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• W2012606436 abstract "Transcription in eukaryotic genomes depends on enzymes that regulate the degree of histone H3 lysine 4 (H3K4) methylation. The mixed lineage leukemia protein-1 (MLL1) is a member of the SET1 family of H3K4 methyltransferases and is frequently rearranged in acute leukemias. Despite sequence comparisons that predict that SET1 family enzymes should only monomethylate their substrates, mono-, di-, and trimethylation of H3K4 has been attributed to SET1 family complexes in vivo and in vitro. To better understand this paradox, we have biochemically reconstituted and characterized a five-component 200-kDa MLL1 core complex containing human MLL1, WDR5, RbBP5, Ash2L, and DPY-30. We demonstrate that the isolated MLL1 SET domain is a slow monomethyltransferase and that tyrosine 3942 of MLL1 prevents di- and trimethylation of H3K4. In contrast, a complex containing the MLL1 SET domain, WDR5, RbBP5, Ash2L, and DPY-30, displays a marked ∼600-fold increase in enzymatic activity but only to the dimethyl form of H3K4. Single turnover kinetic experiments reveal that the reaction leading to H3K4 dimethylation involves the transient accumulation of a monomethylated species, suggesting that the MLL1 core complex uses a non-processive mechanism to catalyze multiple lysine methylation. We have also discovered that the non-SET domain components of the MLL1 core complex possess a previously unrecognized methyltransferase activity that catalyzes H3K4 dimethylation within the MLL1 core complex. Our results suggest that the mechanism of multiple lysine methylation by the MLL1 core complex involves the sequential addition of two methyl groups at two distinct active sites within the complex. Transcription in eukaryotic genomes depends on enzymes that regulate the degree of histone H3 lysine 4 (H3K4) methylation. The mixed lineage leukemia protein-1 (MLL1) is a member of the SET1 family of H3K4 methyltransferases and is frequently rearranged in acute leukemias. Despite sequence comparisons that predict that SET1 family enzymes should only monomethylate their substrates, mono-, di-, and trimethylation of H3K4 has been attributed to SET1 family complexes in vivo and in vitro. To better understand this paradox, we have biochemically reconstituted and characterized a five-component 200-kDa MLL1 core complex containing human MLL1, WDR5, RbBP5, Ash2L, and DPY-30. We demonstrate that the isolated MLL1 SET domain is a slow monomethyltransferase and that tyrosine 3942 of MLL1 prevents di- and trimethylation of H3K4. In contrast, a complex containing the MLL1 SET domain, WDR5, RbBP5, Ash2L, and DPY-30, displays a marked ∼600-fold increase in enzymatic activity but only to the dimethyl form of H3K4. Single turnover kinetic experiments reveal that the reaction leading to H3K4 dimethylation involves the transient accumulation of a monomethylated species, suggesting that the MLL1 core complex uses a non-processive mechanism to catalyze multiple lysine methylation. We have also discovered that the non-SET domain components of the MLL1 core complex possess a previously unrecognized methyltransferase activity that catalyzes H3K4 dimethylation within the MLL1 core complex. Our results suggest that the mechanism of multiple lysine methylation by the MLL1 core complex involves the sequential addition of two methyl groups at two distinct active sites within the complex. Lysine methylation of histones is an important epigenetic indexing system for transcriptionally active and inactive chromatin domains in eukaryotic genomes (1.Litt M.D. Simpson M. Gaszner M. Allis C.D. Felsenfeld G. Science. 