Effects of Mutation or Deletion

Drosophila reptin interacts with PcG mutants

The Drosophila reptin allele l(3)06945 contains a lethal P-element transposon insertion in the 5' 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. Whether reptin mutants genetically interact with mutants of PcG genes was tested by creating flies trans-heterozygous for l(3)06945 and PcG genes and male progeny were examined for the presence of ectopic sex combs on the second and third pair of legs. The trans-heterozygous males were compared with their brothers that do not contain mutations in reptin and to PcG mutants crossed to wild-type males. Under the original culture conditions, heterozygotes of the Pc11 allele do not contain extra sex combs, nor do reptin heterozygous flies. However, 47% of males trans-heterozygous for Pc and reptin contain sex combs on the second pair of legs (T2), and 5% additionally contain sex combs on the third pair of legs (T3). Another Pc allele, Df(3L)Pc, causes sex comb development on T2 in 37% of the flies and on T3 in 6% of the flies. The phenotype can be enhanced by reptin to 41% T2 and 25% T3. This genetic interaction indicates that Reptin and Pc participate in the same pathway in vivo (Qi, 2006).

To investigate whether the genetic interaction with PcG genes is restricted to members of the PRC1 complex, trans-heterozygous combinations were examined of reptin with two additional PRC1 complex components, Psc and ph, and with two components of the E(z)–Esc complex, namely esc and Polycomb like (Pcl). 40% of Psc1/+; reptin/+ trans-heterozygotes contain ectopic sex combs on T2 and 2% on T3, whereas 18% of the Psc1/+; TM3/+ brothers have sex combs on T2 and none on T3. Psc1/+ heterozygotes do not contain any extra sex combs. A deficiency that removes Psc, Df(2R)vg-D, only weakly interacts with reptin, and a P-element-induced Psc allele, Psck07834, does not interact with reptin at all. In the ph allele ph-p410, 90% of the males contain sex combs on T2, but none on T3. When crossed to the reptin mutant stock, sex combs were found on both T2 and T3 in all of the Ph410; reptin/+ mutant males, whereas their Ph410 mutant brothers receiving the TM3 balancer chromosome contain extra sex combs on T2 in 100% and on T3 in 22% of the cases, suggesting a specific phreptin interaction. In summary, some alleles of three different PcG genes in the PRC1 complex genetically interact with reptin (Qi, 2006).

Two members of the E(z)–Esc complex, esc and Pcl, were also tested. 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. This shows that the ability of reptin to genetically interact with PcG genes is not restricted to components of the PRC1 complex (Qi, 2006).

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 chromosome, precise excisions of the P element were generated. Such excision lines are viable and do not interact with PcG genes. Furthermore, expression of the reptin cDNA using an actin-Gal4 driver transgene could rescue the reptinPc interaction. 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. 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. From these data, it is concluded that genetic interactions with PcG genes are specifically due to reduced reptin expression in l(3)06945 mutant flies (Qi, 2006).

Sex comb development is under the control of the Hox gene Sex combs reduced (Scr). To determine whether Reptin is involved in regulation of Scr expression, leg imaginal discs of third instar larvae were stained with anti-Scr antibody 6H4.1. Scr protein is found in the first thoracic (T1), but not in the T2 and T3 leg discs in wild-type larvae. reptin/Pc11 and Psc1/+; reptin/+ larvae express Scr protein ectopically in T2 and T3 discs, but reptin, Psc, or Pc heterozygous larvae do not. In conclusion, genetic data show that Reptin interacts with PcG gene products to control Scr expression (Qi, 2006).

A PRE is derepressed in reptin mutant clones

PcG proteins regulate target gene expression through PREs. When linked to the mini-white reporter gene, many PREs show variegated white expression in the eye that is sensitive to PcG gene dosage. Several PREs were examined in a reptin heterozygous background and a PRE from the second intron of the Scr gene (8.2 XbaI) was used, as well as the Fab7 and Mcp PREs from the bithorax complex. Although Pc heterozygotes interacted with all PREs, no interactions were observed between reptin heterozygotes and any of the PREs tested. reptin homozygous mutant cells were generated by mitotic recombination and expression of a lacZ reporter gene was examined in wing imaginal discs. This lacZ transgene contains the IDE enhancer and a 1.6-kb PRE from the Ultrabithorax (Ubx) gene. The Ubx gene is repressed in wing imaginal discs by PcG genes, but expressed in haltere discs to prevent acquisition of wing fate. reptin mutant clones in wing discs lack reptin mRNA and express the PRE-lacZ reporter. This indicates that Reptin is necessary for PcG function at this PRE (Qi, 2006).

