Drosophila Reptin and Other TIP60 Complex Components Promote Generation of Silent Chromatin

Dai Qi, Haining Jin,1 Tobias Lilja and Mattias Mannervik2 Department of Developmental Biology, Wenner-Gren Institute, Arrhenius Laboratories E3, Stockholm University, S-106 91 Stockholm, Sweden Manuscript received April 26, 2006 Accepted for publication June 22, 2006

ABSTRACT Histone acetyltransferase (HAT) complexes have been linked to activation of transcription. Reptin is a subunit of different chromatin-remodeling complexes, including the TIP60 HAT complex. In Drosophila, Reptin also copurifies with the Polycomb group (PcG) complex PRC1, which maintains genes in a transcriptionally silent state. We demonstrate genetic interactions between reptin mutant flies and PcG mutants, resulting in misexpression of the homeotic gene Scr. Genetic interactions are not restricted to PRC1 components, but are also observed with another PcG gene. In reptin homozygous mutant cells, a Polycomb response-element-linked reporter gene is derepressed, whereas endogenous homeotic gene expression is not. Furthermore, reptin mutants suppress position-effect variegation (PEV), a phenomenon resulting from spreading of heterochromatin. These features are shared with three other components of TIP60 complexes, namely Enhancer of Polycomb, Domino, and dMRG15. We conclude that Drosophila Reptin participates in epigenetic processes leading to a repressive chromatin state as part of the fly TIP60 HAT complex rather than through the PRC1 complex. This shows that the TIP60 complex can promote the generation of silent chromatin.

fundamental regulatory step in transcription and the related Pontin (TIP49, TIP49a, or RUVBL1) pro- A other DNA-dependent processes in eukaryotes is tein, possess intrinsic ATPase and helicase activities and the control of chromatin structure, which regulates can heterodimerize (Kanemaki et al. 1999; Makino et al. access of proteins to DNA. Histone acetylation and the 1999). In yeast, both Reptin and Pontin are part of the protein complexes that mediate this modification have INO80 chromatin-remodeling complex (Shen et al. been linked to activation of transcription (Struhl 2000), as well as the Swr1 complex that can exchange 1998). It is believed that lysine acetylation of histone N histone H2A with the variant histone H2A.Z (Krogan termini results in less compact chromatin by neutral- et al. 2003; Kobor et al. 2004; Mizuguchi et al. 2004). izing the positive charge of histones and that the acetyl Reptin and Pontin appear to play antagonistic roles groups are recognized by regulatory proteins that in development by regulating Wnt signaling (Bauer promote transcription (Workman and Kingston 1998; et al. 2000) and heart growth in zebrafish embryos Strahl and Allis 2000). However, it is becoming clear (Rottbauer et al. 2002). Mammalian Reptin and Pontin that histone acetyltransferases (HATs) can have func- are present in TIP60 HATcomplexes, which are involved tions other than facilitating transcription (Carrozza in induction of apoptosis in response to DNA damage et al. 2003). For example, the TIP60 HAT complex has and which interact with the c-Myc protein to promote its been implicated in DNA repair in yeast, flies, and oncogenic activity (Ikura et al. 2000; Wood et al. 2000; mammals (Ikura et al. 2000; Bird et al. 2002; Kusch Fuchs et al. 2001; Cai et al. 2003; Frank et al. 2003; et al. 2004). We have investigated the role of Drosophila Doyon et al. 2004). Reptin and other TIP60 components in chromatin TIP60 is a HAT of the MYST family (Utley and Cote regulation in vivo. 2003). The homologous yeast protein Esa1 is the The Reptin protein, also known as TIP48, TIP49b, or catalytic subunit of the nucleosome acetyltransferase RUVBL2, is related to bacterial RuvB, an ATP-depen- of H4 (NuA4) complex, which acetylates lysines in dent DNA helicase that promotes branch migration in histone H4 and H2A (Doyon and Cote 2004). In Holliday junctions (Kanemaki et al. 1999). Reptin, and Drosophila, the TIP60 complex acetylates the phos- phorylated variant histone H2Av after DNA double- strand breaks and exchanges it with unmodified H2Av 1Present address: Department of Genetics, University of Wisconsin, (Kusch et al. 2004). The composition of TIP60 and Madison, WI 53706. NuA4 complexes has recently been determined 2Corresponding author: Department of Developmental Biology, Wenner- oyon ote Gren Institute, Arrhenius Laboratories E3, Stockholm University, S-106 (D and C 2004). TIP60 (yeast Esa1), ING3 91 Stockholm, Sweden. E-mail: [email protected] (Yng2), and Enhancer of Polycomb (EPC1, yeast Epl1)

