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• W2091983770 abstract "The mixed lineage leukemia protein-1 (MLL1) belongs to the SET1 family of histone H3 lysine 4 methyltransferases. Recent studies indicate that the catalytic subunits of SET1 family members are regulated by interaction with a conserved core group of proteins that include the WD repeat protein-5 (WDR5), retinoblastoma-binding protein-5 (RbBP5), and the absent small homeotic-2-like protein (Ash2L). It has been suggested that WDR5 functions to bridge the interactions between the catalytic and regulatory subunits of SET1 family complexes. However, the molecular details of these interactions are unknown. To gain insight into the interactions among these proteins, we have determined the biophysical basis for the interaction between the human WDR5 and MLL1. Our studies reveal that WDR5 preferentially recognizes a previously unidentified and conserved arginine-containing motif, called the “Win” or WDR5 interaction motif, which is located in the N-SET region of MLL1 and other SET1 family members. Surprisingly, our structural and functional studies show that WDR5 recognizes arginine 3765 of the MLL1 Win motif using the same arginine binding pocket on WDR5 that was previously shown to bind histone H3. We demonstrate that WDR5's recognition of arginine 3765 of MLL1 is essential for the assembly and enzymatic activity of the MLL1 core complex in vitro. The mixed lineage leukemia protein-1 (MLL1) belongs to the SET1 family of histone H3 lysine 4 methyltransferases. Recent studies indicate that the catalytic subunits of SET1 family members are regulated by interaction with a conserved core group of proteins that include the WD repeat protein-5 (WDR5), retinoblastoma-binding protein-5 (RbBP5), and the absent small homeotic-2-like protein (Ash2L). It has been suggested that WDR5 functions to bridge the interactions between the catalytic and regulatory subunits of SET1 family complexes. However, the molecular details of these interactions are unknown. To gain insight into the interactions among these proteins, we have determined the biophysical basis for the interaction between the human WDR5 and MLL1. Our studies reveal that WDR5 preferentially recognizes a previously unidentified and conserved arginine-containing motif, called the “Win” or WDR5 interaction motif, which is located in the N-SET region of MLL1 and other SET1 family members. Surprisingly, our structural and functional studies show that WDR5 recognizes arginine 3765 of the MLL1 Win motif using the same arginine binding pocket on WDR5 that was previously shown to bind histone H3. We demonstrate that WDR5's recognition of arginine 3765 of MLL1 is essential for the assembly and enzymatic activity of the MLL1 core complex in vitro. Eukaryotes have evolved a complex system to regulate access to genomic information by packaging DNA into chromatin. Chromatin can adopt various levels of organization that regulate essential cellular activities such as transcription, DNA replication, recombination, and repair (1Jenuwein T. Allis C.D. Science. 2001; 293: 1074-1080Crossref PubMed Scopus (7538) Google Scholar). The fundamental repeating unit of chromatin is the nucleosome, in which 147 base pairs of genomic DNA is wrapped around a disc-shaped octamer of histone proteins H2A, H2B, H3, and H4 (2Luger K. Curr. Opin. Genet. Dev. 2003; 13: 127-135Crossref PubMed Scopus (229) Google Scholar, 3Kornberg R.D. Lorch Y. Cell. 1999; 98: 285-294Abstract Full Text Full Text PDF PubMed Scopus (1422) Google Scholar). Nucleosome positioning on DNA is fundamentally involved in controlling gene access and is regulated in part by a diverse array of enzymes that introduce covalent post-translational modifications on histone proteins (4Cosgrove M.S. Boeke J.D. Wolberger C. Nat. Struct. Mol. Biol. 2004; 11: 1037-1043Crossref PubMed Scopus (283) Google Scholar, 5Cosgrove M.S. Wolberger C. Biochem. Cell Biol. 2005; 83: 468-476Crossref PubMed Scopus (167) Google Scholar). An extensive array of histone modifications have been characterized, including lysine acetylation, lysine and arginine methylation, serine and threonine phosphorylation, and lysine ubiquitylation (6Cosgrove M.S. Expert Rev. Proteomics. 