Expression of the endogenous Ubx gene was examined in reptin mutant clones. PcG genes are necessary for silencing of the Ubx gene in wing discs, resulting in ectopic Ubx expression in PcG mutant clones. Wing imaginal discs containing homozygous mutant clones of either reptin or the PcG gene Su(z)12 were stained with a Ubx antibody. In wing discs containing Su(z)12 mutant cells, Ubx is ectopically expressed, but in wing discs with reptin mutant cells, it is not. As expected, Ubx was found in all haltere discs. It appears that although Reptin is necessary for the activity of the 1.6-kb Ubx PRE, loss of Reptin is not sufficient to derepress endogenous Ubx expression (Qi, 2006).

Expression of the homeotic genes Scr and Ubx was examined in reptin homozygous mutant embryos. However, no misexpression was observed. Reptin is maternally contributed to the embryo, and its mRNA is consequently present ubiquitously in early embryos. The maternal contribution may mask a regulatory role for reptin in embryonic Hox gene expression. To address this possibility, attempts were made to remove the maternal reptin contribution by use of germline clones. However, reptin l(3)06945 homozygous germ cells fail to produce embryos. reptin mutants were also found to affect PEV, a phenomenon resulting in clonal silencing of genes juxtaposed to heterochromatin (Qi, 2006).

Reptin shares with other TIP60 complex components interactions with PcG genes and effects on PEV

Drosophila Reptin has recently been found in the TIP60 HAT complex (Kusch, 2004). In addition to Reptin, this complex contains two other subunits that have been genetically characterized, namely Enhancer of Polycomb [E(Pc)] and Domino. Interestingly, like reptin mutants, E(Pc) and domino mutants enhance PcG phenotypes and suppress PEV. Whether an additional TIP60 component, the chromodomain containing protein MRG15 displays similar phenotypes was tested. A strain containing a P-element insertion in the second exon of Drosophila MRG15, dMRG15j6A3, was tested, and a second-site lethal mutation on the chromosome was removed by recombination. The cleaned chromosome was designated dMRG15P and crossed it to T(1;4)wm258-21 and to PcG mutant flies. Interestingly, dMRG15 mutants suppress Notch variegation and interact with PcG genes, whereas a precise excision of the P element does not (Qi, 2006).

It was confirmed that Reptin can physically interact with TIP60 complex components. For this purpose, tagged proteins were expressed in Drosophila S2 tissue-culture cells. A fraction of V5-tagged Reptin is co-immunoprecipitated with FLAG-tagged dTIP60. These results are consistent with a recent report (Kusch, 2004) and show that Reptin can be found in association with the Drosophila TIP60 protein (Qi, 2006).


Reference names in red indicate recommended papers.

Search PubMed for articles about Drosophila Reptin

Andersen, E. C., Lu. X. and Horvitz, H. R. (2006). C. elegans ISWI and NURF301 antagonize an Rb-like pathway in the determination of multiple cell fates. Development 133(14): 2695-704. Medline abstract: 16774993

Bauer, A., et al. (2000). Pontin52 and reptin52 function as antagonistic regulators of beta-catenin signalling activity. EMBO J. 19(22): 6121-30. Medline abstract: 11080158

Bellosta, P., et al. (2005). Myc interacts genetically with Tip48/Reptin and Tip49/Pontin to control growth and proliferation during Drosophila development. Proc. Natl. Acad. Sci. 102(33): 11799-804. Medline abstract: 16087886

Bird, A. W., et al. (2002). Acetylation of histone H4 by Esa1 is required for DNA double-strand break repair. Nature 419(6905): 411-5. Medline abstract: 12353039

Boudreault, A. A., et al. (2003). Yeast Enhancer of Polycomb defines global Esa1-dependent acetylation of chromatin. Genes Dev. 17: 1415-1428. Medline abstract: 12782659

Cai, Y., et al. (2003). Identification of new subunits of the multiprotein mammalian TRRAP/TIP60-containing histone acetyltransferase complex. J. Biol. Chem. 278(44): 42733-6. Medline abstract: 12963728