Genetics 174: 241–251 (September 2006) 242 D. Qi et al. form a core complex that is sufficient for acetylation of homozygous and trans-heterozygous mutant embryos, larvae, histones in nucleosomes (Boudreault et al. 2003; and adults. The reptl(3)06945 allele was recombined to a FRT3L-2A Doyon et al. 2004). Mammalian and Drosophila TIP60 [w1] chromosome. The P element in rept was mobilized using a transposase complexes contain four subunits not present in yeast source, and progeny were scored for loss of the rosy marker NuA4 (Doyon et al. 2004; Kusch et al. 2004): Brd8, gene. reptl(3)06945 [ry1] ry/D2-3 Dr ry males were crossed to Ki ry/ Reptin, Pontin, and Domino (also known as p400), the TM3 ry virgin females and rosy-eyed P* ry/Ki ry male progeny homolog of yeast Swr1. were crossed to CxD/TM3, Sb females to balance the excision Polycomb group (PcG) proteins are evolutionarily chromosome over CxD. Both viable and lethal strains were obtained. Genomic DNA from two of the viable strains was conserved chromatin regulators that maintain appro- sequenced to confirm that the excisions were precise. One of priate expression patterns of developmental control these, reptex1, was used as a control in the genetic interaction genes, such as the Hox genes (Ringrose and Paro tests. 2004). PcG proteins are generally repressors that To make a precise excision of the P[lacW] insertion in j6A3 maintain the off state of genes and exist in at least two dMRG15, we first crossed the dMRG15 allele with a transposase source and selected progeny that had lost the mini- distinct protein complexes. The Esc–E(z) complex is a white marker gene. No homozygous viable w minus strain was histone methyltransferase that includes the catalytic obtained, indicating the presence of a second-site lethal subunit Enhancer of zeste [E(z)], as well as the extra sex mutation on the chromosome. We crossed the w strains to combs (esc) and suppressor of zeste 12 [Su(z)12] Df(3R)ea, which uncovers the dMRG15 locus, and found one irve ao zermin viable precise excision strain. Next, the dMRG15j6A3 [w1] al- subunits (B et al. 2001; C et al. 2002; C 1118 uzmichev uller lele was outcrossed to w , and recombinants that had lost et al. 2002; K et al. 2002; M et al. 2002). the second-site lethal mutation were selected by crossing Another complex purified from Drosophila embryos, to the dMRG15 precise excision [wÀ] strain (that still con- Polycomb repressive complex 1 (PRC1) has a mass of tained the second-site lethal mutation). Viable dMRG15j6A3 .1MDa(Shao et al. 1999). In addition to genetically [w1]/dMRG15 precise excision [wÀ] males were selected and j6A3 identified PcG proteins, it includes TFIID subunits, the crossed to w; CxD/TM3, Sb to establish a clean dMRG15 aurin stock, designated dMRG15P. To remove the lethal mutation Reptin protein, and other polypeptides (S et al. from the dMRG15 precise excision [wÀ] chromosome, it was 2001). The PRC1 complex can block chromatin remod- recombined with dMRG15P [w1]. Putative w- recombinants eling by the SWI/SNF complex in vitro (Levine et al. balanced over CxD were crossed with the original dMRG15 2004). A core PRC1 complex consisting of Polycomb precise excision/TM3, Sb stock, and viable nonbalancer flies (Pc), Posterior sex combs (Psc), Polyhomeotic (Ph), selected. Stocks were established from their Sb siblings, and one of the strains, dMRG15ex1, was used in the genetic inter- and dRING1/Sex combs extra (Sce) is sufficient for the evine action experiments. in vitro activities of PRC1 (L et al. 2004). Recently, it Genetic interactions with PcG genes were scored by crossing was shown that dRing1/Sce as well as its mammalian PcG mutant stocks to wild-type, rept ex1, reptl(3)06945/TM3, Sb, orthologs are E3 ubiquitin ligases that monoubiquity- dMRG15P/TM3, Sb,ordMRG15ex1 flies at 25°. Male progeny late histone H2A (de Napoles et al. 2004; Wang et al. were examined for the presence of sex combs on the second 2004). and third pair of legs. During the course of these experiments, the penetrance of the extra sex combs phenotype in PcG Here, we investigate the role of Drosophila Reptin in alleles increased, perhaps because propionic acid was added to chromatin regulation. We show that it interacts genet- the fly food during later stages of this work. However, ically with PcG gene products and suppresses position- enhancement of the phenotype by rept or dMRG15 alleles effect variegation (PEV), properties shared by other was always compared to control crosses performed in parallel. Drosophila TIP60 complex components. We suggest Rescue of the genetic interaction was obtained with a reptin cDNA cloned into the pUASp vector (Rorth 1998). The that the fly TIP60 complex regulates epigenetic pro- expressed sequence tag (EST) clone LD12420 was PCR cesses leading to a repressive chromatin state. This is a amplified with Pfu polymerase and ligated into the XbaI and novel activity of a HATcomplex that has previously been blunted KpnI sites of the pUASp vector. The construct was implicated in transcription activation and DNA repair. 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; l(3)06945 MATERIALS AND METHODS UASp-reptin; rept /TM3, Sb stock, which was crossed to a w; actin5C-Gal4/1; Pc11/TM3, Sb stock. w; UASp-reptin/ Drosophila stocks and genetics: The following alleles used actin5C-Gal4; Pc11/reptl(3)06945 males could be distinguished in this study were obtained from the Bloomington or Kyoto from w; UASp-reptin/1; Pc11/reptl(3)06945 males by their stronger stock centers, from Jan Larsson, or from A˚ sa Rasmusson- eye color. As a control, we crossed w; UASp-reptin; reptl(3)06945/ Lestander: Pc11, Df(3L)Pc, Psc1, Pscvg-D, Psck07834, ph-p410, Pcl7, Pcl11, TM3, Sb flies to a Pc11/TM3, Sb Ser stock. w; UASp-reptin/1; esc1, esc21, Su(z)124, reptl(3)06945, dMRG15j6A3, Su(var)3-91, In(1)wmottled4h Pc11/reptl(3)06945 males were compared to their w; UASp-reptin/ (wm4h), and T(1;4)wm258-21. reptl(3)06945 contains a P[ry1] inser- 1; Pc11/TM3, Sb brothers. tion 17 nucleotides upstream from the ATG within the 59- Pc and rept mutant stocks were crossed to transgenic flies UTR, whereas in the dMRG15j6A3 allele, a P[lacW] insertion is containing Polycomb response elements (PRE) linked to the present in the dMRG15 second exon, 174 nucleotides down- mini-white reporter gene and the eye color observed in stream from the ATG. Oregon-R was used as the wild-type progeny. Different insertions of 8.2 XbaI derived from the strain. Pc and rept stocks were balanced over TM3 Sb Ubx-lacZ, Scr gene (Gindhart and Kaufman 1995), two constructs from over TM6B Tb, or over TM3 Sb to enable identification of the Mcp element (44.16.1 and 43.36.B11, construct nos. 19 Drosophila Reptin in Chromatin Regulation 243 and 12 described in Muller et al. 1999), and 5F24 from the primary antibodies. Discs were washed in block solution and Fab7 element of the bithorax complex (Cavalli and Paro incubated with Cy3-conjugated anti-rabbit (1:500), and Cy2- 1999) were tested. conjugated anti-mouse (1:200) secondary antibodies (Jackson Effects on position-effect variegation was determined by Laboratories) at room temperature for 2 hr. After being crossing wm4h females or females carrying P elements with washed in PBT solution, wing discs were rinsed in PBS and in variegating mini-white expression (kindly provided by