2001; 293: 2453-2455Crossref PubMed Scopus (521) Google Scholar). Lysine residues can be mono-, di-, or trimethylated at the ϵ-amino group, with each state correlating with a distinct functional outcome (2.Santos-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 (1608) Google Scholar). For example, trimethylation of histone H3 lysine 4 (H3K4me3) is enriched at the 5′ ends of actively transcribed genes in a wide range of eukaryotes (3.Liu C.L. Kaplan T. Kim M. Buratowski S. Schreiber S.L. Friedman N. Rando O.J. PLoS Biol. 2005; 3: e328Crossref PubMed Scopus (401) Google Scholar, 4.Pokholok D.K. Harbison C.T. Levine S. Cole M. Hannett N.M. Lee T.I. Bell G.W. Walker K. Rolfe P.A. Herbolsheimer E. Zeitlinger J. Lewitter F. Gifford D.K. Young R.A. Cell. 2005; 122: 517-527Abstract Full Text Full Text PDF PubMed Scopus (1105) Google Scholar) and is thought to regulate transcription through the recruitment of proteins that activate (5.Wysocka J. Swigut T. Xiao H. Milne T.A. Kwon S.Y. Landry J. Kauer M. Tackett A.J. Chait B.T. Badenhorst P. Wu C. Allis C.D. Nature. 2006; 442: 86-90Crossref PubMed Scopus (891) Google Scholar, 6.Santos-Rosa H. Schneider R. Bernstein B.E. Karabetsou N. Morillon A. Weise C. Schreiber S.L. Mellor J. Kouzarides T. Mol. Cell. 2003; 12: 1325-1332Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar, 7.Pray-Grant M.G. Daniel J.A. Schieltz D. Yates 3rd, J.R. Grant P.A. Nature. 2005; 433: 434-438Crossref PubMed Scopus (409) Google Scholar) or repress transcription (8.Shi X. Hong T. Walter K.L. Ewalt M. Michishita E. Hung T. Carney D. Peña P. Lan F. Kaadige M.R. Lacoste N. Cayrou C. Davrazou F. Saha A. Cairns B.R. Ayer D.E. Kutateladze T.G. Shi Y. Côté J. Chua K.F. Gozani O. Nature. 2006; 442: 96-99Crossref PubMed Scopus (2) Google Scholar). In contrast, H3K4 monomethylation (H3K4me1) is enriched in nucleosomes at the 3′-ends of genes (2.Santos-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 (1608) Google Scholar, 4.Pokholok D.K. Harbison C.T. Levine S. Cole M. Hannett N.M. Lee T.I. Bell G.W. Walker K. Rolfe P.A. Herbolsheimer E. Zeitlinger J. Lewitter F. Gifford D.K. Young R.A. Cell. 2005; 122: 517-527Abstract Full Text Full Text PDF PubMed Scopus (1105) Google Scholar, 9.Ng H.H. Robert F. Young R.A. Struhl K. Mol. Cell. 2003; 11: 709-719Abstract Full Text Full Text PDF PubMed Scopus (857) Google Scholar) or in the distal enhancer sequences of active genes (10.Heintzman N.D. Stuart R.K. Hon G. Fu Y. Ching C.W. Hawkins R.D. Barrera L.O. Van Calcar S. Qu C. Ching K.A. Wang W. Weng Z. Green R.D. Crawford G.E. Ren B. Nat. Genet. 2007; 39: 311-318Crossref PubMed Scopus (2453) Google Scholar). In addition, H3K4 monomethylation in Saccharomyces cerevisiae (11.Nislow C. Ray E. Pillus L. Mol. Biol. Cell. 1997; 8: 2421-2436Crossref PubMed Scopus (202) Google Scholar, 12.Briggs 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 (481) Google Scholar, 13.Schneider J. Wood A. Lee J.S. Schuster R. Dueker J. Maguire C. Swanson S.K. Florens L. Washburn M.P. Shilatifard A. Mol. Cell. 2005; 19: 849-856Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar) and Chlamydomonas reinhardtii (14.van Dijk K. Marley K.E. Jeong B.R. Xu J. Hesson J. Cerny R.L. Waterborg J.H. Cerutti H. Plant Cell. 2005; 17: 2439-2453Crossref PubMed Scopus (68) Google Scholar) is associated with gene silencing. These results suggest that distinct strategies have evolved to regulate the degree of H3K4 methylation in eukaryotic genomes. Although numerous histone lysine methyltransferases and demethylases have been identified in recent years (15.Agger K. Christensen J. Cloos P.A. Helin K. Curr. Opin. Genet. Dev. 2008; 18: 159-168Crossref PubMed Scopus (186) Google Scholar, 16.Dillon S.C. Zhang X. Trievel R.C. Cheng X. Genome Biol. 2005; 6: 227Crossref PubMed Scopus (567) Google Scholar), relatively little is understood about how the different states of lysine methylation are achieved and regulated. The majority of histone lysine methyltransferases that have been identified