Functional Interaction between the Drosophila Knirps Short Range Transcriptional Repressor and RPD3 Histone Deacetylase
2005; Elsevier BV; Volume: 280; Issue: 49 Linguagem: Inglês
10.1074/jbc.m506819200
ISSN1083-351X
AutoresPaolo Struffi, David N. Arnosti,
Tópico(s)Genomics and Chromatin Dynamics
ResumoKnirps and other short range transcriptional repressors play critical roles in patterning the early Drosophila embryo. These repressors are known to bind the C-terminal binding protein corepressor, but their mechanism of action is poorly understood. We purified functional recombinant Knirps protein from transgenic embryos to identify possible cofactors that contribute to the activity of this protein. The protein migrates in a complex of ∼450 kDa and was found to copurify with the Rpd3 histone deacetylase protein during a double affinity purification procedure. Association of Rpd3 with Knirps was dependent on the presence of the C-terminal binding protein-dependent repression domain of Knirps. Previous studies of an rpd3 mutant had not shown defects in the pattern of expression of even-skipped, a target of the Knirps repressor. However, in embryos doubly heterozygous for knirps and rpd3, a marked increase in the frequency of defects in the Knirps-regulated posterior domain of even-skipped expression was found, indicating that Rpd3 contributes to Knirps repression activity in vivo. This finding implicates deacetylation in the mechanism of short range repression in Drosophila. Knirps and other short range transcriptional repressors play critical roles in patterning the early Drosophila embryo. These repressors are known to bind the C-terminal binding protein corepressor, but their mechanism of action is poorly understood. We purified functional recombinant Knirps protein from transgenic embryos to identify possible cofactors that contribute to the activity of this protein. The protein migrates in a complex of ∼450 kDa and was found to copurify with the Rpd3 histone deacetylase protein during a double affinity purification procedure. Association of Rpd3 with Knirps was dependent on the presence of the C-terminal binding protein-dependent repression domain of Knirps. Previous studies of an rpd3 mutant had not shown defects in the pattern of expression of even-skipped, a target of the Knirps repressor. However, in embryos doubly heterozygous for knirps and rpd3, a marked increase in the frequency of defects in the Knirps-regulated posterior domain of even-skipped expression was found, indicating that Rpd3 contributes to Knirps repression activity in vivo. This finding implicates deacetylation in the mechanism of short range repression in Drosophila. Transcriptional repression is critical for the patterned expression of developmentally regulated genes in Drosophila and other metazoans. Transcriptional repressors can exhibit a range of effects; repression can be transient or persist over long periods of time, and repressors can work locally (short range) or influence the activity of cis regulatory elements over 1 kbp away (long range). Repressors act through multiple biochemical pathways, including interference with activation domains, remodeling of chromatin, and direct interactions with the basal transcriptional machinery (1Arnosti D.N. Gossen M. Kaufmann J. Triezenberg S.J. Handbook of Experimental Pharmacology. Springer-Verlag, Heidelberg2004: 33-67Google Scholar, 2Gaston K. Jayaraman P.S. Cell. Mol. Life Sci. 2003; 60: 721-741Crossref PubMed Scopus (97) Google Scholar, 3Thiel G. Lietz M. Hohl M. Eur. J. Biochem. 