Cai, Y., et al. (2005). The mammalian YL1 protein is a shared subunit of the TRRAP/TIP60 histone acetyltransferase and SRCAP complexes. J. Biol. Chem. 280(14): 13665-70. Medline abstract: 15647280

Chauvet, S., Usseglio, F., Aragnol, D. and Pradel, J. (2005). Analysis of paralogous pontin and reptin gene expression during mouse development. Dev. Genes Evol. 215(11): 575-9. Medline abstract: 16003523

Cho, S. G., et al. (2001). TIP49b, a regulator of activating transcription factor 2 response to stress and DNA damage. Mol. Cell. Biol. 21: 8398-8413. Medline abstract: 11713276

Doyon, Y. and Cote, J. (2004). The highly conserved and multifunctional NuA4 HAT complex. Curr. Opin Genet. Dev. 14(2): 147-54. Medline abstract: 15196461

Ducat, D., Kawaguchi, S., Liu, H., Yates, J. R. and Zheng, Y. (2008). Regulation of microtubule assembly and organization in mitosis by the AAA+ ATPase Pontin. Mol. Biol. Cell 19(7): 3097-110. PubMed Citation: 18463163

Eissenberg, J. C., Wong, M. and Chrivia, J. C. (2005). Human SRCAP and Drosophila melanogaster DOM are homologs that function in the notch signaling pathway. Mol. Cell. Biol. 25(15): 6559-69. Medline abstract: 16024792

Etard, C., et al. (2005). Pontin and Reptin regulate cell proliferation in early Xenopus embryos in collaboration with c-Myc and Miz-1. Mech. Dev. 122(4): 545-56. Medline abstract: 15804567

Frank, S. R., et al. (2003). MYC recruits the TIP60 histone acetyltransferase complex to chromatin. EMBO Rep. 4(6): 575-80. Medline abstract: 12776177

Fuchs, M., et al. (2001). The p400 complex is an essential E1A transformation target. Cell 106(3): 297-307. Medline abstract: 11509179

Gause, M., Eissenberg, J. C., Macrae, A. F., Dorsett, M., Misulovin, Z., Dorsett, D. (2006). Nipped-A, the Tra1/TRRAP subunit of the Drosophila SAGA and Tip60 complexes, has multiple roles in Notch signaling during wing development. Mol. Cell. Biol. 26(6): 2347-59. Medline abstract: 16508010

Holmes, A. M., Weedmark, K. A. and Gloor, G. B. (2006). Mutations in the extra sex combs and Enhancer of Polycomb genes increase homologous recombination in somatic cells of Drosophila melanogaster. Genetics 172(4): 2367-77. Medline abstract: 16452150

Ikura, T., et al (2000). Involvement of the TIP60 histone acetylase complex in DNA repair and apoptosis. Cell 102(4): 463-73. Medline abstract: 10966108

Jin, J., et al. (2005). A mammalian chromatin remodeling complex with similarities to the yeast INO80 complex. J. Biol. Chem. 280: 41207-41212. Medline abstract: 16230350

Jonsson, Z. O., et al. (2001). Rvb1p and Rvb2p are essential components of a chromatin remodeling complex that regulates transcription of over 5% of yeast genes. J. Biol. Chem. 276: 16279-16288. Medline abstract: 11278922

Jonsson, Z. O., et al. (2004). Rvb1p/Rvb2p recruit Arp5p and assemble a functional Ino80 chromatin remodeling complex. Mol. Cell 16: 465-477. Medline abstract: 15525518

Kanemaki, M., et al. (1999). TIP49b, a new RuvB-like DNA helicase, is included in a complex together with another RuvB-like DNA helicase, TIP49a. J. Biol. Chem. 274(32): 22437-44. Medline abstract: 10428817

Kim, J. H., et ak, (2005). Transcriptional regulation of a metastasis suppressor gene by Tip60 and beta-catenin complexes. Nature 434(7035): 921-6. Medline abstract: 15829968

Kim, J. H., et al. (2006). Roles of sumoylation of a reptin chromatin-remodelling complex in cancer metastasis. Nat. Cell Biol. 8: 631-639. Medline abstract: 16699503