Lori PBS/50% glycerol and then mounted in PBS/80% glycerol. A Wallrath and Steve Henikoff) to wild-type, Su(var)3-91/TM3 Sb laser-scanning microscope (Zeiss) was used for confocal imag- Ser, rept ex1,orreptl(3)06945/TM3 Sb males at room temperature. ing. Acquired images were processed with LSM510 software The eye color of male progeny was examined, and representa- (Zeiss). tive eyes for each genotype were photographed. Modification Reptin mRNA expression in wing discs was determined by of Notch variegation was examined by crossing T(1;4)wm258-21 y1 fluorescent in situ hybridization using tyramide signal ampli- wa/FM4 females to wild-type, Su(var)3-91/TM3 Sb Ser, rept ex1, fication (TSA; Perkin-Elmer, Norwalk, CT). Larvae at the late reptl(3)06945/TM3 Sb, dMRG15ex1,ordMRG15P/TM3, Sb males. third instar stage containing reptin mutant clones were The number of notches per wing was scored in female progeny dissected, fixed in 1 ml 4% paraformaldehyde (PFA) in PBS without the FM4 balancer chromosome. at room temperature for 20 min. Samples were then washed In situ hybridization and immunohistochemistry: Embryos with PBS, 50% methanol, and methanol and stored at À20° were collected from the reptl(3)06945 stock balanced over TM3, overnight. After five washes in ethanol, samples were in- Ubx-lacZ, and from Oregon-R flies (wild-type controls) on cubated in a mixture of xylene and ethanol (1:1) for 60 min, apple juice plates and aged appropriately. RNA in situ washed five times in ethanol, and rehydrated by immersion in hybridization using a digoxigenin-labeled antisense rept RNA a graded methanol series (80, 50, and 25% v/v in H2O) and autz probe was performed as previously described (T and finally in H2O. After treatment with acetone (80%) at À20° for Pfeifle 1989; Jiang et al. 1991). 10 min, samples were washed four times with PBT, fixed again Immunohistochemistry was performed essentially as de- in 4% PFA in PBS for 20 min, and washed in PBT. Digoxigenin- scribed previously. In brief, reptl(3)06945/TM6 Tb, Pc11/TM3 Sb Ser, labeled reptin and fluorescein-labeled GFP RNA probes were and Psc1/CyO mutant stocks were used to generate rept, Pc, and simultaneously added to the samples and hybridized at 55° Psc heterozygous as well as rept/Pc and Psc/1; rept/1 mutant overnight. After hybridization, samples were washed in PBT third instar larvae. The larvae were dissected in phosphate- and incubated with peroxidase-conjugated antifluorescein buffered saline (PBS), transferred to ice-cold 4% paraformal- antibody (Roche, 1:10,000) for 1.5 hr. FITC-labeled dinitro- dehyde in PBS, and fixed for 20 min at room temperature, phenyl (DNP) amplification reagent was used to develop the followed by washes in PBS and PBT (PBS 1 0.1% Tween 20). signal. To wash away the antibody, samples were incubated They were blocked in PBSBT (PBS 1 0.1% Triton X-100 1 in 0.01 m HCl twice for 5 min each. Following washes in PBT, 0.5% BSA) and incubated with anti-Scr monoclonal antibody peroxidase-coupled antidigoxigenin antibody (Roche, 1:5000) 6H4.1 (1:10 dilution, Develpmental Studies Hybridoma Bank) was added to the samples. Cy3-labeled DNP amplification re- at 4° overnight. After washing with PBSBT, they were de- agent was used to detect the antibody. Discs were dissected, veloped with the Vectastain ABC Elite kit. After being washed mounted in 80% glycerol, and imaged with a confocal micro- in PBT solution, embryos were rinsed in PBS/50% glycerol, scope (Zeiss). followed by mounting and dissection of leg imaginal discs in Immunoprecipitation: A Reptin expression plasmid was 80% glycerol. constructed through amplification of the Reptin open reading Induction of clones and immunofluorescence: Since reptin frame by PCR from ESTclone LD12420. The PCR product was mutant clones are very small (data not shown), we used a cloned into the EcoRI–XbaI sites of the pAc5.1/V5-His vector to Minute background. reptl(3)06945 FRT2A/TM6b Tb flies were generate a V5-tagged Reptin protein under control of the actin i55 2A crossed to yw hsFLP; M(3) hs-nGFP FRT /TM6b Tb and F1 promoter. Similarly, PCR was performed to obtain the dTIP60 larvae were heat-shocked for 1 hr at 37° as first and second open reading frame from EST clone LD31064, and the instar larvae. Prior to dissection of non-Tb larvae, they were product was ligated into the EcoRI-digested pRmHa-C-FLAG- subjected to one more 1-hr heat shock followed by a 1-hr His vector. This results in a plasmid expressing the FLAG- recovery to induce expression of the GFP protein. For de- tagged dTIP60 protein from the metallothionein promoter. tection of GFP mRNA, wing imaginal discs of wandering third A stable cell line expressing V5-tagged Reptin was generated instar larvae were dissected immediately after a 30-min heat as follows. Drosophila Schneider S2 cells were transfected by shock. As a control, we used Su(z)124 FRT2A/TM3 Sb flies means of calcium phosphate (DES transfection kit, Invitrogen, crossed to hsFLP; ubi-GFP FRT2A to generate hsFLP; Su(z)124 San Diego) using a DNA ratio of 19:1 (9.5 mg pAc-Reptin- FRT2A/ubi-GFP FRT2A larvae that were heat-shocked for 1 hr at V5:0.5 mg pCoBlast) and grown in the presence of 50 mg/ml 37° as first and second instar larvae. blasticidin. After 2 weeks, resistant clones were replated. The A Ubx 1.6-kb PRE was recombined onto the reptl(3)06945 FRT2A cell line was maintained in medium (Schneider’s Drosophila chromosome. A yw; .PBX.-PRE1.6-IDE-Ubx-nlacZ [y1] strain medium with 10% fetal calf serum) containing 10 mg/ml (Fritsch et al. 1999) was crossed with reptl(3)06945 FRT2A flies, and blasticidin. y1 w1 recombinants were selected. These were crossed to a FLAG-tagged dTIP60 was transfected into the cells stably reptl(3)06945/TM6b Tb stock to test for the presence of the reptin expressing Reptin-V5 in six-well plates using calcium phos- l(3)06945 2A mutation. The resulting PRE1.6-lacZ rept FRT /TM6b Tb phate. For each well, 2 mg plasmid DNA and 3 ml culture stock was crossed to yw hsFLP; M(3)i55 hs-nGFP FRT2A/TM6b Tb containing 3 3 106 cells were used. Ten microliters of 100 mm flies and larval progeny were heat-shocked as described above. copper sulfate stock was added on day 3 to induce dTIP60- Wing imaginal discs of wandering third instar larvae were FLAG expression. Cells were harvested on day 4 and lysed in a dissected, fixed in 4% paraformaldehyde, blocked in block 500-ml lysis buffer (10 mm Tris pH 8.0, 140 mm NaCl, 1.5 mm solution (1% BSA in PBT), and incubated with a rabbit b-gal MgCl2, 1% NP40, and protease inhibitor cocktail). The pro- antibody (Cappel) diluted 1:150 or with a monoclonal anti- tein concentration was determined with Bradford reagent. A Ubx antibody (White and Wilcox 1984) diluted 1:75 at 4° fraction of the extract was saved as input. The remaining overnight. To identify homozygous mutant clones, a rabbit extract was precleared by incubating with 30 ml protein A anti-GFP (1:1000, Promega, Madison, WI) or a mouse anti- sepharose for 1 hr at 4°. Precleared extract was incubated with GFP (1:500, Sigma, St. Louis) antibody was mixed with the 2.5 mg rabbit polyclonal anti-FLAG antibody (Sigma F7425) 244 D. Qi et al. 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 ml 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, Buckingham- shire, 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.