2004; 271: 2855-2862Crossref PubMed Scopus (94) Google Scholar). One of the best characterized systems of developmental gene regulation involving transcriptional repressors is that present in the Drosophila blastoderm embryo, where repressors encoded by "gap genes" control the presegmental expression of pair rule genes, including even-skipped (eve). This pair rule gene is controlled by five modular enhancers present 5′ and 3′ of the transcription unit, each of which is regulated by short range repressors, including Giant, Krüppel, and Knirps (4Fujioka M. Emi-Sarker Y. Yusibova G.L. Goto T. Jaynes J.B. Development (Camb.). 1999; 126: 2527-2538Crossref PubMed Google Scholar). These short range repressors all interact with the C-terminal binding protein (CtBP) 3The abbreviations used are: CtBPC-terminal binding proteinHDAChistone deacetylaseNi-NTANi2+-nitrilotriacetic acidHRPhorseradish peroxidaseDTTdithiothreitolPMSFphenylmethylsulfonyl fluorideTBPTATA-binding proteindCtBPDrosophila CtBP.3The abbreviations used are: CtBPC-terminal binding proteinHDAChistone deacetylaseNi-NTANi2+-nitrilotriacetic acidHRPhorseradish peroxidaseDTTdithiothreitolPMSFphenylmethylsulfonyl fluorideTBPTATA-binding proteindCtBPDrosophila CtBP. corepressor and exhibit similar functional properties, including range of action, generally less than 100 bp from activators or the basal promoter (5Nibu Y. Zhang H. Bajor E. Barolo S. Small S. Levine M. EMBO J. 1998; 17: 7009-7020Crossref PubMed Scopus (171) Google Scholar, 6Hewitt G.F. Strunk B.S. Margulies C. Priputin T. Wang X.D. Amey R. Pabst B.A. Kosman D. Reinitz J. Arnosti D.N. Development (Camb.). 1999; 126: 1201-1210Crossref PubMed Google Scholar, 7Keller S.A. Mao Y. Struffi P. Margulies C. Yurk C.E. Anderson A.R. Amey R.L. Moore S. Ebels J.M. Foley K. Corado M. Arnosti D.N. Mol. Cell. Biol. 2000; 20: 7247-7258Crossref PubMed Scopus (38) Google Scholar). C-terminal binding protein histone deacetylase Ni2+-nitrilotriacetic acid horseradish peroxidase dithiothreitol phenylmethylsulfonyl fluoride TATA-binding protein Drosophila CtBP. C-terminal binding protein histone deacetylase Ni2+-nitrilotriacetic acid horseradish peroxidase dithiothreitol phenylmethylsulfonyl fluoride TATA-binding protein Drosophila CtBP. Knirps is a member of the nuclear receptor superfamily, possessing this family's characteristic zinc finger DNA binding domain, which is coupled to a unique C terminus that interacts with CtBP (8Nibu Y. Zhang H. Levine M. Science. 1998; 280: 101-104Crossref PubMed Scopus (222) Google Scholar). Knirps represses genes via CtBP-dependent and -independent activities. The combined repression activities play an especially important role on enhancers that are less sensitive to Knirps (7Keller S.A. Mao Y. Struffi P. Margulies C. Yurk C.E. Anderson A.R. Amey R.L. Moore S. Ebels J.M. Foley K. Corado M. Arnosti D.N. Mol. Cell. Biol. 2000; 20: 7247-7258Crossref PubMed Scopus (38) Google Scholar, 9Struffi P. Corado M. Kulkarni M. Arnosti D.N. Development (Camb.). 2004; 131: 2419-2429Crossref PubMed Scopus (22) Google Scholar, 10Sutrias-Grau M. Arnosti D.N. Mol. Cell. Biol. 2004; 24: 5953-5966Crossref PubMed Scopus (27) Google Scholar, 11Ryu J.R. Arnosti D.N. Nucleic Acids Res. 2003; 31: 4654-4662Crossref PubMed Scopus (23) Google Scholar). A gradient of the Knirps protein in the posterior of the embryo is responsible for simultaneously setting the boundaries of eve stripes 3, 4, 6, and 7. These multiple thresholds are achieved by differential sensitivity of the enhancers to Knirps, such that the domains of expression of eve stripe 4/6 are wholly contained within a region of the embryo that has sufficient Knirps protein to repress the eve stripe 3/7 (12Clyde D.E. Corado M.S. Wu X. Pare A. Papatsenko D. Small S. Nature. 2003; 426: 849-853Crossref PubMed Scopus (157) Google Scholar). CtBP is a widely conserved dimeric corepressor that interacts with a variety of transcription factors, recognizing a short peptide motif (PMDLS in Knirps) in the binding partner (reviewed in Ref. 13Chinnadurai G. BioEssays. 