture of xylene and ethanol (1:1) for 60 min, washed five times in ethanol, and rehydrated by immersion in a graded methanol series (80, 50, and 25% v/v in H2O) and finally in H2O. After treatment with acetone (80%) at –20° for 10 min, samples were washed four times with PBT, fixed again in 4% PFA in PBS for 20 min, and washed in PBT. Digoxigenin-labeled reptin and fluorescein-labeled GFP RNA probes were simultaneously added to the samples and hybridized at 55° overnight. After hybridization, samples were washed in PBT and incubated with peroxidase-conjugated antifluorescein antibody (Roche, 1:10,000) for 1.5 hr. FITC-labeled dinitrophenyl (DNP) amplification reagent was used to develop the signal. To wash away the antibody, samples were incubated in 0.01 M HCl twice for 5 min each. Following washes in PBT, peroxidase-coupled antidigoxigenin antibody (Roche, 1:5000) was added to the samples. Cy3-labeled DNP amplification reagent was used to detect the antibody. Discs were dissected, mounted in 80% glycerol, and imaged with a confocal microscope (Zeiss).
Immunoprecipitation:
A Reptin expression plasmid was constructed through amplification of the Reptin open reading frame by PCR from EST clone LD12420. The PCR product was cloned into the EcoRI–XbaI sites of the pAc5.1/V5-His vector to generate a V5-tagged Reptin protein under control of the actin promoter. Similarly, PCR was performed to obtain the dTIP60 open reading frame from EST clone LD31064, and the product was ligated into the EcoRI-digested pRmHa-C-FLAG-His vector. This results in a plasmid expressing the FLAG-tagged dTIP60 protein from the metallothionein promoter.
A stable cell line expressing V5-tagged Reptin was generated as follows. Drosophila Schneider S2 cells were transfected by means of calcium phosphate (DES transfection kit, Invitrogen, San Diego) using a DNA ratio of 19:1 (9.5 µg pAc-Reptin-V5:0.5 µg pCoBlast) and grown in the presence of 50 µg/ml blasticidin. After 2 weeks, resistant clones were replated. The cell line was maintained in medium (Schneider's Drosophila medium with 10% fetal calf serum) containing 10 µg/ml blasticidin.
FLAG-tagged dTIP60 was transfected into the cells stably expressing Reptin-V5 in six-well plates using calcium phosphate. For each well, 2 µg plasmid DNA and 3 ml culture containing 3 x 106 cells were used. Ten microliters of 100 mM copper sulfate stock was added on day 3 to induce dTIP60-FLAG expression. Cells were harvested on day 4 and lysed in a 500-µl lysis buffer (10 mM Tris pH 8.0, 140 mM NaCl, 1.5 mM MgCl2, 1% NP40, and protease inhibitor cocktail). The protein concentration was determined with Bradford reagent. A fraction of the extract was saved as input. The remaining extract was precleared by incubating with 30 µl protein A sepharose for 1 hr at 4°. Precleared extract was incubated with 2.5 µg rabbit polyclonal anti-FLAG antibody (Sigma F7425) overnight in a cold room on a rotator. Protein aggregates were removed by spinning the samples at full speed for 10 min, and the supernatants were incubated with 20 µl protein A sepharose for 1 hr at 4°. Beads were washed twice in 1 ml dilution buffer (lysis buffer with 0.1% NP40), once in TSA buffer (10 mM Tris pH 8.0, 140 mM NaCl), and once in 50 mM Tris pH 6.8. Samples were separated on a SDS–10% PAGE gel and blotted onto PVDF membrane (Amersham, Buckinghamshire, UK) overnight. A Western blot was performed using mouse monoclonal anti-V5 antibody (1:5000, Invitrogen) followed by a HRP-linked secondary antibody diluted 1:5000. Enhanced chemiluminescence detection was performed as described by the manufacturer (Amersham). The membrane was reprobed with mouse monoclonal anti-FLAG antibody M2 (1:400, Sigma), which showed that dTIP60-FLAG was expressed at low levels.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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Two members of the E(z)–Esc complex, esc and Pcl, were also tested (Table 1). The esc alleles esc1 and esc21 showed either no or a very weak interaction with reptin mutants. However, the number of flies with extra sex combs in one of the two Pcl alleles, Pcl11, was increased by the reptin mutation (Table 1). This shows that the ability of reptin to genetically interact with PcG genes is not restricted to components of the PRC1 complex.