RESULTS Drosophila reptin interacts with PcG mutants: The Drosophila reptin allele l(3)06945 contains a lethal P-element transposon insertion in the 59 untranslated region of the gene. This insertion causes a dramatic reduction in reptin mRNA levels, as determined by in situ hybridization to l(3)06945 homozygous mutant embryos (Figure 1). We tested whether reptin mutants genetically interact with mutants of PcG genes by creating flies Figure 1.—Reptin mRNA expression in wild-type and trans-heterozygous for l(3)06945 and PcG genes and mutant embryos. Embryos were hybridized with a digoxigenin- examined male progeny for the presence of ectopic sex labeled antisense reptin RNA probe and are shown with ante- rior to the left and dorsal up. (A) A precellular wild-type (wt) combs on the second and third pair of legs (Table 1). We embryo shows ubiquitous reptin mRNA expression due to its compared the trans-heterozygous males with their maternal contribution. (B) At stage 13, reptin is expressed brothers that do not contain mutations in reptin and to in most tissues in a wild-type embryo. (C) In a stage 13 reptin PcG mutants crossed to wild-type males. Under our homozygous mutant embryo, no reptin mRNA can be de- original culture conditions, heterozygotes of the Pc11 tected. Mutant embryos were identified with the help of a balancer chromosome expressing a Ubx-lacZ reporter gene. allele do not contain extra sex combs, nor do reptin heterozygous flies (Table 1). However, 47% of males trans-heterozygous for Pc and reptin contain sex combs When crossed to the reptin mutant stock, we found sex on the second pair of legs (T2), and 5% additionally combs on both T2 and T3 in all of the Ph410; reptin/1 contain sex combs on the third pair of legs (T3). mutant males, whereas their Ph410 mutant brothers Another Pc allele, Df(3L)Pc, causes sex comb develop- receiving the TM3 balancer chromosome contain extra ment on T2 in 37% of the flies and on T3 in 6% of the sex combs on T2 in 100% and on T3 in 22% of the cases flies. The phenotype can be enhanced by reptin to 41% (Table 1), suggesting a specific ph–reptin interaction. In T2 and 25% T3. This genetic interaction indicates that summary, some alleles of three different PcG genes in Reptin and Pc participate in the same pathway in vivo. the PRC1 complex genetically interact with reptin. To investigate whether the genetic interaction with Two members of the E(z)–Esc complex, esc and Pcl, PcG genes is restricted to members of the PRC1 com- were also tested (Table 1). The esc alleles esc1 and esc21 plex, we examined trans-heterozygous combinations of showed either no or a very weak interaction with reptin reptin with two additional PRC1 complex components, mutants. However, the number of flies with extra sex Psc and ph, and with two components of the E(z)–Esc combs in one of the two Pcl alleles, Pcl11, was increased by complex, namely esc and Polycomb like (Pcl). As shown the reptin mutation (Table 1). This shows that the ability in Table 1, 40% of Psc1/1; reptin/1 trans-heterozygotes of reptin to genetically interact with PcG genes is not contain ectopic sex combs on T2 and 2% on T3, whereas restricted to components of the PRC1 complex. 18% of the Psc1/1; TM3/1 brothers have sex combs on To confirm that the interactions observed are caused T2 and none on T3. Psc1/1 heterozygotes do not con- by the P-element insertion in reptin, and not due to tain any extra sex combs. A deficiency that removes Psc, unidentified second-site mutations on the l(3)06945 Df(2R)vg-D, only weakly interacts with reptin, and a P- chromosome, we generated precise excisions of the element-induced Psc allele, Psck07834, does not interact P element. Such excision lines are viable and do not in- with reptin at all (Table 1). In the ph allele ph-p410, 90% of teract with PcG genes (Table 1). Furthermore, expres- the males contain sex combs on T2, but none on T3. sion of the reptin cDNA using an actin-Gal4 driver Drosophila Reptin in Chromatin Regulation 245