2003; 25: 9-12Crossref PubMed Scopus (82) Google Scholar). CtBP proteins bear striking similarities to α-hydroxy acid dehydrogenases and like these enzymes bind to NAD/NADH (14Kumar V. Carlson J.E. Ohgi K.A. Edwards T.A. Rose D.W. Escalante C.R. Rosenfeld M.G. Aggarwal A.K. Mol. Cell. 2002; 10: 857-869Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar, 15Nardini M. Spanò S. Cericola C. Pesce A. Massaro A. Millo E. Luini A. Corda D. Bolognesi M. EMBO J. 2003; 22: 3122-3130Crossref PubMed Scopus (126) Google Scholar). Binding of the cofactor has been observed to stimulate dimer or higher order complex formation in vitro and has been suggested to function as a metabolic switch in vivo, allowing cells to tune patterns of gene expression to nuclear levels of NADH (16Zhang Q. Piston D.W. Goodman R.H. Science. 2002; 295: 1895-1897PubMed Google Scholar). Loss of NADH binding through mutagenesis of conserved glycines of the dinucleotide binding cleft compromises repression activity by this protein, preventing dimerization, and interaction with target transcription factors (10Sutrias-Grau M. Arnosti D.N. Mol. Cell. Biol. 2004; 24: 5953-5966Crossref PubMed Scopus (27) Google Scholar, 14Kumar V. Carlson J.E. Ohgi K.A. Edwards T.A. Rose D.W. Escalante C.R. Rosenfeld M.G. Aggarwal A.K. Mol. Cell. 2002; 10: 857-869Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar). In addition, residues important for enzymatic function in the related dehydrogenases are conserved in metazoan CtBP proteins, although the possible enzymatic function of CtBP is poorly understood. Two reports find a weak dehydrogenase activity associated with the protein, but the conserved histidine residue of the catalytic triad is not required for transcriptional repression in vivo (10Sutrias-Grau M. Arnosti D.N. Mol. Cell. Biol. 2004; 24: 5953-5966Crossref PubMed Scopus (27) Google Scholar, 14Kumar V. Carlson J.E. Ohgi K.A. Edwards T.A. Rose D.W. Escalante C.R. Rosenfeld M.G. Aggarwal A.K. Mol. Cell. 2002; 10: 857-869Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar, 17Balasubramanian P. Zhao L.J. Chinnadurai G. FEBS Lett. 2003; 537: 157-160Crossref PubMed Scopus (70) Google Scholar). Mammalian CtBP physically associates with a variety of chromatin-modifying proteins, including histone deacetylases, histone methyltransferases, and a polyamine oxidase that was recently found to mediate lysine demethylation (18Shi Y. Sawada J. Sui G. Affar el B. Whetstine J.R. Lan F. Ogawa H. Luke M.P. Nakatani Y. Shi Y. Nature. 2003; 422: 735-738Crossref PubMed Scopus (632) Google Scholar, 19Shi Y. Lan F. Matson C. Mulligan P. Whetstine J.R. Cole P.A. Casero R.A. Shi Y. Cell. 2004; 119: 941-953Abstract Full Text Full Text PDF PubMed Scopus (3121) Google Scholar). These associated factors may be the main mediators of the repression activity for CtBP, although how a particular suite of CtBP associated activities may combine to regulate gene expression on endogenous circuits is not currently understood. The physiological role of one CtBP-associated histone deacetylase enzyme, HDAC1 (also termed Rpd3 in yeast and Drosophila), has been extensively characterized with respect to gene regulation. In addition to association with CtBP, Rpd3 proteins have been found in complexes that include the Sin3, Sds3, SAP18, and SAP30 proteins (20Kasten M.M. Dorland S. Stillman D.J. Mol. Cell. Biol. 1997; 17: 4852-4858Crossref PubMed Scopus (114) Google Scholar, 21Lechner T. Carrozza M.J. Yu Y. Grant P.A. Eberharter A. Vannier D. Brosch G. Stillman D.J. Shore D. Workman J.L. J. Biol. Chem. 2000; 275: 40961-40966Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 22Dorland S. Deegenaars M.L. Stillman D.J. Genetics. 