2007; 4: 465-478Crossref PubMed Scopus (61) Google Scholar, 7Peterson C.L. Laniel M.A. Curr. Biol. 2004; 14: R546-R551Abstract Full Text Full Text PDF PubMed Scopus (962) Google Scholar). The large number of potential histone modification patterns provides cells with an enormous combinatorial potential for the precise regulation of gene function. This potential is summarized by the histone code hypothesis (8Strahl B.D. Allis C.D. Nature. 2000; 403: 41-45Crossref PubMed Scopus (6507) Google Scholar, 9Turner B.M. BioEssays. 2000; 22: 836-845Crossref PubMed Scopus (954) Google Scholar, 10Fischle W. Wang Y. Allis C.D. Nature. 2003; 425: 475-479Crossref PubMed Scopus (541) Google Scholar), which predicts that patterns of histone modifications are recognized by specialized “effector” domains found in numerous chromatin regulators. It is thought that effector domains help recruit the activities of proteins that either stabilize or remodel specific chromatin states (5Cosgrove M.S. Wolberger C. Biochem. Cell Biol. 2005; 83: 468-476Crossref PubMed Scopus (167) Google Scholar, 11Taverna S.D. Li H. Ruthenburg A.J. Allis C.D. Patel D.J. Nat. Struct. Mol. Biol. 2007; 14: 1025-1040Crossref PubMed Scopus (1153) Google Scholar, 12Ruthenburg A.J. Li H. Patel D.J. Allis C.D. Nat. Rev. Mol. Cell Biol. 2007; 8: 983-994Crossref PubMed Scopus (811) Google Scholar).The combinatorial complexity of histone modifications is further increased by histone lysine residues that can be mono-, di-, or trimethylated, which are correlated with distinct functional outcomes. For example, chromatin immunoprecipitation experiments suggest that nucleosomes occupying the 5′ ends of active genes are trimethylated at H3 lysine 4 (H3K4me3), whereas the body of active genes tend to possess nucleosomes dimethylated at H3K4 (13Santos-Rosa H. Schneider R. Bannister A.J. Sherriff J. Bernstein B.E. Emre N.C. Schreiber S.L. Mellor J. Kouzarides T. Nature. 2002; 419: 407-411Crossref PubMed Scopus (1580) Google Scholar, 14Krogan N.J. Dover J. Wood A. Schneider J. Heidt J. Boateng M.A. Dean K. Ryan O.W. Golshani A. Johnston M. Greenblatt J.F. Shilatifard A. Mol. Cell. 2003; 11: 721-729Abstract Full Text Full Text PDF PubMed Scopus (565) Google Scholar, 15Ng H.H. Robert F. Young R.A. Struhl K. Mol. Cell. 2003; 11: 709-719Abstract Full Text Full Text PDF PubMed Scopus (852) Google Scholar, 16Bernstein B.E. Kamal M. Lindblad-Toh K. Bekiranov S. Bailey D.K. Huebert D.J. McMahon S. Karlsson E.K. Kulbokas III, E.J. Gingeras T.R. Schreiber S.L. Lander E.S. Cell. 2005; 120: 169-181Abstract Full Text Full Text PDF PubMed Scopus (1171) Google Scholar, 17Schubeler D. MacAlpine D.M. Scalzo D. Wirbelauer C. Kooperberg C. van Leeuwen F. Gottschling D.E. O'Neill L.P. Turner B.M. Delrow J. Bell S.P. Groudine M. Genes Dev. 2004; 18: 1263-1271Crossref PubMed Scopus (634) Google Scholar). H3K4me3 is thought to activate transcription through the recruitment of proteins that mobilize nucleosomes by ATP-dependent nucleosome-remodeling enzymes, such as CHD1 (18Sims III, R.J. Chen C.F. Santos-Rosa H. Kouzarides T. Patel S.S. Reinberg D. J. Biol. Chem. 2005; 280: 41789-41792Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar) or NURF (19Li H. Ilin S. Wang W. Duncan E.M. Wysocka J. Allis C.D. Patel D.J. Nature. 