TABLE 1 was enhanced by the reptin mutation (Table 2). In- Genetic interactions between reptin and PcG mutants troduction of actin-Gal4 and UAS-reptin transgenes into the Pc11/reptinl(3)06945 trans-heterozygous flies reduced the % phenotype with sex combs number of sex combs to below the number observed with Pc11 over the balancer chromosome (Table 2). Genotype T2 T3 n From these data, we conclude that genetic interactions reptl(3)06945/1 0046with PcG genes are specifically due to reduced reptin Pc11/1 0070expression in l(3)06945 mutant flies. 11 l(3)06945 Pc /rept 47 5 67 Sex comb development is under the control of the Df(3L)Pc/1 37 6 51 Hox gene Sex combs reduced (Scr). To determine whether Df(3L)Pc/reptl(3)06945 41 25 32 Psc1/1 0063Reptin is involved in regulation of Scr expression, we Psc1/1; TM3/1 18 0 39 stained leg imaginal discs of third instar larvae with anti- Psc1/1; reptl(3)06945/1 40 2 53 Scr antibody 6H4.1. Scr protein is found in the first Pscvg-D/1 0052thoracic (T1), but not in the T2 and T3 leg discs in wild- Pscvg-D/1; TM3/1 0029type larvae. As shown in Figure 2, reptin/Pc11 and Psc1/1; vg-D l(3)06945 Psc /1; rept /1 6251reptin/1 larvae express Scr protein ectopically in T2 and Psck07834/1 3038 T3 discs, which reptin, Psc,orPc heterozygous larvae do Psck07834/1; TM3/1 0030 Psck07834/1; reptl(3)06945/1 0060not. In conclusion, our genetic data show that Reptin in- ph-p410/Y 90 0 34 teracts with PcG gene products to control Scr expression. ph-p410/Y; TM3/1 100 22 37 A PRE is derepressed in reptin mutant clones: PcG ph-p410/Y; reptl(3)06945/1 100 100 68 proteins regulate target gene expression through PREs. esc1/1 0062When linked to the mini-white reporter gene, many PREs 1 esc /1; TM3/1 0062show variegated white expression in the eye that is esc1/1; reptl(3)06945/1 0083 sensitive to PcG gene dosage. We examined several esc21/1 0047 esc21/1; TM3/1 0049PREs in a reptin heterozygous background and used a esc21/1; reptl(3)06945/1 2050PRE from the second intron of the Scr gene (8.2 XbaI), Pcl7/1 0058as well as the Fab7 and Mcp PREs from the bithorax Pcl7/1; TM3/1 0034complex. Although Pc heterozygotes interacted with all Pcl7/1; reptl(3)06945/1 2045PREs, we did not observe interactions between reptin 11 Pcl /1 17 0 81 heterozygotes and any of the PREs tested (data not Pcl11/1; TM3/1 30 7 27 shown). We generated reptin homozygous mutant cells Pcl11/1; reptl(3)06945/1 47 4 57 Pc11/reptex1 0033by mitotic recombination and examined expression of a Psc1/1; reptex1/1 0045lacZ reporter gene in wing imaginal discs. This lacZ transgene contains the IDE enhancer and a 1.6-kb PRE Percentage of flies containing ectopic sex combs on the from the Ultrabithorax (Ubx) gene (Fritsch et al. 1999). second (T2) and third (T3) pair of legs. n, number of flies counted. The Ubx gene is repressed in wing imaginal discs by PcG genes, but expressed in haltere discs to prevent acqui- transgene could rescue the reptin–Pc interaction (Table sition of wing fate. As shown in Figure 3A, reptin mutant 2). Under these culture conditions, Pc11 flies contained clones in wing discs lack reptin mRNA and express the extra sex combs even in the absence of the reptin PRE-lacZ reporter (Figure 3B). This indicates that mutation. However, the number of sex combs per fly Reptin is necessary for PcG function at this PRE.

