Daxx-like Protein of Drosophila Interacts with Dmp53 and Affects Longevity and Ark mRNA Level
2007; Elsevier BV; Volume: 282; Issue: 50 Linguagem: Inglês
10.1074/jbc.m705547200
ISSN1083-351X
AutoresLászló Bodai, Norbert Pardi, Zsuzsanna Újfaludi, Orsolya Bereczki, Orbán Komonyi, Éva Bálint, Imre Boros,
Tópico(s)DNA Repair Mechanisms
ResumoDaxx-like protein (DLP), the Drosophila homolog of Daxx, binds Drosophila melanogaster p53 (Dmp53) through its C-terminal region. We generated DLP mutants and found that although DLP expression is developmentally regulated, it is not essential for the execution of the developmental program. The effects DLP mutations show in the loss of heterozygosity assay and on phenotypes resulting from Dmp53 overexpression indicate a genetic interaction between DLP and Dmp53. In contrast to Dmp53 mutants, however, loss of DLP does not result in radiosensitivity indicating that it does not play an essential role in the activation of Dmp53-dependent response after ionizing radiation, and DLP is also not required for the irradiation-induced activation of reaper. In contrast, DLP is involved in the transcriptional regulation of Ark, because Ark mRNA level is decreased in DLP mutants and increased upon ectopic overexpression of DLP. Interestingly, DLP mutants have reduced longevity and reduced female fertility. Altogether, our data suggest complex functions for DLP, which include an anti-apoptotic effect exerted through repression of some Dmp53 functions, and activation of some proapoptotic genes. Daxx-like protein (DLP), the Drosophila homolog of Daxx, binds Drosophila melanogaster p53 (Dmp53) through its C-terminal region. We generated DLP mutants and found that although DLP expression is developmentally regulated, it is not essential for the execution of the developmental program. The effects DLP mutations show in the loss of heterozygosity assay and on phenotypes resulting from Dmp53 overexpression indicate a genetic interaction between DLP and Dmp53. In contrast to Dmp53 mutants, however, loss of DLP does not result in radiosensitivity indicating that it does not play an essential role in the activation of Dmp53-dependent response after ionizing radiation, and DLP is also not required for the irradiation-induced activation of reaper. In contrast, DLP is involved in the transcriptional regulation of Ark, because Ark mRNA level is decreased in DLP mutants and increased upon ectopic overexpression of DLP. Interestingly, DLP mutants have reduced longevity and reduced female fertility. Altogether, our data suggest complex functions for DLP, which include an anti-apoptotic effect exerted through repression of some Dmp53 functions, and activation of some proapoptotic genes. Mammalian p53 is a transcription factor that plays a fundamental role in cellular response to genotoxic stress (1Coates P.J. Lorimore S.A. Wright E.G. J. Pathol. 2005; 205: 221-235Crossref PubMed Scopus (57) Google Scholar). Activated p53 induces cell cycle arrest, DNA repair, or apoptosis by both transcription-dependent and independent ways (2Schuler M. Green D.R. Trends Genet. 2005; 21: 182-187Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). The observation that p53 is the most commonly mutated gene in human cancer (3Hainaut P. Hernandez T. Robinson A. Rodriguez-Tome P. Flores T. Hollstein M. Harris C.C. Montesano R. Nucleic Acids Res. 1998; 26: 205-213Crossref PubMed Scopus (416) Google Scholar) underlines the importance of these activities in preserving genome integrity and eliminating transformed cells that pose a risk at the organism level. Although a great body of knowledge has accumulated about p53, its regulation and activity is still not understood in full detail. Furthermore, the existence of two paralogs of