2006; 442: 91-95Crossref PubMed Scopus (181) Google Scholar). Several enzymes that regulate H3K4 methylation have been identified, all possessing the evolutionarily conserved SET domain, which is required for lysine methylation (20Rea S. Eisenhaber F. O'Carroll D. Strahl B.D. Sun Z.W. Schmid M. Opravil S. Mechtler K. Ponting C.P. Allis C.D. Jenuwein T. Nature. 2000; 406: 593-599Crossref PubMed Scopus (2152) Google Scholar, 21Dillon S.C. Zhang X. Trievel R.C. Cheng X. Genome Biol. 2005; 6: 227Crossref PubMed Scopus (547) Google Scholar). Members of the SET1 family of SET domain proteins assemble into multisubunit complexes that regulate mono-, di-, and trimethylation of H3K4. In budding yeast, evidence suggests that SET1p is the sole H3K4 methyltransferase (22Briggs S.D. Bryk M. Strahl B.D. Cheung W.L. Davie J.K. Dent S.Y. Winston F. Allis C.D. Genes Dev. 2001; 15: 3286-3295Crossref PubMed Scopus (473) Google Scholar), which exists in a multisubunit complex called COMPASS (23Roguev A. Schaft D. Shevchenko A. Pijnappel W.W. Wilm M. Aasland R. Stewart A.F. EMBO J. 2001; 20: 7137-7148Crossref PubMed Scopus (453) Google Scholar, 24Miller T. Krogan N.J. Dover J. Erdjument-Bromage H. Tempst P. Johnston M. Greenblatt J.F. Shilatifard A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 12902-12907Crossref PubMed Scopus (445) Google Scholar, 25Nagy P.L. Griesenbeck J. Kornberg R.D. Cleary M.L. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 90-94Crossref PubMed Scopus (263) Google Scholar). In contrast, humans encode at least six different SET1 family members: the mixed lineage leukemia proteins MLL1–4 and the SET1a and SET1b proteins (26Hughes C.M. Rozenblatt-Rosen O. Milne T.A. Copeland T.D. Levine S.S. Lee J.C. Hayes D.N. Shanmugam K.S. Bhattacharjee A. Biondi C.A. Kay G.F. Hayward N.K. Hess J.L. Meyerson M. Mol. Cell. 2004; 13: 587-597Abstract Full Text Full Text PDF PubMed Scopus (494) Google Scholar, 27Wysocka J. Myers M.P. Laherty C.D. Eisenman R.N. Herr W. Genes Dev. 2003; 17: 896-911Crossref PubMed Scopus (316) Google Scholar, 28Yokoyama A. Wang Z. Wysocka J. Sanyal M. Aufiero D.J. Kitabayashi I. Herr W. Cleary M.L. Mol. Cell Biol. 2004; 24: 5639-5649Crossref PubMed Scopus (528) Google Scholar, 29Milne T.A. Briggs S.D. Brock H.W. Martin M.E. Gibbs D. Allis C.D. Hess J.L. Mol. Cell. 2002; 10: 1107-1117Abstract Full Text Full Text PDF PubMed Scopus (860) Google Scholar, 30Steward M.M. Lee J.S. O'Donovan A. Wyatt M. Bernstein B.E. Shilatifard A. Nat. Struct. Mol. Biol. 2006; 13: 852-854Crossref PubMed Scopus (254) Google Scholar, 31Glaser S. Schaft J. Lubitz S. Vintersten K. van der Hoeven F. Tufteland K.R. Aasland R. Anastassiadis K. Ang S.L. Stewart A.F. Development (Camb.). 2006; 133: 1423-1432Crossref PubMed Scopus (224) Google Scholar, 32Lee J.H. Skalnik D.G. J. Biol. Chem. 2005; 280: 41725-41731Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, 33Lee J.H. Tate C.M. You J.S. Skalnik D.G. J. Biol. Chem. 2007; 282: 13419-13428Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar, 34Prasad R. Zhadanov A.B. Sedkov Y. Bullrich F. Druck T. Rallapalli R. Yano T. Alder H. Croce C.M. Huebner K. Mazo A. Canaani E. Oncogene. 1997; 15: 549-560Crossref PubMed Scopus (80) Google Scholar). The most studied human SET1 family member is MLL1 (ALL1, HRX, Htrx), which is required for the regulation of hox genes in hematopoiesis and development (29Milne T.A. Briggs S.D. Brock H.W. Martin M.E. Gibbs D. Allis C.D. Hess J.L. Mol. Cell. 2002; 10: 1107-1117Abstract Full Text Full Text PDF PubMed Scopus (860) Google Scholar, 35Terranova R. Agherbi H. Boned A. Meresse S. Djabali M. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 6629-6634Crossref PubMed Scopus (157) Google Scholar, 36Yu B.D. Hess J.L. Horning S.E. Brown G.A. Korsmeyer S.J. Nature. 