2000; 154: 573-586Crossref PubMed Google Scholar, 23Zhang Y. Sun Z.W. Iratni R. Erdjument-Bromage H. Tempst P. Hampsey M. Reinberg D. Mol. Cell. 1998; 1: 1021-1031Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar). As a complex or individually, Rpd3/HDAC1 proteins are recruited to promoters to mediate repression by a variety of DNA-binding transcription factors, including Ume6 in yeast and YY1 and Mad/Max in mammals (24Kadosh D. Struhl K. Cell. 1997; 89: 365-371Abstract Full Text Full Text PDF PubMed Scopus (457) Google Scholar, 25Yang W.M. Inouye C. Zeng Y. Bearss D. Seto E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12845-12850Crossref PubMed Scopus (482) Google Scholar, 26Sommer A. Hilfenhaus S. Menkel A. Kremmer E. Seiser C. Loidl P. Luscher B. Curr. Biol. 1997; 7: 357-365Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Rpd3 also associates with corepressors, including Groucho, SMRTER, retinoblastoma, and E(z)-containing Polycomb complexes (27Chen G. Fernandez J. Mische S. Courey A.J. Genes Dev. 1999; 13: 2218-2230Crossref PubMed Scopus (353) Google Scholar, 28Pile L.A. Schlag E.M. Wassarman D.A. Mol. Cell. Biol. 2002; 22: 4965-4976Crossref PubMed Scopus (55) Google Scholar, 29Lewis P.W. Beall E.L. Fleischer T.C. Georlette D. Link A.J. Botchan M.R. Genes Dev. 2004; 18: 2929-2940Crossref PubMed Scopus (221) Google Scholar, 30Magnaghi-Jaulin L. Groisman R. Naguibneva I. Robin P. Lorain S. Le V.J.P. Troalen F. Trouche D. Harel-Bellan A. Nature. 1998; 391: 601-605Crossref PubMed Scopus (802) Google Scholar, 31Tie F. Prasad-Sinha J. Birve A. Rasmuson-Lestander A. Harte P.J. Mol. Cell. Biol. 2003; 23: 3352-3362Crossref PubMed Scopus (115) Google Scholar). Loss of Rpd3 leads to increased acetylation at target promoters and increased transcription of many genes (32Robyr D. Suka Y. Xenarios I. Kurdistani S.K. Wang A. Suka N. Grunstein M. Cell. 2002; 109: 437-446Abstract Full Text Full Text PDF PubMed Scopus (373) Google Scholar, 33Kurdistani S.K. Robyr D. Tavazoie S. Grunstein M. Nat. Genet. 2002; 31: 248-254Crossref PubMed Scopus (222) Google Scholar, 34Sabet N. Volo S. Yu C. Madigan J.P. Morse R.H. Mol. Cell. Biol. 2004; 24: 8823-8833Crossref PubMed Scopus (45) Google Scholar). In yeast and in metazoans, rpd3 mutants show defects in transcriptional regulation of cell cycle genes and firing of the origins of replication (35Aggarwal B.D. Calvi B.R. Nature. 2004; 430: 372-376Crossref PubMed Scopus (229) Google Scholar, 36Vogelauer M. Rubbi L. Lucas I. Brewer B.J. Grunstein M. Mol. Cell. 2002; 10: 1223-1233Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar, 37Aparicio J.G. Viggiani C.J. Gibson D.G. Aparicio O.M. Mol. Cell. Biol. 2004; 24: 4769-4780Crossref PubMed Scopus (139) Google Scholar). The Rpd3 protein can deacetylate histone H4 and H3 in vitro, and in mutant backgrounds increases in histone H3 and H4 acetylation is observed (38Rundlett S.E. Carmen A.A. Suka N. Turner B.M. Grunstein M. Nature. 1998; 392: 831-835Crossref PubMed Scopus (363) Google Scholar, 39Rundlett S.E. Carmen A.A. Kobayashi R. Bavykin S. Turner B.M. Grunstein M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14503-14508Crossref PubMed Scopus (517) Google Scholar, 40Kadosh D. Struhl K. Genes Dev. 1998; 12: 797-805Crossref PubMed Scopus (204) Google Scholar). The extent of histone deacetylation in yeast appears to be very local, extending over a range of 1-2 nucleosomes (41Kadosh D. Struhl K. Mol. Cell. Biol. 1998; 18: 5121-5127Crossref PubMed Scopus (267) Google Scholar). Rpd3 has also been suggested to function positively, suppressing the repressive effects of heterochromatin and in certain cases directly activating the transcription of specific genes, although in most cases this regulation is thought to be indirect (42de Nadal E. Zapater M. Alepuz P.M. Sumoy L. Mas G. Posas F. Nature. 