• Abstract and Figures
• Public Full-text

Copyright Ó 2006 by the Genetics Society of America DOI: 10.1534/genetics.106.059980 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 trans- posase 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 in- sertion 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.

Load more

  11

Copy Link