p53 (p63 and p73) in mammalian cells with partly overlapping functions, makes the dissection of the cellular role of p53 difficult, and underlines the importance of simple models for studying p53 function. p53 and its only Drosophila homolog Dmp53 share limited conservation at the sequence level, yet the two proteins are surprisingly similar in domain structure and their residues critical for DNA binding are well preserved (4Ollmann M. Young L.M. Di Como C.J. Karim F. Belvin M. Robertson S. Whittaker K. Demsky M. Fisher W.W. Buchman A. Duyk G. Friedman L. Prives C. Kopczynski C. Cell. 2000; 101: 91-101Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar, 5Jin S. Martinek S. Joo W.S. Wortman J.R. Mirkovic N. Sali A. Yandell M.D. Pavletich N.P. Young M.W. Levine A.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7301-7306Crossref PubMed Scopus (170) Google Scholar). Importantly, Dmp53 is able to bind to human p53 recognition sites and activate transcription in vitro (5Jin S. Martinek S. Joo W.S. Wortman J.R. Mirkovic N. Sali A. Yandell M.D. Pavletich N.P. Young M.W. Levine A.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7301-7306Crossref PubMed Scopus (170) Google Scholar, 6Brodsky M.H. Nordstrom W. Tsang G. Kwan E. Rubin G.M. Abrams J.M. Cell. 2000; 101: 103-113Abstract Full Text Full Text PDF PubMed Scopus (385) Google Scholar). Although Dmp53 is nonessential under normal circumstances, null mutants show genomic instability and radiosensitivity (7Lee J.H. Lee E. Park J. Kim E. Kim J. Chung J. FEBS Lett. 2003; 550: 5-10Crossref PubMed Scopus (61) Google Scholar, 8Sogame N. Kim M. Abrams J.M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 4696-4701Crossref PubMed Scopus (133) Google Scholar). In response to genotoxic stimuli, activated Dmp53 induces DNA repair or programmed cell death by activating the expression of its target genes, such as the pro-apoptotic reaper (rpr), Hid and sickle, and the DNA repair genes Ku70 and Ku80 (9Brodsky M.H. Weinert B.T. Tsang G. Rong Y.S. McGinnis N.M. Golic K.G. Rio D.C. Rubin G.M. Mol. Cell Biol. 2004; 24: 1219-1231Crossref PubMed Scopus (242) Google Scholar). Loss of Dmp53 abolishes radiation-induced apoptosis in larval imaginal discs (6Brodsky M.H. Nordstrom W. Tsang G. Kwan E. Rubin G.M. Abrams J.M. Cell. 2000; 101: 103-113Abstract Full Text Full Text PDF PubMed Scopus (385) Google Scholar, 8Sogame N. Kim M. Abrams J.M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 4696-4701Crossref PubMed Scopus (133) Google Scholar), and overexpression induces cell death (5Jin S. Martinek S. Joo W.S. Wortman J.R. Mirkovic N. Sali A. Yandell M.D. Pavletich N.P. Young M.W. Levine A.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7301-7306Crossref PubMed Scopus (170) Google Scholar, 9Brodsky M.H. Weinert B.T. Tsang G. Rong Y.S. McGinnis N.M. Golic K.G. Rio D.C. Rubin G.M. Mol. Cell Biol. 2004; 24: 1219-1231Crossref PubMed Scopus (242) Google Scholar). Interestingly, a disturbed level of p53 affects aging and longevity both in mouse and Drosophila (7Lee J.H. Lee E. Park J. Kim E. Kim J. Chung J. FEBS Lett. 2003; 550: 5-10Crossref PubMed Scopus (61) Google Scholar, 10Bauer J.H. Poon P.C. Glatt-Deeley H. Abrams J.M. Helfand S.L. Curr. Biol. 2005; 15: 2063-2068Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 11Maier B. Gluba W. Bernier B. Turner T. Mohammad K. Guise T. Sutherland A. Thorner M. Scrable H. Genes Dev. 2004; 18: 306-319Crossref PubMed Scopus (496) Google Scholar, 12Tyner S.D. Venkatachalam S. Choi J. Jones S. Ghebranious N. Igelmann H. Lu X. Soron G. Cooper B. Brayton C. Hee Park S. Thompson T. Karsenty G. Bradley A. Donehower L.A. Nature. 