King, T. H., et al. (2001). A well-connected and conserved nucleoplasmic helicase is required for production of box C/D and H/ACA snoRNAs and localization of snoRNP proteins. Mol. Cell. Biol. 21: 7731-7746. Medline abstract: 11604509

Kobor, M. S., et al. (2004). A protein complex containing the conserved Swi2/Snf2-related ATPase Swr1p deposits histone variant H2A.Z into euchromatin. PLoS Biol. 2(5): E131. Medline abstract: 15045029

Krogan, N. J., et al. (2003). A Snf2 family ATPase complex required for recruitment of the histone H2A variant Htz1. Mol. Cell 12(6): 1565-76. Medline abstract: 14690608

Kusch, T., et al. (2004). Acetylation by Tip60 is required for selective histone variant exchange at DNA lesions. Science 306(5704): 2084-7. Medline abstract: 15528408

Lim, C. R., et al. (2000). The Saccharomyces cerevisiae RuvB-like protein, Tih2p, is required for cell cycle progression and RNA polymerase II-directed transcription. J. Biol. Chem. 275: 22409-22417. Medline abstract: 10787406

Makino, Y., Kanemaki, M., Kurokawa, Y., Koji, T. and Tamura, T. (1999). A rat RuvB-like protein, TIP49a, is a germ cell-enriched novel DNA helicase. J. Biol. Chem. 274(22): 15329-35. Medline abstract: 10336418

Mizuguchi, G., Shen, X., Landry, J., Wu, W. H., Sen, S. and Wu, C. (2004). ATP-driven exchange of histone H2AZ variant catalyzed by SWR1 chromatin remodeling complex. Science 303(5656): 343-8. Medline abstract: 14645854

Ohdate, H., et al. (2003). Impairment of the DNA binding activity of the TATA-binding protein renders the transcriptional function of Rvb2p/Tih2p, the yeast RuvB-like protein, essential for cell growth. J. Biol. Chem. 278: 14647-14656. Medline abstract: 12576485

Olson, L. E., et al. (2006). Homeodomain-mediated beta-catenin-dependent switching events dictate cell-lineage determination. Cell 125(3): 593-605. Medline abstract: 16678101

Park, J., et al. (2002). BAF53 forms distinct nuclear complexes and functions as a critical c-Myc-interacting nuclear cofactor for oncogenic transformation. Mol. Cell Biol. 22: 1307-1316. Medline abstract: 11839798

Puri, T., et al. (2007). Dodecameric structure and ATPase activity of the human TIP48/TIP49 complex. J. Mol. Biol. 366: 179-192. Medline abstract: 17157868

Qi, D., Jin, H., Lilja, T. and Mannervik, M. (2006). Drosophila Reptin and other TIP60 complex components promote generation of silent chromatin. Genetics 174(1): 241-51. Medline abstract: 16816423

Rottbauer, W., et al. (2002). Reptin and pontin antagonistically regulate heart growth in zebrafish embryos. Cell 111(5): 661-72. Medline abstract: 12464178

Shen, X., et al. (2000). A chromatin remodelling complex involved in transcription and DNA processing. Nature 406: 541-544. Medline abstract: 10952318

Sigala, B., et al. (2005). Relocalization of human chromatin remodeling cofactor TIP48 in mitosis. Exp. Cell Res. 310: 357-369. Medline abstract: 16157330

Swaminathan J., Baxter, E. M. and Corces, V. G. (2005). The role of histone H2Av variant replacement and histone H4 acetylation in the establishment of Drosophila heterochromatin. Genes Dev. 19(1): 65-76. Medline abstract: 15630020

Utley, R. T. and Cote, J. (2003). The MYST family of histone acetyltransferases. Curr. Top. Microbiol. Immunol. 274: 203-36. Review. Medline abstract: 12596909

Weiske, J. and Huber, O. (2005). The histidine triad protein Hint1 interacts with Pontin and Reptin and inhibits TCF-beta-catenin-mediated transcription. J. Cell. Sci. 118(Pt 14): 3117-29. Medline abstract: 16014379

Wood, M. A., McMahon, S. B. and Cole, M. D. (2000). An ATPase/helicase complex is an essential cofactor for oncogenic transformation by c-Myc. Mol. Cell 5(2): 321-30. Medline abstract: 10882073

reptin: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation

date revised: 25 November 2008

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