2004; 427: 370-374Crossref PubMed Scopus (251) Google Scholar, 43De Rubertis F. Kadosh D. Henchoz S. Pauli D. Nature. 1996; 384: 589-591Crossref PubMed Scopus (194) Google Scholar, 44Robert F. Pokholok D.K. Hannett N.M. Rinaldi N.J. Chandy M. Rolfe A. Workman J.L. Gifford D.K. Young R.A. Mol. Cell. 2004; 16: 199-209Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). Unlike the nonlethal phenotype observed in yeast, homozygous null rpd3 mutations in Drosophila are lethal in the larval stage (45Mottus R. Sobel R.E. Grigliatti T.A. Genetics. 2000; 154: 657-668Crossref PubMed Google Scholar). Many aspects of pair rule gene regulation in the embryo that are under the control of gap repressor proteins such as Knirps are not affected by loss of Rpd3. This finding prompted Mannervik and Levine (46Mannervik M. Levine M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6797-6801Crossref PubMed Scopus (96) Google Scholar) to suggest that these short range repressors do not require Rpd3 for their activity. However, Rpd3 has been linked to a number of repressors active in the Drosophila embryo. Loss of rpd3 activity is observed to lead to misregulation of ftz and odd, genes regulated by the Even-skipped repressor, suggesting that Rpd3 might serve as a cofactor for Eve (46Mannervik M. Levine M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6797-6801Crossref PubMed Scopus (96) Google Scholar). In addition, the Runt repressor requires Rpd3 for repression of the engrailed gene during the later "maintenance" phase of repression (47Wheeler J.C. VanderZwan C. Xu X. Swantek D. Tracey W.D. Gergen J.P. Nat. Genet. 2002; 32: 206-210Crossref PubMed Scopus (45) Google Scholar). Biochemical studies showed that the long range repressor Hairy associates with Rpd3 through the Groucho corepressor (48Chen G. Courey A.J. Gene (Amst.). 2000; 249: 1-16Crossref PubMed Scopus (324) Google Scholar). Although rpd3 germ line mutants do not have the dramatic embryonic phenotypes of gro and hairy mutants, gro shows strong dosage effect interactions with rpd3, suggesting that Rpd3 protein may play a partially redundant role in the function of this cofactor (48Chen G. Courey A.J. Gene (Amst.). 2000; 249: 1-16Crossref PubMed Scopus (324) Google Scholar). Rpd3 is also found to copurify with a large complex containing the Polycomb group (PcG) gene products E(z) and Pcl, and misregulation in homeotic genes controlled by PcG proteins have been reported in an rpd3 mutant background (31Tie F. Prasad-Sinha J. Birve A. Rasmuson-Lestander A. Harte P.J. Mol. Cell. Biol. 2003; 23: 3352-3362Crossref PubMed Scopus (115) Google Scholar, 49Chang Y.L. Peng Y.H. Pan I.C. Sun D.S. King B. Huang D.H. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9730-9735Crossref PubMed Scopus (49) Google Scholar). These lines of evidence indicate that Rpd3 plays central roles in a number of repression activities in the Drosophila embryo. Work from our laboratory has underscored the role of multiple repression activities that Knirps and other short range repressors deploy in specific developmental settings (7Keller S.A. Mao Y. Struffi P. Margulies C. Yurk C.E. Anderson A.R. Amey R.L. Moore S. Ebels J.M. Foley K. Corado M. Arnosti D.N. Mol. Cell. Biol. 2000; 20: 7247-7258Crossref PubMed Scopus (38) Google Scholar, 9Struffi P. Corado M. Kulkarni M. Arnosti D.N. Development (Camb.). 2004; 131: 2419-2429Crossref PubMed Scopus (22) Google Scholar, 10Sutrias-Grau M. Arnosti D.N. Mol. Cell. Biol. 2004; 24: 5953-5966Crossref PubMed Scopus (27) Google Scholar, 50Strunk B. Struffi P. Wright K. Pabst B. Thomas J. Qin L. Arnosti D.N. Dev. Biol. 2001; 239: 229-240Crossref PubMed Scopus (29) Google Scholar). These repression activities respond to a complex cis regulatory grammar relating to binding site number, affinity, and arrangement that dictates how a repressor functions in specific contexts (12Clyde D.E. Corado M.S. Wu X. Pare A. Papatsenko D. Small S. Nature. 