1995; 378: 505-508Crossref PubMed Scopus (714) Google Scholar). MLL1 assembles into distinct multisubunit complexes that share several proteins that are conserved among SET1 family complexes from yeast to humans (28Yokoyama A. Wang Z. Wysocka J. Sanyal M. Aufiero D.J. Kitabayashi I. Herr W. Cleary M.L. Mol. Cell Biol. 2004; 24: 5639-5649Crossref PubMed Scopus (528) Google Scholar, 37Nakamura T. Mori T. Tada S. Krajewski W. Rozovskaia T. Wassell R. Dubois G. Mazo A. Croce C.M. Canaani E. Mol. Cell. 2002; 10: 1119-1128Abstract Full Text Full Text PDF PubMed Scopus (591) Google Scholar, 38Dou Y. Milne T.A. Tackett A.J. Smith E.R. Fukuda A. Wysocka J. Allis C.D. Chait B.T. Hess J.L. Roeder R.G. Cell. 2005; 121: 873-885Abstract Full Text Full Text PDF PubMed Scopus (522) Google Scholar).Recent evidence suggests that the enzymatic activity of SET1 family complexes is regulated by specific protein-protein interactions (30Steward M.M. Lee J.S. O'Donovan A. Wyatt M. Bernstein B.E. Shilatifard A. Nat. Struct. Mol. Biol. 2006; 13: 852-854Crossref PubMed Scopus (254) Google Scholar, 39Dou Y. Milne T.A. Ruthenburg A.J. Lee S. Lee J.W. Verdine G.L. Allis C.D. Roeder R.G. Nat. Struct. Mol. Biol. 2006; 13: 713-719Crossref PubMed Scopus (557) Google Scholar). For example, it was recently shown that the minimal complex required for di- and trimethylation of H3K4 includes MLL1, WDR5, RbBP5, and Ash2L, which together form the MLL1 core complex (39Dou Y. Milne T.A. Ruthenburg A.J. Lee S. Lee J.W. Verdine G.L. Allis C.D. Roeder R.G. Nat. Struct. Mol. Biol. 2006; 13: 713-719Crossref PubMed Scopus (557) Google Scholar). Evidence suggests that WDR5, RbBP5, and Ash2L form a stable subcomplex that is capable of interacting with the different members of the SET1 family of SET domain proteins (39Dou Y. Milne T.A. Ruthenburg A.J. Lee S. Lee J.W. Verdine G.L. Allis C.D. Roeder R.G. Nat. Struct. Mol. Biol. 2006; 13: 713-719Crossref PubMed Scopus (557) Google Scholar). The WD40 repeat protein WDR5 is critical for these interactions, as it has been shown to bridge the interactions between the catalytic SET domain of MLL1 and the RbBP5 and Ash2L regulatory components of the MLL1 core complex (39Dou Y. Milne T.A. Ruthenburg A.J. Lee S. Lee J.W. Verdine G.L. Allis C.D. Roeder R.G. Nat. Struct. Mol. Biol. 2006; 13: 713-719Crossref PubMed Scopus (557) Google Scholar). RNA interference knockdown of WDR5 in mammalian cells results in the global loss of H3K4me3 (39Dou Y. Milne T.A. Ruthenburg A.J. Lee S. Lee J.W. Verdine G.L. Allis C.D. Roeder R.G. Nat. Struct. Mol. Biol. 2006; 13: 713-719Crossref PubMed Scopus (557) Google Scholar, 40Wysocka J. Swigut T. Milne T.A. Dou Y. Zhang X. Burlingame A.L. Roeder R.G. Brivanlou A.H. Allis C.D. Cell. 2005; 121: 859-872Abstract Full Text Full Text PDF PubMed Scopus (640) Google Scholar), which is correlated with decreased expression of Hox genes and defects in hematopoiesis and development (40Wysocka J. Swigut T. Milne T.A. Dou Y. Zhang X. Burlingame A.L. Roeder R.G. Brivanlou A.H. Allis C.D. Cell. 2005; 121: 859-872Abstract Full Text Full Text PDF PubMed Scopus (640) Google Scholar). Similar phenotypes are observed in MLL1–/– and MLL1ΔSET mice (35Terranova R. Agherbi H. Boned A. Meresse S. Djabali M. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 6629-6634Crossref PubMed Scopus (157) Google Scholar, 36Yu B.D. Hess J.L. Horning S.E. Brown G.A. Korsmeyer S.J. Nature. 