share a conserved SET domain motif, named for its presence in diverse Drosophila chromatin regulators: SU(VAR)3–9, E(z), and Trx (17.Jones R.S. Gelbart W.M. Mol. Cell. Biol. 1993; 13: 6357-6366Crossref PubMed Scopus (202) Google Scholar, 18.Stassen M.J. Bailey D. Nelson S. Chinwalla V. Harte P.J. Mech. Dev. 1995; 52: 209-223Crossref PubMed Scopus (116) Google Scholar, 19.Tschiersch B. Hofmann A. Krauss V. Dorn R. Korge G. Reuter G. EMBO J. 1994; 13: 3822-3831Crossref PubMed Scopus (475) Google Scholar). SET domain proteins can be classified into different families based on sequence similarities, substrate specificity, and other structural features and include the SUV39, SET1, SET2, E(z), RIZ, SMYD, and SUV2–20 families (20.Kouzarides T. Curr. Opin. Genet. Dev. 2002; 12: 198-209Crossref PubMed Scopus (748) Google Scholar). It has been suggested that SET1p, the founding member of SET1 class of SET domain proteins, is the sole H3K4 methyltransferase in S. cerevisiae (12.Briggs 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 (481) Google Scholar), and its deletion results in defects in growth, transcriptional silencing, and telomere maintenance (11.Nislow C. Ray E. Pillus L. Mol. Biol. Cell. 1997; 8: 2421-2436Crossref PubMed Scopus (202) Google Scholar, 12.Briggs 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 (481) Google Scholar). In mammals, six SET1 family members have been identified: SET1a and SET1b (21.Lee J.H. Skalnik D.G. J. Biol. Chem. 2005; 280: 41725-41731Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar, 22.Lee 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 (187) Google Scholar) and four mixed lineage leukemia (MLL) 2The abbreviations used are: MLLmixed lineage leukemia proteinAdoMetS-adenosyl methionineMALDI-TOFmatrix-assisted laser desorption/ionization-time of flight. family H3K4 methyltransferases, MLL1, MLL2, MLL3, and MLL4 (23.Nakamura 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 (603) Google Scholar, 24.Dou 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 (533) Google Scholar, 25.Hughes 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 (509) Google Scholar, 26.Goo Y.H. Sohn Y.C. Kim D.H. Kim S.W. Kang M.J. Jung D.J. Kwak E. Barlev N.A. Berger S.L. Chow V.T. Roeder R.G. Azorsa D.O. Meltzer P.S. Suh P.G. Song E.J. Lee K.J. Lee Y.C. Lee J.W. Mol. Cell. Biol. 2003; 23: 140-149Crossref PubMed Scopus (188) Google Scholar, 27.Wysocka J. Myers M.P. Laherty C.D. Eisenman R.N. Herr W. Genes Dev. 2003; 17: 896-911Crossref PubMed Scopus (321) Google Scholar). Evidence suggests that the enzymatic activity of SET1 family members is regulated by interaction with a conserved subcomplex of proteins that include WDR5, RbBP5, and Ash2L (28.Dou 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 (572) Google Scholar, 29.Steward 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 (263) Google Scholar, 30.Cho 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 (419) Google Scholar). The best characterized mammalian SET1 family member is MLL1 (also known as ALL1, HRX, and Htrx), which has been shown to be important for the maintenance of HOX gene expression patterns in hematopoiesis and development (24.Dou 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 (533) Google Scholar, 31.Milne T.A. Dou Y. Martin M.E. Brock H.W. Roeder R.G. Hess J.L. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 14765-14770Crossref PubMed Scopus (169) Google Scholar, 32.Milne 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 (872) Google Scholar, 33.Yu B.D. Hess J.L. Horning S.E. Brown G.A. Korsmeyer S.J. Nature. 1995; 378: 505-508Crossref PubMed Scopus (727) Google Scholar, 34.Terranova R. Agherbi H. Boned A. Meresse S. Djabali M. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 6629-6634Crossref PubMed Scopus (159) Google Scholar). The C-terminal SET domain is responsible for the H3K4 methylation activity of MLL1, which is thought to be a general mechanism for MLL1-mediated transcriptional regulation (32.Milne 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 (872) Google Scholar). mixed lineage leukemia protein S-adenosyl methionine matrix-assisted laser desorption/ionization-time of flight. SET domain proteins differ in their ability to utilize mono- and dimethylated lysine side chains as substrates for further methylation, a phenomenon known as product specificity (35.Cheng X. Zhang X. Mutat. Res. 2007; 618: 102-115Crossref PubMed Scopus (57) Google Scholar). Structure-function studies suggest that product specificity is determined by the presence of a tyrosine or phenylalanine at a key position in the SET domain active site, called the “Phe/Tyr switch” position (36.Qian C. Wang X. Manzur K. Sachchidanand Farooq A. Zeng L. Wang R. Zhou M.M. J. Mol. Biol. 2006; 359: 86-96Crossref PubMed Scopus (50) Google Scholar, 37.Trievel R.C. Flynn E.M. Houtz R.L. Hurley J.H. Nat. Struct. Biol. 2003; 10: 545-552Crossref PubMed Scopus (102) Google Scholar, 38.Xiao B. Jing C. Kelly G. Walker P.A. Muskett F.W. Frenkiel T.A. Martin S.R. Sarma K. Reinberg D. Gamblin S.J. Wilson J.R. Genes Dev. 2005; 19: 1444-1454Crossref PubMed Scopus (155) Google Scholar, 39.Xiao B. Jing C. Wilson J.R. Walker P.A. Vasisht N. Kelly G. Howell S. Taylor I.A. Blackburn G.M. Gamblin S.J. Nature. 2003; 421: 652-656Crossref PubMed Scopus (306) Google Scholar, 40.Zhang 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 (274) Google Scholar). SET domain enzymes with a phenylalanine at the switch position, like Dim5 and G9a (40.Zhang 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 (274) Google Scholar, 41.Rice J.C. Briggs S.D. Ueberheide B. Barber C.M. Shabanowitz J. Hunt D.F. Shinkai Y. Allis C.D. Mol. Cell. 2003; 12: 1591-1598Abstract Full Text Full Text PDF PubMed Scopus (635) Google Scholar), have larger active site volumes that can accommodate the side chain rotation required for the processive addition of more than one methyl group. In contrast, SET domain enzymes with a tyrosine at the switch position, like SET7/9 and SET8 (39.Xiao B. Jing C. Wilson J.R. Walker P.A. Vasisht N. Kelly G. Howell S. Taylor I.A. Blackburn G.M. Gamblin S.J. Nature. 2003; 421: 652-656Crossref PubMed Scopus (306) Google Scholar, 42.Couture J.F. Collazo E. Brunzelle J.S. Trievel R.C. Genes Dev. 2005; 19: 1455-1465Crossref PubMed Scopus (192) Google Scholar), have a relatively limited active site volume and are predominantly monomethyltransferases. Although mutagenesis experiments have validated the Phe/Tyr switch hypothesis for several SET domain enzymes (40.Zhang 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 (274) Google Scholar, 43.Collins R.E. Tachibana M. Tamaru H. Smith K.M. Jia D. Zhang X. Selker E.U. Shinkai Y. Cheng X. J. Biol. Chem. 2005; 280: 5563-5570Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar), enzymes from the SET1 family appear to contradict this rule (43.Collins R.E. Tachibana M. Tamaru H. Smith K.M. Jia D. Zhang X. Selker E.U. Shinkai Y. Cheng X. J. Biol. Chem. 2005; 280: 5563-5570Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). This is because SET1 family enzymes are predicted to monomethylate their substrates based on the presence of a conserved tyrosine at the switch position. However, chromatin immunoprecipitation experiments suggest that SET1 family enzymes are capable of mono-, di-, and trimethylation in vivo (2.Santos-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 (1608) Google Scholar, 9.Ng H.H. Robert F. Young R.A. Struhl K. Mol. Cell. 2003; 11: 709-719Abstract Full Text Full Text PDF PubMed Scopus (857) Google Scholar). In addition, a purified MLL1 complex was shown to catalyze mono-, di, and trimethylation