To confirm that the interactions observed are caused by the P-element insertion in reptin, and not due to unidentified second-site mutations on the l(3)06945 chrom osome, we generated precise excisions of the P element. Such excision lines are viable and do not interact with PcG genes (Table 1). Furthermore, expression of the reptin cDNA using an actin-Gal4 driver transgene could rescue the reptin–Pc interaction (Table 2). Under these culture conditions, Pc11 flies contained extra sex combs even in the absence of the reptin mutation. However, the number of sex combs per fly was enhanced by the reptin mutation (Table 2). Introduction of actin-Gal4 and UAS-reptin transgenes into the Pc11/reptinl(3)06945 trans-heterozygous flies reduced the number of sex combs to below the number observed with Pc11 over the balancer chromosome (Table 2). From these data, we conclude that genetic interactions with PcG genes are specifically due to reduced reptin expression in l(3)06945 mutant flies.
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We also examined expression of the homeotic genes Scr and Ubx in reptin homozygous mutant embryos. However, no misexpression was observed (data not shown). Reptin is maternally contributed to the embryo, and its mRNA is consequently present ubiquitously in early embryos (Figure 1A). The maternal contribution may mask a regulatory role for reptin in embryonic Hox gene expression. To address this possibility, we attempted to remove the maternal reptin contribution by use of germline clones. However, reptin l(3)06945 homozygous germ cells fail to produce embryos.
Reptin is a suppressor of position-effect variegation:
We then tested whether reptin mutants affect PEV, a phenomenon resulting in clonal silencing of genes juxtaposed to heterochromatin (SCHOTTA et al. 2003). In In(1)wm4h flies, an inversion on the X chromosome positions the white gene close to pericentric heterochromatin, resulting in a variegated eye color (REUTER and WOLFF 1981). We crossed reptin mutants to wm4h flies and compared the progeny to a cross of wm4h with Su(var)3-9 flies. Methylation of histone H3 lysine 9 by the Su(var)3-9 protein is necessary for heterochromatin formation (REA et al. 2000; SCHOTTA et al. 2002), and consequently, Su(var)3-9 mutants strongly suppress PEV (Figure 4E; compare with Figure 4, A and F). We found that reptin mutants also suppress variegation of wm4h (Figure 4B; compare with Figure 4, C and D). By contrast, most PcG genes do not affect PEV (SINCLAIR et al. 1998).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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Drosophila Reptin copurifes with the Polycomb complex PRC1 (SAURIN et al. 2001). This prompted us to investigate whether the biochemical interaction with PRC1 was accompanied by a genetic interaction. Table 1 and Figure 2 show that Reptin and PRC1 components genetically interact to regulate expression of the Hox gene Scr. However, Reptin also interacts with a PcG gene product not associated with the PRC1 complex, Pcl. Although we did not detect interactions between reptin heterozygous mutants and several PREs tested, a PRE from the Ubx gene is derepressed in reptin homozygous mutant cells (Figure 3B). This shows that Reptin contributes an essential function to the activity of this PRE. However, unlike most PcG genes, reptin homozygous mutants do not derepress endogenous Hox gene expression (Figure 3 and data not shown). It appears that repression of endogenous Hox genes is more complex and not as sensitive to the loss of Reptin as the Ubx PRE. In contrast to most PcG genes, reptin mutants suppress PEV (Figure 4 and Table 3). Interestingly, derepression of the Ubx PRE also occurs in embryos mutant for other suppressors of PEV (CHAN et al. 1994), indicating that this PRE may be highly sensitive to the chromatin environment in its vicinity. Since reptin mutants suppress PEV and fail to derepress endogenous Hox gene expression, we do not consider reptin a bona fide PcG gene, and we find it unlikely that Reptin protein contributes an essential function to the PRC1 complex. In fact, the biochemical activities ascribed to PRC1 can be reconstituted either with recombinant dRing1/Sce (WANG et al. 2004) or with four core components whose activity can be further enhanced by the DNA-binding proteins zeste and GAGA (MULHOLLAND et al. 2003).