1995; 378: 505-508Crossref PubMed Scopus (714) Google Scholar), suggesting that WDR5 and MLL1 function together to regulate Hox gene expression in vivo (41Ruthenburg A.J. Wang W. Graybosch D.M. Li H. Allis C.D. Patel D.J. Verdine G.L. Nat. Struct. Mol. Biol. 2006; 13: 704-712Crossref PubMed Scopus (186) Google Scholar). In this and the accompanying investigation (58Patel A. Dharmarajan V. Cosgrove M.S. J. Biol. Chem. 2008; 283: 32158-32161Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar), we identify and characterize a highly conserved WDR5 interaction motif, or “Win” motif, which is located in the N-SET region of MLL1 and other SET1 family members. In addition, we demonstrate that the Win motif is critical for the assembly and enzymatic activity of the MLL1 core complex in vitro.EXPERIMENTAL PROCEDURESProtein Expression and Purification—A human spleen c-DNA library was used to amplify an MLL1 C-terminal fragment encoding residues 3592–3969. PCR subcloning was used to amplify MLL1 SET domain constructs consisting of residues 3745–3969 (MLL3745) and residues 3811–3969 (MLL3811). MLL3745 and MLL3811 were ligated into the pGST and pMBP parallel vectors, respectively (42Sheffield P. Garrard S. Derewenda Z. Protein Expression Purif. 1999; 15: 34-39Crossref PubMed Scopus (525) Google Scholar). pMCGS7 plasmids encoding full-length WDR5 as N-terminal His6 fusions were obtained as generous gifts from Alexander Ruthenburg and David Allis. A plasmid containing the RbBP5 clone was obtained as a generous gift from Yali Dou and was subcloned into the pET3a vector without affinity tags. The ash2L gene (Clone ID 3921999) was purchased from Open Biosystems and subcloned into the pHis Parallel vector, which encodes tobacco etch virus (TEV) 3The abbreviations used are: TEV, tobacco etch virus; MALDI-TOF, matrix-assisted laser desorption/ionization-time of flight; TCEP, tris(2-carboxyethyl)phosphine. cleavable N-terminal His6 fusion.All recombinant proteins were overexpressed in Escherichia coli (Rosetta II, Novagen) by growing cells containing the plasmids at 37 °C in Terrific Broth medium containing 50 μg/ml carbenicillin to an A600 of 1.0. The temperature was then lowered to 16 °C, and cells were induced with isopropyl-1-thio-β-d-galactopyranoside (0.1–1 mm) for 16–18 h. RBBP5 and Ash2L were induced with 0.1 mm and 0.25 mm isopropyl-1-thio-β-d-galactopyranoside, respectively. Cells were harvested, resuspended in lysis buffer (50 mm Tris, pH 7.3, 300 mm NaCl, 10% glycerol, 3 mm dithiothreitol, 0.1 mm phenylmethylsulfonyl fluoride, and EDTA-free protease inhibitor mixture (Roche Applied Science)), lysed with a microfluidizer cell disrupter, and clarified by centrifugation. Cells containing the RbBP5 plasmid were lysed instead using the same lysis buffer containing 1× BugBuster® (Novagen), which minimized RbBP5 degradation upon cell lysis. Clarified supernatants containing the GST-MLL3745 protein were passed over a glutathione-Sepharose column (GSTrap™ FF column, GE-Healthcare), and GST-MLL3745 was eluted with a gradient of reduced glutathione. Fractions containing GST-MLL3745 were combined, treated with TEV protease, and dialyzed with three changes against lysis buffer (without protease inhibitors). MLL3745 was further purified over a glutathione-Sepharose column followed by gel filtration chromatography. MBP-MLL3811 fusion protein was purified by amylose affinity chromatography followed by TEV protease treatment and two rounds of gel filtration chromatography. Full-length WDR5 protein was expressed and purified as described previously (43Couture, J., Collazo, E., and Trievel, R. C. (2006) Nat. Struct. Mol. Biol.Google Scholar). RbBP5 protein was purified by ion exchange and gel filtration chromatography. Ash2L was purified by nickel affinity chromatography (HisTrap column, GE Healthcare), dialysis and TEV cleavage to remove imidazole and the His6 tag, and repurification over a His-trap column. As a final step of purification and for buffer exchange, all