TABLE 2 Rescue of the genetic interaction between reptin and Polycomb mutants

Phenotype % with sex combs Extra sex combs per ﬂy Genotype T2 T3 T2 T3 n UAS-rept; reptl(3)06945/TM3, Sb 3 act-Gal4/1; Pc11/TM3, Sb UAS-rept/1; Pc11/reptl(3)06945 93 73 4.0 1.9 15 UAS-rept/act-Gal4; Pc11/reptl(3)06945 69 25 1.3 0.3 16

UAS-rept; reptl(3)06945/TM3, Sb 3 Pc11/TM3, Sb Ser UAS-rept/1; Pc11/TM3, Sb 89 89 2.2 1.8 9 UAS-rept/1; Pc11/reptl(3)06945 100 90 4.9 2.0 10 246 D. Qi et al.

Figure 2.—Reptin genetically interacts with Polycomb and Posterior sex combs to induce ectopic expression of Scr in leg imaginal discs. Third instar larvae leg imaginal discs were stained with an antibody recognizing the homeotic protein Scr. In wild-type larvae, Scr is expressed in the ﬁrst thoracic discs (T1), but not in the second or third thoracic leg discs (T2 and T3). (A– C, G–I, M–O) Pc11, Psc1, and reptl(3)06945 heterozygous larvae have a wild-type Scr expression pattern, being present in T1 but absent from T2 and T3 leg discs. (D–F, J–L) Pc11/reptl(3)06945 and Psc1/1; reptl(3)06945/1 trans-heterozygous larvae ectopically express Scr in T2 and T3 leg discs (arrows in E, F, K, and L) in addition to the normal expression in T1 discs.

We also examined expression of the endogenous Ubx matin, resulting in a variegated eye color (Reuter and gene in reptin mutant clones. PcG genes are necessary Wolff 1981). We crossed reptin mutants to wm4h flies and for silencing of the Ubx gene in wing discs, resulting in compared the progeny to a cross of wm4h with Su(var)3-9 ectopic Ubx expression in PcG mutant clones (Beuchle flies. Methylation of histone H3 lysine 9 by the Su(var)3-9 et al. 2001). We stained wing imaginal discs containing protein is necessary for heterochromatin formation homozygous mutant clones of either reptin or the PcG (Rea et al. 2000; Schotta et al. 2002), and consequently, gene Su(z)12 with a Ubx antibody. Figure 3 shows that in Su(var)3-9 mutants strongly suppress PEV (Figure 4E; wing discs containing Su(z)12 mutant cells, Ubx is compare with Figure 4, A and F). We found that reptin ectopically expressed, but in wing discs with reptin mutants also suppress variegation of wm4h (Figure 4B; mutant cells, it is not. As expected, Ubx was found in compare with Figure 4, C and D). By contrast, most PcG all haltere discs (data not shown). It appears that al- genes do not affect PEV (Sinclair et al. 1998). though Reptin is necessary for the activity of the 1.6-kb We investigated whether reptin mutants also suppress Ubx PRE, loss of Reptin is not sufficient to derepress en- other instances of PEV. We examined P-element inser- dogenous Ubx expression. tions in telomeric and centric heterochromatin that We also examined expression of the homeotic genes show variegated mini-white expression (Wallrath and Scr and Ubx in reptin homozygous mutant embryos. Elgin 1995; Cryderman et al. 1998, 1999). As shown in However, no misexpression was observed (data not Figure 4, G–I, and Table 3, reptin mutants suppress PEV shown). Reptin is maternally contributed to the embryo, of most insertions that respond to classic modifiers of and its mRNA is consequently present ubiquitously in PEV, such as Su(var)3-9. Reptin mutant flies also sup- early embryos (Figure 1A). The maternal contribution press variegation caused by a mini-white transgene array may mask a regulatory role for reptin in embryonic (DX1, Figure 4, J–L), and variegation of the notched Hox gene expression. To address this possibility, we at- wing phenotype caused by a translocation that posi- tempted to remove the maternal reptin contribution by tions Notch near pericentric heterochromatin [Table 3, use of germline clones. However, reptin l(3)06945 ho- T(1;4)wm258-21]. We conclude that Reptin contributes mozygous germ cells fail to produce embryos. to generation of silent chromatin at many loci in Reptin is a suppressor of position-effect variegation: Drosophila. We then tested whether reptin mutants affect PEV, a Reptin shares with other TIP60 complex compo- phenomenon resulting in clonal silencing of genes nents interactions with PcG genes and effects on PEV: juxtaposed to heterochromatin (Schotta et al. 2003). Drosophila Reptin has recently been found in the TIP60 In In(1)wm4h flies, an inversion on the X chromosome HATcomplex (Kusch et al. 2004). In addition to Reptin, positions the white gene close to pericentric heterochro- this complex contains two other subunits that have been Drosophila Reptin in Chromatin Regulation 247

Figure 3.—PRE-controlled gene expression is derepressed in reptin mutant cells, whereas endogenous homeotic gene expression is not. Wing imaginal discs with clones of cells that Figure 4.—Reptin mutants suppress PEV. (A–F) In(1)wm4h are homozygous mutant for reptin (A–C) or the PcG gene (wm4h) flies contain an inversion on the X chromosome that Su(z)12 (D) were stained for reptin mRNA (A) or with antibod- positions the white gene close to pericentric heterochromatin, b ies against GFP (green, B–D) and -galactosidase (red, B)or resulting in a variegated eye color. A known suppressor of var- Ubx (red, C and D). Clones are marked by the absence of GFP iegation, Su(var)3-9, was compared to reptin (rept) mutants. expression. The reptin clones were generated in a Minute back- (A) wm4h females were crossed to wild-type males and the ground. (A) reptin mRNA is absent from reptin mutant clones eye color of a representative male progeny was photographed. (GFP mRNA staining is not shown). (B) A lacZ reporter gene (B and C) Eye color of male progeny from a cross of wm4h fe- driven by a Ubx enhancer (IDE) and containing a 1.6-kb Ubx males with rept/TM3 males. The reptin mutation (B) sup- PRE is expressed in haltere discs but repressed in wing discs ritsch presses PEV as compared to the same chromosome with a (F et al. 1999). Strong misexpression of the reporter precise excision of the P element (D), as well as compared gene is observed in reptin clones, but not in control wing discs to the TM3 balancer chromosome (C). (D) Males containing without clones (not shown). (C and D) Misexpression of Ubx a precise excision of the P element in reptin (reptex1) were is seen in Su(z)12 mutant clones, but not in reptin mutant crossed to wm4h females. The eye color of a male progeny is clones. As expected, Ubx expression was found in all haltere shown. (E and F) A Su(var)3-9/TM3 stock was crossed to discs (not shown), demonstrating that the staining procedure wm4h females and males receiving the balancer chromosome was successful. The arrowheads in A and C point to some of (F) were compared with their Su(var)3-9 mutant brothers the clones. (E). (G–I) A mini-white transgene (118E-10) inserted into centric heterochromatin results in a strongly variegating eye color genetically characterized, namely Enhancer of Poly- when crossed with wild-type males (G). Variegation is strongly suppressed by the reptin mutation (H), but not by the TM3 comb [E(Pc)] and Domino. Interestingly, like reptin balancer chromosome (I). (J–L) A mini-white transgene array mutants, E(Pc) and domino mutants enhance PcG phe- consisting of six copies (DX1) produces a variegating eye notypes and suppress PEV (Sinclair et al. 1998; Ruhf color when crossed to wild-type flies (J). When crossed to a et al. 2001). We tested whether an additional TIP60 rept/TM3 stock, reptin mutant flies suppress variegation (K) component, the chromodomain containing protein compared to their brothers receiving the TM3 balancer chromosome (L). MRG15 (Bertram and Pereira-Smith 2001), displays similar phenotypes. We used a strain containing a P- element insertion in the second exon of Drosophila MRG15, dMRG15j6A3, and removed a second-site lethal We confirmed that Reptin can physically interact with mutation on the chromosome by recombination (see TIP60 complex components. For this purpose, we ex- materials and methods). We designated the cleaned pressed tagged proteins in Drosophila S2 tissue-culture chromosome dMRG15P and crossed it to T(1;4)wm258-21 cells. As shown in Figure 5, a fraction of V5-tagged and to PcG mutant flies. Interestingly, dMRG15 mutants Reptin is co-immunoprecipitated with FLAG-tagged suppress Notch variegation (Table 3) and interact with dTIP60. These results are consistent with a recent re- PcG genes (Table 4), whereas a precise excision of the port (Kusch et al. 2004) and show that Reptin can be P element does not. found in association with the Drosophila TIP60 protein. 248 D. Qi et al.