2003; 426: 849-853Crossref PubMed Scopus (157) Google Scholar, 51Kulkarni M.M. Arnosti D.N. Mol. Cell. Biol. 2005; 25: 3411-3420Crossref PubMed Scopus (55) Google Scholar). We currently lack a deeper understanding of the molecular mechanisms underlying these phenomena, however. Therefore, to understand better the activity of Knirps repression, we have undertaken a biochemical and genetic study of Knirps-associated proteins, and we find contrary to previous indications that the Rpd3 protein may play an important role in mediating Knirps repression activity. Transgenic Flies Carrying Inducible, Double-tagged Knirps Genes—Details on the generation of transgenic flies expressing either full-length Knirps-(1-429) or the N-terminal, CtBP-independent repression domain of Knirps-(1-330) were reported previously (9Struffi P. Corado M. Kulkarni M. Arnosti D.N. Development (Camb.). 2004; 131: 2419-2429Crossref PubMed Scopus (22) Google Scholar). Each protein is double-tagged, carrying an N-terminal hexahistidine tag and a C-terminal double FLAG tag, and is expressed under the control of the hsp70 promoter. Recombinant proteins are functional and can be expressed in embryos older than ∼2 h (see Ref. 9Struffi P. Corado M. Kulkarni M. Arnosti D.N. Development (Camb.). 2004; 131: 2419-2429Crossref PubMed Scopus (22) Google Scholar; data not shown). To induce expression of recombinant Knirps proteins, transgenic embryos collected on apple juice plates at room temperature (22-23 °C) were incubated for 30 min at 38 °C in a 10-liter water bath to ensure rapid and even heating. After induction, embryos were immediately dechorionated and sonicated within 15 min from the end of heat shock. Western Blotting Analysis—Immunoblotting was performed using a tank transfer system (Mini Trans-Blot® Cell; 170-3930, Bio-Rad) and Immun-Blot™ PVDF membranes (162-0177, Bio-Rad). Antibody incubation was in TBST (20 mm Tris-HCl, pH 7.5, 120 mm NaCl, 0.1% Tween 20) supplemented with 5% (w/v) nonfat dry milk as blocking agent. SuperSignal® West Pico chemiluminescent substrate (34080, Pierce) was used for detecting horseradish peroxidase (HRP) on immunoblots. FLAG M2 monoclonal antibody (F3165, Sigma) was used at a 1:10,000 dilution. Rabbit polyclonal antiserum against Drosophila Rpd3 (kindly provided by D. Wassarman, University of Wisconsin) (52Pile L.A. Wassarman D.A. EMBO J. 2000; 19: 6131-6140Crossref PubMed Scopus (71) Google Scholar) was used at 1:5,000 dilution. Rabbit polyclonal antiserum against Drosophila CtBP (dCtBP) was generated against full-length dCtBP and used at 1:20,000 dilution. To generate this antiserum, recombinant full-length, hexahistidine-tagged Drosophila CtBP (dCtBP-(1-479)) from pET15bCtBPL vector was expressed in Escherichia coli BL21-Codon-Plus™ RIL competent cells (230240, Stratagene) and purified on Ni-NTA-agarose beads (30210, Qiagen) according to the manufacturer's instructions. dCtBP protein (400 μg) in 0.2 ml of PBS buffer (1.9 mm NaH2PO4, 8.1 mm Na2HPO4, 154 mm NaCl, pH 7.2) was mixed with an equal volume of Titermax Gold adjuvant (T2684, Sigma) and injected subcutaneously at multiple sites in a New Zealand female rabbit. Two secondary boosts were performed similarly after 4 and 12 weeks. ImmunoPure® goat anti-mouse HRP-conjugated antibody (31430, Pierce) was used at 1:20,000 dilution. Goat anti-rabbit HRP-conjugated antibody (170-6515, Bio-Rad) was used at 1:10,000 dilution. Drosophila Embryo Nuclear Extract Preparation—0-12-h-old embryos from the hskni1-429.3 line were collected on grape juice plates from two population cages. For each extraction, two 0-12-h collections (the first one stored 12 h at 13 °C) were pooled together. 