2002; 415: 45-53Crossref PubMed Scopus (1176) Google Scholar). Although little is known about the regulation of Dmp53, the information accumulated so far suggests that Dmp53 is regulated in a partially conserved, ancestral way. MDM2 does not have a Drosophila homolog, and the amino acid residues critical for its binding are not preserved in Dmp53 (4Ollmann M. Young L.M. Di Como C.J. Karim F. Belvin M. Robertson S. Whittaker K. Demsky M. Fisher W.W. Buchman A. Duyk G. Friedman L. Prives C. Kopczynski C. Cell. 2000; 101: 91-101Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar). In contrast, similar to its human counterpart, Dmp53 is phosphorylated, and this modification is necessary for the induction of Dmp53-dependent apoptosis (9Brodsky M.H. Weinert B.T. Tsang G. Rong Y.S. McGinnis N.M. Golic K.G. Rio D.C. Rubin G.M. Mol. Cell Biol. 2004; 24: 1219-1231Crossref PubMed Scopus (242) Google Scholar, 13Peters M. DeLuca C. Hirao A. Stambolic V. Potter J. Zhou L. Liepa J. Snow B. Arya S. Wong J. Bouchard D. Binari R. Manoukian A.S. Mak T.W. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11305-11310Crossref PubMed Scopus (78) Google Scholar). The death domain-associated factor 6, Daxx, is one of the cofactors modulating p53 functions. Daxx was initially identified as a cytoplasmic Fas receptor-binding protein that potentiates apoptosis (14Chang H.Y. Nishitoh H. Yang X. Ichijo H. Baltimore D. Science. 1998; 281: 1860-1863Crossref PubMed Scopus (534) Google Scholar, 15Yang X. Khosravi-Far R. Chang H.Y. Baltimore D. Cell. 1997; 89: 1067-1076Abstract Full Text Full Text PDF PubMed Scopus (832) Google Scholar). More recent studies found that Daxx resides primarily in the nucleus, and participates in transcriptional regulation (16Emelyanov A.V. Kovac C.R. Sepulveda M.A. Birshtein B.K. J. Biol. Chem. 2002; 277: 11156-11164Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 17Li H. Leo C. Zhu J. Wu X. O'Neil J. Park E.J. Chen J.D. Mol. Cell Biol. 2000; 20: 1784-1796Crossref PubMed Scopus (309) Google Scholar, 18Salomoni P. Khelifi A.F. Trends Cell Biol. 2006; 16: 97-104Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). Loss of Daxx results in extensive apoptosis and early embryonic death in mice (19Michaelson J.S. Bader D. Kuo F. Kozak C. Leder P. Genes Dev. 1999; 13: 1918-1923Crossref PubMed Scopus (199) Google Scholar), suggesting that it may also bear anti-apoptotic functions. This view is supported by the finding that the expression of p53, similarly to that of several other proapoptotic proteins, is down-regulated by Daxx in myelocytes (20Boehrer S. Nowak D. Schaaf S. Bergmann M. Brieger A. Hoelzer D. Mitrou P.S. Weidmann E. Chow K.U. Hematol. J. 2004; 5: 513-518Crossref PubMed Scopus (2) Google Scholar). Several groups demonstrated the binding of Daxx to p53 but the consequences of this interaction are not clearly elucidated. Tumorigenic mutant forms of p53 were found to bind Daxx and inhibit the activation of the Daxx-dependent ASK1/JNK pathway (21Ohiro Y. Usheva A. Kobayashi S. Duffy S.L. Nantz R. Gius D. Horikoshi N. Mol. Cell Biol. 2003; 23: 322-334Crossref PubMed Scopus (29) Google Scholar). Other studies showed that Daxx also binds wild-type p53 and modulates its transcriptional activator function in vitro (22Gostissa M. Morelli M. Mantovani F. Guida E. Piazza S. Collavin L. Brancolini C. Schneider C. Del Sal G. J. Biol. Chem. 2004; 279: 48013-48023Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 23Kim E.J. Park J.S. Um S.J. Nucleic Acids Res. 2003; 31: 5356-5367Crossref PubMed Scopus (61) Google Scholar, 24Zhao L.Y. Liu J. Sidhu G.S. Niu Y. Liu Y. Wang R. Liao D. J. Biol. Chem. 2004; 279: 50566-50579Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar), although these experiments provided partially conflicting results. Several recent reports indicate that Daxx bears both pro- and anti-cell death