TABLE 3 Reptin and Drosophila MRG15 modify position-effect variagation

Responsive to Suppressed by Transgene Insertion classic modiﬁersa reptin mutants 39C-4 Centric Yes Yes 118E-10 Centric Yes Yes 118E-12 Centric Yes Yes 39C-72 Telomeric Yes Yes 39C-12 Chromosome 4 Yes No 39C-5 Telomeric No No 39C-27 Telomeric No No

Genotype % with wild-type wings Wing notches per ﬂy n T(1;4)wm258-21/1; 1 0 2.9 22 T(1;4)wm258-21/1; Su(var)3-91/1 55 0.8 20 T(1;4)wm258-21/1; reptl(3)06945/1 52 0.9 29 T(1;4)wm258-21/w; 1 0 4.6 12 T(1;4)wm258-21/w; dMRG15P/1 67 0.6 18 T(1;4)wm258-21/w; dMRG15ex1/1 0 4.5 16 a Cryderman et al. (1998, 1999); Wallrath and Elgin (1995).

DISCUSSION Although we did not detect interactions between reptin heterozygous mutants and several PREs tested, a PRE The Reptin protein is present in several chromatin- from the Ubx gene is derepressed in reptin homozygous remodeling complexes. We have shown that, in vivo, mutant cells (Figure 3B). This shows that Reptin con- Drosophila Reptin is involved in formation of silent tributes an essential function to the activity of this PRE. chromatin. We propose that Reptin acts as a subunit of However, unlike most PcG genes, reptin homozygous mu- the TIP60 HAT complex to generate a repressive chro- tants do not derepress endogenous Hox gene expres- matin state. This is a novel activity of a HAT complex sion (Figure 3 and data not shown). It appears that previously shown to promote transcription (Doyon and repression of endogenous Hox genes is more complex Cote 2004). and not as sensitive to the loss of Reptin as the Ubx PRE. Drosophila Reptin copurifes with the Polycomb In contrast to most PcG genes, reptin mutants suppress complex PRC1 (Saurin et al. 2001). This prompted us PEV (Figure 4 and Table 3). Interestingly, derepression to investigate whether the biochemical interaction with of the Ubx PRE also occurs in embryos mutant for other PRC1 was accompanied by a genetic interaction. Table 1 suppressors of PEV (Chan et al. 1994), indicating that and Figure 2 show that Reptin and PRC1 components this PRE may be highly sensitive to the chromatin en- genetically interact to regulate expression of the Hox vironment in its vicinity. Since reptin mutants suppress gene Scr. However, Reptin also interacts with a PcG gene product not associated with the PRC1 complex, Pcl.

TABLE 4 Genetic interaction of Drosophila MRG15 with PcG genes

% phenotype with sex combs Genotype T2 T3 n dMRG15P/1 0061 Pc11/TM3 57 32 37 Pc11/dMRG15P 85 54 39 Figure 5.—Reptin interacts with dTIP60. A Drosophila S2 Pc11/dMRG15ex1 54 33 39 cell line stably expressing V5-tagged Reptin protein was trans- Psc1/1; TM3/1 5038fected with a plasmid expressing FLAG-tagged dTIP60 under Psc1/1; dMRG15P/1 38 11 37 control of the metallothionein promoter. Untransfected cells Psc1/1; dMRG15ex1/1 8049and transfected cells treated with CuSO4 to induce dTIP60- esc1/1; TM3/1 0044FLAG expression were immunoprecipitated with an anti-FLAG esc1/1; dMRG15P/1 0041antibody. The presence of Reptin-V5 in the cell extract and in the immunoprecipitate was detected in a Western blot with esc1/1; dMRG15ex1/1 0055 anti-V5 antibody. Drosophila Reptin in Chromatin Regulation 249