of H3 peptides in vitro (24.Dou 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 (533) Google Scholar). These results suggest that either the product specificity of SET1 family enzymes is established by a mechanism distinct from the Phe/Tyr switch hypothesis or the product specificity of these enzymes is regulated by specific protein-protein interactions in the cell. An understanding of this paradox has been limited by the absence of a well defined system to examine the product specificity of SET1 family enzymes in the presence and absence of interacting proteins. To better understand how product specificity is regulated in SET1 family enzymes, we have developed an in vitro system to identify the molecular mechanisms of H3K4 methylation by the human MLL1 core complex. We previously reported the identification of a minimal MLL1 SET domain fragment that is required for the interaction between the MLL1 and WDR5 components of the MLL1 core complex (44.Patel A. Dharmarajan V. Cosgrove M.S. J. Biol. Chem. 2008; 283: 32158-32161Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 45.Patel A. Vought V.E. Dharmarajan V. Cosgrove M.S. J. Biol. Chem. 2008; 283: 32162-32175Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). In this investigation, we used this MLL1 SET domain fragment to reconstitute and characterize the hydrodynamic and kinetic properties of a five-component 200-kDa MLL1 core complex including MLL1, WDR5, RbBP5, Ash2L, and DPY-30. Our results confirm that the isolated MLL1 SET domain is an H3K4 monomethyltransferase, consistent with the predictions of the Phe/Tyr switch hypothesis. In contrast, when the MLL1 SET domain fragment is assembled with a complex containing WDR5, RbBP5, Ash2L, and DPY-30, the rate of lysine methylation is dramatically increased but only to the dimethyl form of H3K4, suggesting that the MLL1 core complex is predominantly a dimethyltransferase. Unexpectedly, we demonstrate that the H3K4 dimethylation activity of the MLL1 core complex is catalyzed by a previously unrecognized methyltransferase activity conferred by the non-SET domain components of the MLL1 core complex. These results suggest that SET1 family complexes have evolved a novel mechanism to precisely regulate the degree of H3K4 methylation in eukaryotes. A human MLL1 construct consisting of residues 3745–3969 (MLL3745) as well as full-length human WDR5, RbBP5, and ASH2L proteins were individually expressed in Escherichia coli (Rosetta II, Novagen) and purified as described previously (45.Patel A. Vought V.E. Dharmarajan V. Cosgrove M.S. J. Biol. Chem. 2008; 283: 32162-32175Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). A cDNA clone of the human dpy-30 gene was obtained from Open Biosystems (clone ID LIFESEQ1240436), PCR-subcloned into the pHis parallel vector (46.Sheffield P. Garrard S. Derewenda Z. Protein Expr. Purif. 1999; 15: 34-39Crossref PubMed Scopus (536) Google Scholar), and purified by nickel affinity and gel filtration chromatography as described previously (45.Patel A. Vought V.E. Dharmarajan V. Cosgrove M.S. J. Biol. Chem. 2008; 283: 32162-32175Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). 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 tris(2-carboxyethyl)phosphine, and 1 μm ZnCl2. Analytical ultracentrifugation experiments were carried out using a Beckman Coulter ProteomeLabTM 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 were run at various concentrations and combinations as described under “Results.” Sedimentation boundaries were analyzed by the continuous sedimentation coefficient distribution (c(s)) method using the program SEDFIT (47.Schuck P. Biophys. J. 2000; 78: 1606-1619Abstract Full Text Full Text PDF PubMed Scopus (3089) Google Scholar). Equilibrium dissociation constants for all binary complexes were obtained by globally