activities, and its involvement in malignancies is convincingly demonstrated (18Salomoni P. Khelifi A.F. Trends Cell Biol. 2006; 16: 97-104Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). Here we show both the physical interaction between Dmp53 and the Drosophila homolog of Daxx, DLP, 3The abbreviations used are:DLPDaxx-like proteinANOVAanalysis of varianceGSTglutathione S-transferaseArkApaf-1-related-killerLOHloss-of-heterozygosity. and the genetic interaction of the corresponding genes. Analyzing the effect of DLP mutations we found that, although DLP is required for the transcription of some proapoptotic genes, such as Ark for example, DLP is not required for Dmp53-dependent stress response upon high dose ionizing radiation. Surprisingly, DLP mutants have reduced longevity similarly to Dmp53 mutant flies (7Lee J.H. Lee E. Park J. Kim E. Kim J. Chung J. FEBS Lett. 2003; 550: 5-10Crossref PubMed Scopus (61) Google Scholar). Altogether, our data suggest a role for DLP regulating both pro- and antiapoptotic pathways. Daxx-like protein analysis of variance glutathione S-transferase Apaf-1-related-killer loss-of-heterozygosity Plasmid Constructs—To generate bait plasmids pBTM116-Dmp53ΔN, pBTM116-Dmp53C, pBTM116-Dmp53C1, and pBTM116-Dmp53C2, a Dmp53 cDNA was PCR-amplified using the primer pairs Dmp53F1-Dmp53R1, Dmp53F3-Dmp53R1, Dmp53F3-Dmp53R2, and Dmp53F4-Dmp53R1, respectively. The amplicons were cut with EcoRI and BamHI and inserted into pBTM116. To generate pBTM116-Dmp53ΔNΔC the plasmid pBTM116-Dmp53ΔN was cut with StyI and BamHI, filled in with Klenow and ligated. Human p53 was cloned into pBTM116 using EcoRI and BamHI, then the transactivation domain was deleted by digestion with EcoRI and NcoI, fill-in and religation. The sequences of the oligonucleotide primers were as follows: Dmp53F1: GGGAATTCTTGCAGGGATTAAACTCCG, Dmp53F3: GCGAATTCAGCAAGAAGCGCAAGTCC, Dmp53F4: GAATTCGGCATGATTAAGGAGGCGGC, Dmp53R1: CGGGATCCTCATGGCAGCTCGTAGGC, Dmp53R2: GGATCCAATCATGCCCTCGATGCTCT. To generate P[UASDLP1.7 kb] plasmid 1.7-kbp fragment of DLP was PCR-amplified using the DLPF2 (ACTGAGTACGCGGCACTTCT) and DLPR (ATGATCCGAACGTATTCAGGA) primers. The fragment was cloned into pUAST vector using the EcoRI and ApaI restriction sites. Yeast Two-hybrid Experiments—The yeast two-hybrid screen was performed using MATCHMAKER lexA Two-Hybrid System (Clontech Laboratories, Inc.), following the supplier's recommendations. Positive clones were validated by β-galactosidase colony-lift filter assay (25Breeden L. Nasmyth K. Cold Spring Harb. Symp. Quant. Biol. 1985; 50: 643-650Crossref PubMed Scopus (470) Google Scholar). Plasmid DNA was isolated from colonies proved to be positive in both assays (i.e. complementation of auxotrophy and β-galactosidase assay), sequenced, and cDNAs identified by BLAST homology searches (26Altschul S.F. Madden T.L. Schaffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (60233) Google Scholar). Binding of DLP to different domains of Dmp53 was tested by co-transformation of L40 yeast cells with the plasmid DLP-pACT2, identified in the screen, and pBTM116 plasmids carrying cDNA fragments spanning different regions of the Dmp53 open reading frame. To test the potential interactions, transformants were screened for growth in medium lacking histidine and assayed for β-galactosidase activity, as described above. GST Pull-down Experiments—The C-terminal region of DLP cDNA identified in Y2H screen was inserted into pET28c and transcribed-translated in the presence of [3H]leucine using the TnT T7-coupled reticulocyte lysate system (Promega) following the manufacturer's instructions. Dmp53C was cloned into pGEX-4T-1 vector using EcoRI and SalI sites. Expressed GST-Dmp53C was bound to glutathione-Sepharose beads (Amersham