20-40 g of embryos were either dechorionated and processed immediately or transferred on a 155-mm Petri dish and floated on a 38 °C water bath for 60 min. Heat-shocked embryos were recovered for 30 min at room temperature, prior to dechorionation and homogenization. Drosophila standard nuclear extracts were made according to Soeller et al. (53Soeller W.C. Poole S.J. Kornberg T. Genes Dev. 1988; 2: 68-81Crossref PubMed Scopus (190) Google Scholar). Coimmunoprecipitation Experiments—200 μl of nuclear extracts (30 μg/μl of total protein) from embryos overexpressing full-length Knirps were incubated overnight at 4 °C with 4 μl (4.9 μg/μl) of α-FLAG M2 monoclonal antibody or an equivalent amount of mouse IgG antibody on a rotating wheel. 1 ml of washing buffer (150 mm NaCl, 50 mm Hepes, pH 7.9, 0.5 mm EDTA, 10% glycerol, 1 mm DTT, 1 mm PMSF, 1 mm sodium metabisulfite, 1 mm benzamidine, 10 μm pepstatin A) was added, and each sample was supplemented with 10 μl of pre-equilibrated protein G-agarose beads (16-266, Upstate) and incubated for 3 h at 4 °C on a rotator. Beads were washed for four times (10 min each time) with 1 ml of washing buffer, resuspended in Laemmli buffer, and boiled for 5 min at 95 °C. λ-Protein Phosphatase Treatment—Crude embryo lysates were prepared from embryos expressing full-length Knirps (hskni1-429.3) and subjected to treatment with increasing amounts of λ-phosphatase. For each reaction, 20 μl of crude embryo lysate (280 μg of total protein) from 2- to 4-h hskni1-429.3 embryos subjected to 30 min of heat shock was incubated with 0, 20, 80, 400, or 1600 units of λ-phosphatase (P0753S, New England Biolabs) in 1× λ-phosphatase buffer (50 mm Tris-HCl, pH 7.5, 0.1 mm Na2EDTA, 5 mm DTT, 0.01% Brij 35) supplemented with 2 mm MnCl2, in a final volume of 50 μl. To limit the activity of endogenous phosphatases, reactions were carried out at 4 °C for 1 h. Phosphatase activity was blocked by addition of 20 mm sodium orthovanadate (S-6508, Sigma). Reactions were stopped by the addition of Laemmli buffer followed immediately by incubation at 95 °C for 5 min. Proteins were resolved by 8% SDS-PAGE, and recombinant Knirps was detected by Western blot using anti-FLAG M2 antibody. Double Affinity Purification of Recombinant Knirps Proteins—0-12-h embryos were collected at room temperature (22-23 °C) and heat-shocked for 30 min at 38 °C as described. 2-4 grams of dechorionated embryos were resuspended in 40 ml of lysis buffer (150 mm NaCl, 50 mm Hepes, pH 7.9, 10% glycerol, 10 mm imidazole, 20 mm β-mercaptoethanol, 1 mm PMSF, 1 mm sodium metabisulfite, 1 mm benzamidine, 10 μm pepstatin A) and sonicated using a Branson-250 Sonifier (4 cycles, 20-30 pulses/cycle, output 6, duty cycle 60%, 3 min on ice between cycles, using a medium tip). Lysates were cleared by centrifugation (20 min at 27,000 × g), and 2 ml of washed and pre-equilibrated Ni-NTA-agarose beads (His Select™ HC Nickel, 6611, Sigma) were added to the supernatant. After 6 h at 4°C on a rotating wheel, beads were washed three times with 50 ml of lysis buffer supplemented with 20 mm imidazole and transferred to a 5-ml tube. Proteins were eluted twice using 3 ml of lysis buffer supplemented with 150 mm imidazole. The eluates were pooled and diluted to 50 ml with 150 mm NaCl, 50 mm Hepes, pH 7.9, 10% glycerol, 0.2 mm EDTA, 2 mm DTT, 1 mm sodium metabisulfite, 1 mm benzamidine, 10 μm pepstatin A, 1 mm PMSF. 200-300 μl of protein-G-agarose beads (16-266, Upstate) covalently coupled with anti-FLAG M2 antibody (2 mg of antibody per ml of wet beads) were added to the solution and