PEV and fail to derepress endogenous Hox gene ex- et al. 2003; Doyon et al. 2004), where it directs the pression, we do not consider reptin a bona fide PcG histone deacetylase to coding regions through interac- gene, and we find it unlikely that Reptin protein con- tion of its chromodomain with methylated histone H3 tributes an essential function to the PRC1 complex. In lysine 36 (Carrozza et al. 2005; Joshi and Struhl 2005; fact, the biochemical activities ascribed to PRC1 can be Keogh et al. 2005). We found that MRG15 mutant flies reconstituted either with recombinant dRing1/Sce interact with PcG genes and suppress PEV, just as other (Wang et al. 2004) or with four core components whose TIP60 complex components do. We take this as further activity can be further enhanced by the DNA-binding support of our conclusion that Reptin’s effects on proteins zeste and GAGA (Mulholland et al. 2003). chromatin processes are mediated through its associa- Given that Reptin is present in TIP60 complexes in tion with the fly TIP60 complex. mammals and recently was shown to be a component of a What is the basis for the genetic interaction between Drosophila TIP60 complex (Ikura et al. 2000; Fuchs et al. TIP60 components and PcG genes? One possibility is that 2001; Cai et al. 2003; Doyon et al. 2004; Kusch et al. 2004), the TIP60 complex regulates PcG expression. However, we considered the possibility that the genetic interactions we did not observe reduced Pc expression in reptin mutant observed with PcG genes are due to the presence of embryos (data not shown). Another possibility is that the Reptin in the fly TIP60 complex. The products of two enzymatic activities of the TIP60 complex cooperate with previously characterized Drosophila genes, E(Pc) and PcG genes to mediate transcriptional silencing. Since domino, are also present in the TIP60 complex. Strikingly, binding of Pc to polytene chromosomes is abolished in E(Pc) and domino mutants share with reptin the ability to H2Av mutant animals (Swaminathan et al. 2005), TIP60 genetically interact with PcG genes and suppress PEV. complex-mediated histone variant exchange might cause E(Pc) is an unusual PcG gene that has very minor effects the genetic interaction with PRC1. However, we found on Hox gene expression, and unlike most PcG genes, that binding of PcG proteins to polytene chromosomes is modifies PEV (Soto et al. 1995; Sinclair et al. 1998; unaffected in domino mutant larvae (data not shown). It Stankunas et al. 1998). In both yeast and humans, E(Pc) is possible that PRC1-mediated H2A ubiquitylation helps homologs form a core complex with Esa1 (TIP60) and to recruit the TIP60 complex, whose histone acetylation Yng2 (ING3) that is sufficient for the nucleosomal or histone exchange activity assists in transcriptional acetylation of histones H4 and H2A by the NuA4 complex repression. Alternatively, histone acetylation or exchange (Boudreault et al. 2003; Doyon et al. 2004). That such facilitates binding of the PRC1 complex to PREs. A similar an integral NuA4/TIP60 complex component displays mechanism has been invoked for the cooperation of the phenotypes similar to reptin mutants suggests to us that Esc–E(z) complex and PRC1, where Esc–E(z) trimethy- Reptin functions through the fly TIP60 complex. lates histone H3 lysine 27, which is recognized by the Domino protein is similar to p400 and to SRCAP in chromodomain of Polycomb (Fischle et al. 2003; Min mammals and to Swr1 in yeast (Eissenberg et al. 2005). et al. 2003). Swr1 has recently been shown to exchange the variant We have shown that the Drosophila TIP60 complex histone H2A.Z (Htz1 in yeast) for H2A in nucleosomes plays a role in epigenetic gene silencing in vivo.Asimilar (Krogan et al. 2003; Kobor et al. 2004; Mizuguchi et al. case has been described for the yeast HATcomplex SAGA 2004). Intriguingly, an involvement of Htz1 (H2A.Z) in (Spt-Ada-Gen5-acetyltransferase) that is required for controlling the spreading of silenced chromatin has both activation and repression of the ARG1 gene (Ricci recently been demonstrated in yeast (Meneghini et al. et al. 2002). Two other yeast HATs, Sas2 and Sas3, also pro- 2003; Babiarz et al. 2006). Exchange of variant histones mote gene silencing (Reifsnyder et al. 1996). Interest- may be a conserved feature of chromatin regulation since ingly, the Drosophila HAT Chameau suppresses PEV and a recent report demonstrates that Drosophila H2Av cooperates with PcG genes as well (Grienenberger et al. behaves genetically as a PcG gene and suppresses PEV 2002). TIP60, Sas2, Sas3, and Chameau are HATs that (Swaminathan et al. 2005). Domino exchanges phos- belong to the MYST family (Utley and Cote 2003). phorylated and acetylated H2Av for unmodified H2Av Therefore, MYST family HATs in both yeast and flies can after DNA damage (Kusch et al. 2004). However, we found control epigenetic inheritance of silent chromatin. no change in binding of H2Av to polytene chromosomes prepared from domino mutant larvae (data not shown). We thank Jan Larsson for PcG mutant flies, PRE lines and advice, A˚ sa 4 A P-element insertion was identified in the gene Rasmusson-Lestander for the FRT Su(z)12 stock and comments on the manuscript, Tom Kaufman and Renato Paro for PRE lines, Lori encoding one additional TIP60 complex component, Wallrath and Steve Henikoff for PEV reporters, Ju¨rg Mu¨ller for PRE- the chromodomain-containing protein MRG15. Hu- lacZ and FRT Minute strains, and the Bloomington and Kyoto stock man MRG15 (MORF-related gene on chromosome centers for mutant Drosophila strains. Haojiang Luan constructed the 15) has been implicated in cellular senescence and UASp-reptin plasmid. The Scr antibody was developed by D. Brower regulation of the B-myb promoter (Leung et al. 2001). and obtained from the Developmental Studies Hybridoma Bank at the University of Iowa. We are grateful to the Christos Samakovlis lab for Both human and yeast (Eaf3/Alp13) MRG15 have been sharing reagents. This work has been supported by grants from the found in Sin3/HDAC complexes in addition to the A˚ ke Wiberg Foundation, the Swedish Cancer Society, and the Swedish TIP60 (NuA4) complex (Gavin et al. 2002; Nakayama Research Council to M.M. 250 D. Qi et al.

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