Biosciences) according to the manufacturer's instructions. In vitro translated DLP protein and GST-Dmp53C-bound beads were mixed in PD buffer (20 mm Tris-HCl, pH 7.5, 150 mm NaCl, 3 mm EDTA pH8.0, 1 mm β-mercaptoethanol, 1% Nonidet P40) and kept at 4 °C for 2 h. Beads were washed four times with PD buffer containing 0.1% Nonidet P40, and once with PD buffer lacking Nonidet P40. Interactions were analyzed by SDS-PAGE and fluorography (using Amplify Fluorographic Reagent from Amersham Biosciences). Quantitative RT-PCR—Developmental expression of DLP was determined by measuring DLP transcript levels in synchronized w1118 animals of different developmental stages. Dmp53-dependent transcriptional activation after DNA damage was determined by measuring rpr and Ark mRNA levels of wandering third-instar w1118, Dmp535A-1–4, DLPU26, DLPU32, and DLPU42 larvae without irradiation or 2 h after 4 krad x-ray irradiation (1 krad/min). To measure DLP and Ark transcript levels in flies overexpressing DLP P[hs-GAL4]/EP (2)2108, P[hs-GAL4]/EP (2)2193, P[hs-GAL4]/EP (2)2180, P[hs-GAL4]/P[UASDLP1.7kb], and P[hs-GAL4]/+ (control) adult females were heat-shocked for 60 min at 37 °C. 60 min after heat-shock RNA was isolated and used in Q-PCR. Total RNA was isolated with Qiagen RNeasy mini kit (Qiagen Inc., Valencia, CA) according to the manufacturer's instructions. First-strand cDNA was synthesized from 2 μg of total RNA with random hexamer primers using TaqMan Reverse Transcription Reagent (Applied Biosystems, Foster City, CA). PCR reactions were carried out in duplicates in an ABI Prism 7500 real-time PCR system. cDNAs corresponding 18S rRNA and rpr were amplified using TaqMan Universal PCR Master Mix while SYBR-Green PCR Master Mix (27Price D.M. Jin Z. Rabinovitch S. Campbell S.D. Genetics. 2002; 161: 721-731PubMed Google Scholar) was used for the amplification of DLP and Ark cDNA. The sequence of primers and TaqMan probes were as follows: 18S forward: GCCAGCTAGCAATTGGGTGTA, 18S reverse: CCGGAGCCCAAAAAGCTT, 18S probe: TATGGCTCTCTCAGTCGCTTCCGGG, rpr forward: CCAGTTGTGTAATTCCGAACGA, rpr reverse: TCGCCTGATCGGGTATGTAGA, rpr probe: AAGAAAGATAAACCAATGGCAGTGGCA, Ark forward: TGTCGCCAATCAAGACTGAG, Ark reverse: CATCCAAGGCTACCCAAGTC, DLP forward: TCGCCTACGATGCCTTTAAC, DLP reverse: AGATCTTGCTCGAGGGCATA. mRNA expression levels were evaluated by comparing to appropriate control samples after normalization with 18S RNA levels. In Situ Hybridization—Whole mount in situ hybridization was performed using digoxigenin-labeled, hydrolyzed antisense RNA probes following the manufacturer's recommendations (Roche Applied Science). Probes were prepared from the full-length DLP cDNA clone SD20887 (28Stapleton M. Carlson J. Brokstein P. Yu C. Champe M. George R. Guarin H. Kronmiller B. Pacleb J. Park S. Wan K. Rubin G.M. Celniker S.E. Genome Biol. 2002; 3 (RESEARCH0080)Crossref Google Scholar) received through the Drosophila Genomics Resource Center. Hybridization and pre- and post-hybridization washes were done at 55 °C, RNA hybrids were detected with anti-digoxigenin-AP antibody followed by NBT/BCIP staining. Drosophila Stocks and Crosses—Fly stocks were maintained at 25 °C on standard cornmeal-yeast-agar Drosophila medium. The Dmp535A-1–4 line (29Rong Y.S. Titen S.W. Xie H.B. Golic M.M. Bastiani M. Bandyopadhyay P. Olivera B.M. Brodsky M. Rubin G.M. Golic K.G. Genes Dev. 2002; 16: 1568-1581Crossref PubMed Scopus (271) Google Scholar) was a generous gift from Yikang S. Rong (National Institutes of Health, Bethesda, MD). The P[UASp53] strain was a kind gift from Michael W. Young (5Jin S. Martinek S. Joo W.S. Wortman J.R. Mirkovic N. Sali A. Yandell M.D. Pavletich N.P. Young M.W. Levine A.