proteins were passed through a gel filtration column (Superdex 200, GE Healthcare) pre-equilibrated with 20 mm Tris (pH 7.5), 300 mm NaCl, 1 mm TCEP, and 1 μm ZnCl2.Mutagenesis and Peptides—Point mutations were introduced into MLL1 and WDR5 constructs using the QuikChange site-directed mutagenesis kit (Stratagene). Plasmids were sequenced to verify the presence of the intended mutations and the absence of additional mutations. Peptides were synthesized by Global Peptide and Genscript.Analytical Ultracentrifugation—Analytical ultracentrifugation experiments were carried out using a Beckman Coulter ProteomeLab™ XL-A analytical ultracentrifuge equipped with absorbance optics and an eight-hole An-50 Ti analytical rotor. Sedimentation velocity experiments were carried out at 10 °C and 50,000 rpm (200,000 × g) using 3-mm two-sector charcoal-filled Epon centerpieces with quartz windows. Each sample was scanned at 0-min time intervals for 300 scans. Protein samples in 20 mm Tris-Cl, pH 7.5, 300 mm NaCl, 1 mm TCEP, and 1 μm ZnCl2 were run at various concentrations, and molar ratios were as described under “Results.” Sedimentation boundaries were analyzed by the continuous distribution (c(s)) method using the program SEDFIT (44Schuck P. Biophys. J. 2000; 78: 1606-1619Abstract Full Text Full Text PDF PubMed Scopus (3005) Google Scholar). Equilibrium dissociation constants for WDR5-MLL1 complexes were obtained by global fitting sedimentation velocity data acquired at several different protein concentrations using the single-site hetero-association model (A + B ↔ AB) of SEDPHAT (45Schuck P. Scott D.J. Harding S.E. Rowe A.J. Analytical Ultracentrifugation: Techniques and Methods. Royal Society of Chemistry, Cambridge, UK2005: 26-50Google Scholar, 46Dam J. Velikovsky C.A. Mariuzza R.A. Urbanke C. Schuck P. Biophys. J. 2005; 89: 619-634Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). The program SEDNTERP, version 1.09 (47Laue T.M. Shah B. Ridgeway T.M. Pelletier S.L. Harding S.E. Rowe A.J. Horton L.C. Analytical Ultracentrifugation in Biochemistry and Polymer Science. Royal Society of Chemistry, Cambridge1992: 90-125Google Scholar), was used to correct the experimental s value (s*) to standard conditions at 20 °C in water (s20,w) and to calculate the partial specific volume of each protein.Methyltransferase Assays—Radiolabeling assays were conducted by combining 7 μg of MLL3745 or MLL3811 with 500 μm histone H3 peptide containing residues 1–20 (with GGK-biotin on the C terminus) and 1 μCi of [3H]methyl-S-adenosyl-methionine (GE Healthcare) in 50 mm Tris, pH 8.5, 200 mm NaCl, 3 mm dithiothreitol, 5 mm MgCl2, and 5% glycerol. The reactions were incubated at 15 °C for 2 h, stopped by the addition of SDS-loading buffer to 1×, and separated by SDS-PAGE on a 4–12% gradient gel (Invitrogen). The gel then was soaked in an autoradiography enhancer solution (Enlightning, PerkinElmer Life Sciences), dried, and exposed to film at −80 °C for 24 h.Mass spectrometry assays were conducted by adapting that previously reported for Dim5 and SET7/9 histone methyltransferases (48Zhang X. Yang Z. Khan S.I. Horton J.R. Tamaru H. Selker E.U. Cheng X. Mol. Cell. 2003; 12: 177-185Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar). Six micrograms of MLL3745 or MLL1 core complex was incubated with 250 μm s-adenosyl-methionine and 10 μm histone H3 peptide (amino acid residues 1–20) at 15 °C in 50 mm Tris-Cl, pH 9.0, 200 mm NaCl, 3 mm dithiothreitol, and 5% glycerol. The reactions were quenched at various time points by the addition of trifluoroacetic acid to 0.5%. The quenched samples were diluted 1:4 with α-cyano-4-hydroxycinnamic acid. MALDI-TOF mass spectrometry was performed on a Bruker AutoFlex mass spectrometer (State University