fitting sedimentation velocity data using the single-site hetero-association model (A + B ↔ AB) of SEDPHAT (48.Schuck P. Scott D.J. Harding S.E. Rowe A.J. Analytical Ultracentrifugation: Techniques and Methods. Royal Society of Chemistry, Cambridge, UK2005: 26-49Google Scholar, 49.Dam J. Velikovsky C.A. Mariuzza R.A. Urbanke C. Schuck P. Biophys. J. 2005; 89: 619-634Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). The program SEDNTERP version 1.09 (50.Laue T.M. Shah B.D. Ridgeway T.M. Pelletier S.L. Harding S. Rowe A. Horton J. Analytical Ultracentrifugation in Biochemistry and Polymer Science. Royal Society of Chemistry, Cambridge, UK1992: 19-125Google Scholar) was used to correct the experimental s value to standard conditions at 20 °C in water (s20,w) and to calculate the partial specific volume of each protein. MALDI-TOF mass spectrometry assays were carried out as described previously (45.Patel A. Vought V.E. Dharmarajan V. Cosgrove M.S. J. Biol. Chem. 2008; 283: 32162-32175Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). Briefly, 6 or 30 μg of MLL3745 were mixed with 250 μm S-adenosyl methionine (AdoMet) and 10 μm histone H3 peptide consisting of residues 1–20 in a buffer containing 50 mm Tris-Cl, pH 9.0, 200 mm NaCl, 3 mm dithiothreitol, and 5% glycerol. The total reaction volume was 20 μl. To prevent enzyme inactivation over the duration of the experiment, the reactions were incubated at 15 °C for 24 h, and at various time points, aliquots were removed and quenched by the addition of trifluoroacetic acid to 0.5%. The quenched samples were diluted 1:5 with α-cyano-4-hydroxycinnamic acid. MALDI-TOF mass spectrometry was performed on a Bruker AutoFlex mass spectrometer (State University of New York, Oswego, NY) operated in reflectron mode. Final spectra were the average of 100 shots/position at 10 different positions chosen at random on each spot. Duplicate or triplicate measurements were taken at each time point. Unmodified histone H3 peptide (residues 1–20), or mono-, di-, and trimethylated H3K4 peptides (residues 1–21) were purchased from Global Peptide and Millipore, respectively. All peptides contained a Gly-Gly linker on the C terminus followed by lysine with biotin on the ϵ-amino group. H3K4 methyltransferase assays were conducted by combining 2 μg of MLL3745-WDR5-RbBP5-Ash2L complex with 250 μm histone H3 peptide and 1 μCi of [3H]methyl-S-adenosyl-methionine ([3H]AdoMet, 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 8 h, stopped by the addition of 1× SDS loading buffer, 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 or 48 h. Enzymatic reactions were initiated with substrate quantities of enzyme (12 or 60 μm) and were quenched manually at various time points by the addition of 0.5% trifluoroacetic acid. MALDI-TOF mass spectrometry was used to determine the relative distribution of unmodified, mono-, di-, and trimethylated species in each reaction using a procedure similar to that described by Frey and colleagues (51.Gross J.W. Hegeman A.D. Vestling M.M. Frey P.A. Biochemistry. 2000; 39: 13633-13640Crossref PubMed Scopus (53) Google Scholar). We note that although MALDI-TOF is generally believed to be a qualitative technique, it has been successfully used in several systems to quantitate reaction intermediates in rapid reaction kinetic experiments with results comparable with other rapid reaction techniques (51.Gross J.W. Hegeman A.D. Vestling M.M. Frey P.A. Biochemistry. 