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7301-7306Crossref PubMed Scopus (170) Google Scholar). The RS-element insertion lines UM-8182-3 and CB-6241-3 (30Ryder E. Blows F. Ashburner M. Bautista-Llacer R. Coulson D. Drummond J. Webster J. Gubb D. Gunton N. Johnson G. O'Kane C.J. Huen D. Sharma P. Asztalos Z. Baisch H. Schulze J. Kube M. Kittlaus K. Reuter G. Maroy P. Szidonya J. Rasmuson-Lestander A. Ekstrom K. Dickson B. Hugentobler C. Stocker H. Hafen E. Lepesant J.A. Pflugfelder G. Heisenberg M. Mechler B. Serras F. Corominas M. Schneuwly S. Preat T. Roote J. Russell S. Genetics. 2004; 167: 797-813Crossref PubMed Scopus (271) Google Scholar), the EP insertion lines EP (2)2108, EP (2)2193, and EP (2)2180 were kindly provided by the Szeged Drosophila Stock Centre. The RNAi strains 29374 and 29377 were obtained from Vienna Drosophila RNAi Center. P[act-GAL4] strain (BL-3954) and other stocks were obtained from the Bloomington Drosophila Stock Center. The P[ey-GAL4] strain was kindly provided by J. Mihaly (BRC, Szeged, Hungary). To generate new DLP alleles the RS elements in the viable insertion lines UM-8182-3 and CB-6241-3 were remobilized using the TM3, ryRK Sb1 Ser1 P(Δ2-3)99B transposase source. Excision lines were identified based on the loss of the miniwhite marker gene and stocks were established using the y+CyO balancer chromosome. From each homozygous line genomic DNA was prepared, and PCR reactions were performed with primer pairs amplifying sequences upstream and downstream of the insertion site. (Sequences of primers are available upon request.) Deletion lines were selected on the basis of loss of the PCR products. The sizes of deletions were determined by further PCR reactions and in some selected cases molecular break-points were determined by sequencing. In addition to the deletions, precise excision lines (PE) in which precise jumpout of the P element restored the structure of DLP, were also identified by PCR analysis. Lethal mutations were transferred over a Cy-GFP balancer chromosome, the presence of deletions inspected in GFP-negative embryos as described above and allelism tested by complementation analysis. To measure the reduction of viability after ionizing radiation wandering third-instar w1118, p535A-1–4, DLPU26, DLPU32, and DLPU42 larvae were exposed to 2 krad (1 krad/min) x-ray irradiation and transferred into fresh vials, 20 larvae/vial. The number of pupae and eclosed adults were recorded and viability expressed in the percent of the number of irradiated larvae. At least six independent experiments were performed for each genotype. For the longevity assay freshly eclosed males in groups of 20 were put into vials, and the number of survivors was counted daily. For the fertility assay five 48-h-old females with 2 males were put into a vial, and the number of eggs laid in a 24-h period was recorded for 7 days in five independent experiments of each genotype. To observe the effect of DLP mutation on the phenotype resulting from Dmp53 overexpression we performed the following crosses: DLPU26;P[UASp53]/T (2Schuler M. Green D.R. Trends Genet. 2005; 21: 182-187Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 3Hainaut P. Hernandez T. Robinson A. Rodriguez-Tome P. Flores T. Hollstein M. Harris C.C. Montesano R. Nucleic Acids Res. 1998; 26: 205-213Crossref PubMed Scopus (416) Google Scholar) TSTL, Cy; Tb Hu females were mated to DLPU26; P[act-GAL4]/TSTL males, and DLPU26; P[UASp53]/TSTL females were crossed with PE; P[act-GAL4]/TSTL males as a control. The DLPU26/DLPU26; P[UASp53]/ P[act-GAL4], and DLPU26/PE; P[UASp53]/P[act-GAL4] animals were selected based on the Tb+ phenotype. Three independent experiments were performed and the number of animals reaching specific developmental stages was recorded. Similar experiments were carried out using