Given that Reptin is present in TIP60 complexes in mammals and recently was shown to be a component of a Drosophila TIP60 complex (IKURA et al. 2000; FUCHS et al. 2001; CAI et al. 2003; DOYON et al. 2004; KUSCH et al. 2004), we considered the possibility that the genetic interactions observed with PcG genes are due to the presence of Reptin in the fly TIP60 complex. The products of two previously characterized Drosophila genes, E(Pc) and domino, are also present in the TIP60 complex. Strikingly, E(Pc) and domino mutants share with reptin the ability to genetically interact with PcG genes and suppress PEV. E(Pc) is an unusual PcG gene that has very minor effects on Hox gene expression, and unlike most PcG genes, modifies PEV (SOTO et al. 1995; SINCLAIR et al. 1998; STANKUNAS et al. 1998). In both yeast and humans, E(Pc) homologs form a core complex with Esa1 (TIP60) and Yng2 (ING3) that is sufficient for the nucleosomal acetylation of histones H4 and H2A by the NuA4 complex (BOUDREAULT et al. 2003; DOYON et al. 2004). That such an integral NuA4/TIP60 complex component displays phenotypes similar to reptin mutants suggests to us that Reptin functions through the fly TIP60 complex.
Domino protein is similar to p400 and to SRCAP in mammals and to Swr1 in yeast (EISSENBERG et al. 2005). Swr1 has recently been shown to exchange the variant histone H2A.Z (Htz1 in yeast) for H2A in nucleosomes (KROGAN et al. 2003; KOBOR et al. 2004; MIZUGUCHI et al. 2004). Intriguingly, an involvement of Htz1 (H2A.Z) in controlling the spreading of silenced chromatin has recently been demonstrated in yeast (MENEGHINI et al. 2003; BABIARZ et al. 2006). Exchange of variant histones may be a conserved feature of chromatin regulation since a recent report demonstrates that Drosophila H2Av behaves genetically as a PcG gene and suppresses PEV (SWAMINATHAN et al. 2005). Domino exchanges phosphorylated and acetylated H2Av for unmodified H2Av after DNA damage (KUSCH et al. 2004). However, we found no change in binding of H2Av to polytene chromosomes prepared from domino mutant larvae (data not shown).
A P-element insertion was identified in the gene encoding one additional TIP60 complex component, the chromodomain-containing protein MRG15. Human MRG15 (MORF-related gene on chromosome 15) has been implicated in cellular senescence and regulation of the B-myb promoter (LEUNG et al. 2001). Both human and yeast (Eaf3/Alp13) MRG15 have been found in Sin3/HDAC complexes in addition to the TIP60 (NuA4) complex (GAVIN et al. 2002; NAKAYAMA et al. 2003; DOYON et al. 2004), where it directs the histone deacetylase to coding regions through interaction of its chromodomain with methylated histone H3 lysine 36 (CARROZZA et al. 2005; JOSHI and STRUHL 2005; KEOGH et al. 2005). We found that MRG15 mutant flies interact with PcG genes and suppress PEV, just as other TIP60 complex components do. We take this as further support of our conclusion that Reptin's effects on chromatin processes are mediated through its association with the fly TIP60 complex.
What is the basis for the genetic interaction between TIP60 components and PcG genes? One possibility is that the TIP60 complex regulates PcG expression. However, we did not observe reduced Pc expression in reptin mutant embryos (data not shown). Another possibility is that the enzymatic activities of the TIP60 complex cooperate with PcG genes to mediate transcriptional silencing. Since binding of Pc to polytene chromosomes is abolished in H2Av mutant animals (SWAMINATHAN et al. 2005), TIP60 complex-mediated histone variant exchange might cause the genetic interaction with PRC1. However, we found that binding of PcG proteins to polytene chromosomes is unaffected in domino mutant larvae (data not shown). It is possible that PRC1-mediated H2A ubiquitylation helps to recruit the TIP60 complex, whose histone acetylation or histone exchange activity assists in transcriptional repression. Alternatively, histone acetylation or exchange facilitates binding of the PRC1 complex to PREs. A similar mechanism has been invoked for the cooperation of the Esc–E(z) complex and PRC1, where Esc–E(z) trimethylates histone H3 lysine 27, which is recognized by the chromodomain of Polycomb (FISCHLE et al. 2003; MIN et al. 2003).
We have shown that the Drosophila TIP60 complex plays a role in epigenetic gene silencing in vivo. A similar case has been described for the yeast HAT complex SAGA (Spt-Ada-Gen5-acetyltransferase) that is required for both activation and repression of the ARG1 gene (RICCI et al. 2002). Two other yeast HATs, Sas2 and Sas3, also promote gene silencing (REIFSNYDER et al. 1996). Interestingly, the Drosophila HAT Chameau suppresses PEV and cooperates with PcG genes as well (GRIENENBERGER et al. 2002). TIP60, Sas2, Sas3, and Chameau are HATs that belong to the MYST family (UTLEY and COTE 2003). Therefore, MYST family HATs in both yeast and flies can control epigenetic inheritance of silent chromatin.
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FOOTNOTES |
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