2000; 39: 13633-13640Crossref PubMed Scopus (53) Google Scholar, 52.Houston C.T. Taylor W.P. Widlanski T.S. Reilly J.P. Anal Chem. 2000; 72: 3311-3319Crossref PubMed Scopus (57) Google Scholar). In the present investigation, data were quantitated by determining the percentage of total integrated area for each species and using standard curves to estimate the concentration of each modified form at each time point. Standard curves were constructed by collecting MALDI-TOF spectra on peptide mixtures containing various ratios of synthetic histone H3 peptides that were either unmodified or previously mono- or dimethylated at H3K4. Amino acid analysis was used to determine peptide stock concentrations (Purdue Proteomics Facility). Relative intensity plots of each species in the presence of varying ratios of the other species are linear (supplemental Fig. 1), indicating that ionization and detection of each species is independent of the concentration of the other species. Control experiments indicated that the presence of enzyme did not significantly affect the ionization behavior of the peptides. Single turnover progress curves for the enzyme-catalyzed reactions were fitted to a kinetic model with one or two irreversible consecutive reactions (A→B→C) using (Eq. 1), (Eq. 2), (Eq. 3) as described by Fersht (53.Fersht A. Sturcture and Mechanism in Protein Science. W.H. Freeman and Company, New York1999: 143-146Google Scholar). A=A0exp−k1t(Eq. 1) B=A0k1k2−k1exp−k1t−exp−k2t(Eq. 2) C=A01+1k1−k2k2exp−k1t−k1exp−k2t(Eq. 3) where [A]0 is the concentration of the unmodified peptide at time (t) zero. [B] and [C] represent the concentrations of the monomethylated and dimethylated species in single turnover progress curves, respectively. k1 and k2 represent the pseudo-first-order rate constants for the conversion of A→B and B→C, respectively. For reactions with two irreversible consecutive steps, the program DynaFit (BioKin, Ltd.) (54.Kuzmic P. Anal. Biochem. 1996; 237: 260-273Crossref PubMed Scopus (1360) Google Scholar) was used to globally fit (Eq. 1), (Eq. 2), (Eq. 3) to the data. Previous co-immunoprecipitation studies suggest that the minimal complex required for di- and trimethylation of H3K4 includes MLL1, WDR5, RbBP5, and Ash2L (28.Dou 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 (572) Google Scholar). To begin to test this hypothesis, we individually overexpressed each component in E. coli and purified them to homogeneity (Fig. 1a). We also similarly purified the human DPY-30 protein, which was recently shown to interact with the Ash2L component of the MLL3 core complex and is conserved in SET1 family complexes ranging from yeast to humans (30.Cho 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 (419) Google Scholar). All proteins were full-length human proteins with the exception of human MLL1, for which we used a recombinant fragment containing residues 3745–3969 (MLL3745). This fragment from the extreme C terminus of MLL1 contains the conserved SET and post-SET domains as well as the conserved Win or WDR5 interaction motif in the N-SET region of MLL1 (Fig. 1b). The MLL1 Win motif was previously demonstrated to be required for the interaction between MLL1 and WDR5 (44.Patel A. Dharmarajan V. Cosgrove M.S. J. Biol. Chem. 2008; 283: 32158-32161Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 45.Patel A. Vought V.E. Dharmarajan V. Cosgrove M.S. J. Biol. Chem. 2008; 283: 32162-32175Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 56.Song J.J. Kingston R.E. J. Biol. Chem. 2008; 283: 35258-35264Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar) and for the assembly and dimethylation activity of the MLL1 core complex (45.Patel A. Vought V.E. Dharmarajan V. Cosgrove M.S. J. Biol. Chem. 2008; 283: 32162-32175Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). Sedimentation velocity analytical ultracentrifugation experiments were used to characterize the hydrodynamic properties of each protein in isolation and to characterize intermolecular interactions within the complex. Sedimentation velocity profiles for individual proteins were fitted to a distribution of Lamm equation sol" @default.
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