the eye-specific eyeless-GAL4 driver. For this the crosses were: DLPU26; P[ey-GAL4] females × DLPU26; P[UASp53]/TSTL males and DLPU26; P[ey-GAL4] females × PE; P[UASp53]/TSTL males as a control. The DLPU26/DLPU26; P[UASp53]/P[ey-GAL4], and DLPU26/PE; P[UASp53]/P[ey-GAL4] animals were selected based on the Tb+ phenotype. The largest diameters of the eyes of adult flies (20 in each groups in three parallels) were determined after photography. In loss-of-heterozygosity (LOH) assay DLPU26/DLPU26; mwh Dmp535A-1–4/++, and PE/DLPU26; mwh Dmp535A-1–4/++ (control) late-third-instar (wandering) larvae were x-ray-irradiated with 250 rad (150 kV; 0.5-mm Al filter; 1,000 rad/min). Wings were dissected after eclosion, mounted in 1:1 methyl salicylate/Canada balsam (Sigma) and the number of mwh clones determined. Both genetic combinations were tested in four independent experiments, each involving 8–20 wings. Daxx-like Protein Interacts with Dmp53—In an attempt to identify Dmp53-interacting proteins we employed the yeast two-hybrid (Y2H) method to screen Drosophila embryonic cDNA library using a lexA-Dmp53 fusion lacking the N-terminal transcriptional activation domain of Dmp53 (Dmp53ΔN) as bait. (Fig. 1A) Sequence analysis identified one of the positive clones as a partial cDNA of the Daxx-like protein gene (CG9537). The 810-bp long clone encodes the last 135 amino acids (1524–1659) of DLP indicating that the C-terminal part of the protein mediates the interaction with Dmp53. GST pull-down experiment validated the specific interaction of Dmp53 and the DLP C-terminal region encoded by the cDNA recovered in the Y2H screen (Fig. 1C). To determine which region of Dmp53 is necessary for DLP interaction, we fused various segments of Dmp53 to lexA and tested for interaction with the identified DLP clone in Y2H experiments. These experiments revealed that the DNA binding domain of Dmp53 (Dmp53ΔNΔC) did not, but the C-terminal part of Dmp53 (Dmp53C) showed strong interaction with DLP (Fig. 1, A and B). When we asked whether either of the two functionally distinguishable C-terminal regions alone are able to bind DLP we found that neither the oligomerization domain (Dmp53C1), nor the basic regulatory domain (Dmp53C2) alone showed interaction (Fig. 1, A and B). Consequently, the entire C-terminal region of Dmp53, containing both the oligomerization and the basic regulatory domain, is necessary and sufficient to mediate DLP binding. These results indicate that, similarly to the interaction of p53 and Daxx (22Gostissa M. Morelli M. Mantovani F. Guida E. Piazza S. Collavin L. Brancolini C. Schneider C. Del Sal G. J. Biol. Chem. 2004; 279: 48013-48023Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar), the C-terminal region of Dmp53 is responsible for DLP binding. The similar regions of p53 and Dmp53 involved in Daxx and DLP interaction, respectively, prompted us to test whether DLP can bind human p53. Surprisingly, in Y2H assay we detected strong interaction between the C-terminal region of DLP and human p53 (Fig. 1D). Thus, although the human and fly p53 orthologs have low sequence similarity, the protein features necessary for Daxx/DLP binding are evolutionarily conserved. Daxx-like Proteins Are Conserved in the Drosophila Genus—The DLP gene has a coding capacity for a putative protein of 1659 amino acids, which shows partial similarity to its 740-residue-long human ortholog, Daxx. Comparison of the two proteins revealed that they share 27% sequence identity and 46% similarity in the Daxx-homology region (residues 1125–1472 of DLP), a region conserved in all known Daxx homologs (Fig. 2A). The Dmp53 interacting region of DLP shows 51% similarity to the part of Daxx reported to mediate p53 binding in vitro (22Gostissa M. Morelli