of New York (SUNY), Oswego, NY) operated in reflectron mode. Final spectra were averaged from 100 shots/position at 10 different positions.Circular Dichroism Spectroscopy—CD spectra were collected on an AVIV 62A DS spectropolarimeter equipped with a Neslab CFT-33 refrigerated circulator at 10 °C using a 0.1-mm path length cell. Spectra for each protein at a concentration of 0.2 mg/ml were collected in a buffer containing 10 mm Tris (pH 7.5), 200 mm NaCl, 1 mm TCEP, and 1 μm ZnCl2. The background contribution of the buffer alone was subtracted from each protein spectrum.RESULTSWDR5 Forms a Stable Complex with the N-SET Region of MLL1—MLL1 is a large protein of 3,969 residues containing several conserved domains with functions implicated in transcriptional regulation (49Rasio D. Schichman S.A. Negrini M. Canaani E. Croce C.M. Cancer Res. 1996; 56: 1766-1769PubMed Google Scholar) (Fig. 1). Previous efforts to map the interaction region between MLL1 and WDR5 led to the identification of three distinct WDR5 interaction regions in MLL1 (38Dou Y. Milne T.A. Tackett A.J. Smith E.R. Fukuda A. Wysocka J. Allis C.D. Chait B.T. Hess J.L. Roeder R.G. Cell. 2005; 121: 873-885Abstract Full Text Full Text PDF PubMed Scopus (522) Google Scholar). The most C-terminal MLL1 fragment that interacts with WDR5 includes residues 3301–3969, which includes the N-SET region and the evolutionarily conserved ∼130-amino-acid SET domain at the extreme C terminus. The location of the WDR5 binding site within this region is unknown. Previous immunoprecipitation experiments showed that deletion of the last 149 amino acid residues spanning the MLL1 SET domain prevents co-immunoprecipitation of WDR5, RbBP5, and Ash2L with MLL1 (28Yokoyama A. Wang Z. Wysocka J. Sanyal M. Aufiero D.J. Kitabayashi I. Herr W. Cleary M.L. Mol. Cell Biol. 2004; 24: 5639-5649Crossref PubMed Scopus (528) Google Scholar), suggesting that the conserved SET domain of MLL1 is important for the interaction with the subcomplex. In contrast, domain-mapping experiments with the human SET1a protein suggest that ∼120 amino acid residues in the N-SET region are required for co-immunoprecipitation of WDR5, RbBP5, and Ash2L (33Lee J.H. Tate C.M. You J.S. Skalnik D.G. J. Biol. Chem. 2007; 282: 13419-13428Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). A similar region of the MLL3 protein was shown to interact with WDR5 in GST pulldown experiments (50Cho Y.W. Hong T. Hong S. Guo H. Yu H. Kim D. Guszczynski T. Dressler G.R. Copeland T.D. Kalkum M. Ge K. J. Biol. Chem. 2007; 282: 20395-20406Abstract Full Text Full Text PDF PubMed Scopus (406) Google Scholar). These results suggest that the N-SET region of MLL1 may be recognized by the WDR5 component of the MLL1 core complex. To test this hypothesis, we performed sedimentation velocity analytical ultracentrifugation experiments to compare the hydrodynamic properties of two highly pure MLL1 SET domain constructs in the presence and absence of WDR5.The first MLL1 SET domain construct consisted of residues 3745–3969 of human MLL1 (hereafter referred to as MLL3745), which was shown in previous studies to be the minimal domain required for interaction with the Ini1 tumor suppressor (51Rozenblatt-Rosen O. Rozovskaia T. Burakov D. Sedkov Y. Tillib S. Blechman J. Nakamura T. Croce C.M. Mazo A. Canaani E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4152-4157Crossref PubMed Scopus (213) Google Scholar) and for MLL1 SET domain self-association (52Rozovskaia T. Rozenblatt-Rosen O. Sedkov Y. Burakov D. Yano T. Nakamura T. Petruck S. Ben-Simchon L. Croce C.M. Mazo A. Canaani E. Oncogene. 