incubated for 12-18 h at 4 °C on a rotating wheel. Beads were washed three times with 50 ml of the same buffer and transferred to a microcentrifuge tube, and proteins were eluted with 1.2 ml of buffer containing 0.2% N-lauroyl-sarkosine (L-5777, Sigma). Protein samples were trichloroacetic acid-precipitated using 4 mg/ml sodium deoxycholate (D-6750, Sigma) as carrier, resuspended in Laemmli buffer, and heated 5 min at 95 °C. For each purification experiment, one or two negative controls were used in parallel as follows: either heat-shocked, nontransgenic yellow, white67 (yw67) embryos or transgenic embryos from the hs-kni line that was not heat-shocked. Chromatographic Identification of the Knirps Complex—To determine the apparent molecular size of recombinant Knirps, whole-cell extracts from embryos expressing full-length Knirps were subjected to gel filtration chromatography. 0-12-h-old embryos from hskni1-429.3 were heat-shocked for 30 min, and a crude lysate was prepared essentially as described above, using a lysis buffer containing 100 mm NaCl, 50 mm Hepes, pH 7.9, 5% glycerol, 0.1 mm EDTA, 1 mm DTT, 1 mm sodium metabisulfite, 1 mm benzamidine, 10 μm pepstatin A, and 1 mm PMSF. Lysates were centrifuged at 27,000 × g for 20 min, and 300 μl of cleared lysate (6 mg of total protein) was loaded onto a pre-equilibrated Superdex-200 HR 10/30 column (17-1088-01, Amersham Biosciences) and eluted with 1.5 volumes of lysis buffer at the flow rate of 0.4 ml/min using the ÄKTAexplorer 100 system (Amersham Biosciences). Fractions (0.5 ml) were collected and analyzed by Western blot for the presence of recombinant Knirps, CtBP, and Rpd3. The same column was loaded with size markers (MW-GF-1000, Sigma) and run using identical conditions to determine in which fractions the different markers elute. Genetic Interaction between Knirps and rpd3—To test for a genetic interaction between kni and rpd3, transheterozygous flies for kni and rpd3 were generated, and the expression pattern of the Knirps target gene eve was monitored by in situ hybridization as described previously (9Struffi P. Corado M. Kulkarni M. Arnosti D.N. Development (Camb.). 2004; 131: 2419-2429Crossref PubMed Scopus (22) Google Scholar). kni9 (Bloomington stock 3332) carries a null mutation in Knirps and was previously used to test a genetic interaction between kni and CtBP (8Nibu Y. Zhang H. Levine M. Science. 1998; 280: 101-104Crossref PubMed Scopus (222) Google Scholar). kni7G (Tübingen stock number Z334) is a loss of function mutation caused by a point mutation (C48S) in the DNA binding domain (54Gerwin N. La R.A. Sauer F. Halbritter H.P. Neumann M. Jackle H. Nauber U. Mol. Cell. Biol. 1994; 12: 7899-7908Crossref Scopus (26) Google Scholar). rpd304556 (Bloomington stock 11633) is a strong hypomorphic mutation caused by a P-element insertion in the 5′-untranslated region of rpd3, resulting in a severe reduction in Rpd3 expression (46Mannervik M. Levine M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6797-6801Crossref PubMed Scopus (96) Google Scholar). rpd3def24 (kindly provided by S. Frankel) is a null allele caused by excision of a P-element, resulting in a deletion of ∼870 bp in rpd304556 in the 5′-coding region of the gene (45Mottus R. Sobel R.E. Grigliatti T.A. Genetics. 2000; 154: 657-668Crossref PubMed Google Scholar). Heterozygous phenotypes for each kni and rpd3 allele were noted after crossing balanced lines to a yw67 strain. Half of embryos from outcrosses to wild-type stocks are heterozygous, thus the observed frequency of eve stripe abnormalities in kni heterozygous embryos (14% for kni9 and 21% for kni7G) indicates that the penetrance is 28-42%. Expression and Phosphorylation of Recombinant Full-length Knirps in Drosophila
Referência(s)