M. Mantovani F. Guida E. Piazza S. Collavin L. Brancolini C. Schneider C. Del Sal G. J. Biol. Chem. 2004; 279: 48013-48023Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Beside the regions mentioned above, DLP bears N-terminal glutamine-, proline-, and alanine-rich regions, which seem to be specific for the Drosophila Daxx homolog. Identifiable DLP-like proteins in members of the Drosophila genus are highly homologous in their Daxx homology regions and share significant sequence similarities outside this domain (Fig. 2A). Generation of DLP Mutants—DLP is located on the second chromosome at the cytological position 26D8. The transposons in the P{RS3} element lines UM-8182-3 and CB-6241-3 (30Ryder E. Blows F. Ashburner M. Bautista-Llacer R. Coulson D. Drummond J. Webster J. Gubb D. Gunton N. Johnson G. O'Kane C.J. Huen D. Sharma P. Asztalos Z. Baisch H. Schulze J. Kube M. Kittlaus K. Reuter G. Maroy P. Szidonya J. Rasmuson-Lestander A. Ekstrom K. Dickson B. Hugentobler C. Stocker H. Hafen E. Lepesant J.A. Pflugfelder G. Heisenberg M. Mechler B. Serras F. Corominas M. Schneuwly S. Preat T. Roote J. Russell S. Genetics. 2004; 167: 797-813Crossref PubMed Scopus (271) Google Scholar) reside in the first intron of the gene, +568 bp and +638 bp downstream of the transcriptional start site, respectively. To investigate DLP functions in vivo, we generated mutant alleles of the gene by remobilization of the P element present in these lines. Excision lines obtained following transposon remobilization were identified by loss of the miniwhite eye color marker and characterized molecularly by PCR analysis performed on homozygous embryos. Genetic crosses and molecular analysis of jumpout mutants revealed that in all lethal alleles the deletions extended out of the DLP region into adjacent genes and carriers of either of the 14 deletions mapped by PCR analysis within the DLP were viable. Thus, we concluded that DLP mutations are most probably viable and selected three alleles for further analysis (Fig. 2B). In DLPU26 a 1.7-kbp deletion removes the downstream part of the first intron and 1296 bp of coding sequence from the second exon. DLPU32 carries a 0.3-kbp deletion that removes the first exon-intron junction and 28 nucleotides from the first exon. In DLPU42 a 0.4-kbp deletion removes the translational start site. We believe that the deletions in these DLP alleles interfere with the splicing and/or translation of the DLP message. Using primers located in the vicinity of original P element insertion site DLP specific mRNA in mutant animals cannot be detected. We believe that these represent null alleles of the gene, however, in the lack of DLP specific antibody, we cannot rule out the possibility that they are hypomorph alleles, in which a truncated form of the protein is produced. In addition to the deletions, by PCR analysis we also identified lines in which precise excision of the P element restored the structure of DLP. In further genetic experiments one of these (PE) was used as an isogenic control of DLP mutants. DLP Is Developmentally Regulated but Is Not Required for Normal Development—To determine whether loss of DLP has any effect on Drosophila development we crossed homozygous DLPU26, DLPU32, and DLPU42 females to heterozygous males and determined the ratio of homozygous and heterozygous offsprings. No significant differences were found in any allele (data not shown), proving that DLP is not essential for execution of the developmental program. Lines expressing DLP siRNA obtained from the VDRC (Vienna Drosophila RNAi Center) collection are also viable and develop normally (data not shown). Therefore, we decided to determine whether DLP is expressed during develop
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