2000; 19: 351-357Crossref PubMed Scopus (40) Google Scholar). This construct contains 66 amino acid residues of the N-SET region followed by the conserved SET and post-SET domains (Fig. 1a). We show in a separate investigation that MLL3745 is the minimal MLL SET domain construct required for the assembly of the full MLL1 core complex in vitro. 4A. Patel, V. E. Vought, V. Dharmarajan, and M. S. Cosgrove, manuscript in preparation. In the second MLL1 SET domain construct, the N-terminal residues encompassing the N-SET region were deleted. This construct consists of residues 3811–3969 (MLL3811) and encompasses only the conserved SET and post-SET domains (Fig. 1a). Both constructs are catalytically active in methyltransferase assays with histone H3 peptides encompassing residues 1–20 (Fig. 1b).Sedimentation velocity analyses were first performed with each of the proteins individually and were fitted to a distribution of Lamm equation solutions to determine the diffusion-free sedimentation coefficient distribution (c(s)) (44Schuck P. Biophys. J. 2000; 78: 1606-1619Abstract Full Text Full Text PDF PubMed Scopus (3005) Google Scholar). Fig. 2, a–c, shows that MLL3745, MLL3811, and WDR5 are monodisperse in solution with experimental s* values independent of protein concentration (not shown), suggesting that each is predominantly monomeric over the concentration range used. 5It was previously shown by yeast two-hybrid and GST pulldown experiments that MLL3745 self-associates (52Rozovskaia T. Rozenblatt-Rosen O. Sedkov Y. Burakov D. Yano T. Nakamura T. Petruck S. Ben-Simchon L. Croce C.M. Mazo A. Canaani E. Oncogene. 2000; 19: 351-357Crossref PubMed Scopus (40) Google Scholar). However, we could not detect self-association MLL3745 in this investigation by sedimentation velocity analysis. This is probably because the limited solubility of MLL3745 in the absence of interacting proteins prevents performing sedimentation velocity experiments at a high enough concentration to observe significant self-association. In support of this, control sedimentation equilibrium experiments show that MLL3745 weakly self-associates with a Kd of 12.7 μm (A. Patel and M. S. Cosgrove, unpublished data). When run individually, MLL3745, MLL3811, and WDR5 sediment with s* values of 1.68 ± 0.03 (1.76 s20,w), 1.31 ± 0.02 (1.38 s20,w), and 2.28 ± 0.05 (2.41 s20,w), respectively. The molecular masses calculated from these s20,w values are within error (<3%), similar to that of the expected masses for each protein (Table 1).FIGURE 2Sedimentation velocity analyses of WDR5 and recombinant MLL1 constructs. a–c, diffusion-free sedimentation coefficient distributions c(s) derived from sedimentation velocity data of MLL3745 (a), MLL3811 (b), and WDR5 (c). Each protein was measured at a concentration of 15 μm. d, c(s) distribution of sedimentation velocity data of MLL3745-WDR5 complex at the following concentrations: 7 μm (solid black line), 3.5 μm (dashed line), and 1.5 μm (dotted line). e, upper panel, direct fitting (solid black line) of sedimentation velocity boundary data (open circles) using a hetero-association model (A+B ↔ AB) in the program SEDPHAT. Lower panel, residuals are plotted as a function of radius. f, c(s) distribution from the sedimentation velocity analysis of MLL3811 and WDR5 each at a concentration of 7 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)TABLE 1Summary of sedimentation coefficients derived from sedimentation velocity analysesProteins*aExperimental sedimentation coefficients (s*) ± standard error of measurement determine from three independent expe" @default.
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• W2091983770 title "A Conserved Arginine-containing Motif Crucial for the Assembly and Enzymatic Activity of the Mixed Lineage Leukemia Protein-1 Core Complex" @default.
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