anovelnon-setdomainmulti-subunitmethyltransferase ... · tions were incubated at 15 °c for 8 h and...

A Novel Non-SET Domain Multi-subunit MethyltransferaseRequired for Sequential Nucleosomal Histone H3Methylation by the Mixed Lineage Leukemia Protein-1(MLL1) Core Complex*

Received for publication, August 11, 2010, and in revised form, November 8, 2010 Published, JBC Papers in Press, November 24, 2010, DOI 10.1074/jbc.M110.174524

Anamika Patel, Valarie E. Vought, Venkatasubramanian Dharmarajan, and Michael S. Cosgrove1

From the Department of Biology, Syracuse University, Syracuse, New York 13244

Gene expression within the context of eukaryotic chroma-tin is regulated by enzymes that catalyze histone lysinemethylation. Histone lysine methyltransferases that havebeen identified to date possess the evolutionarily conservedSET or Dot1-like domains. We previously reported the identi-fication of a new multi-subunit histone H3 lysine 4 methyl-transferase lacking homology to the SET or Dot1 family of his-tone lysine methyltransferases. This enzymatic activityrequires a complex that includes WRAD (WDR5, RbBP5,Ash2L, and DPY-30), a complex that is part of the MLL1(mixed lineage leukemia protein-1) core complex but that alsoexists independently of MLL1 in the cell. Here, we report thatthe minimal complex required for WRAD enzymatic activityincludes WDR5, RbBP5, and Ash2L and that DPY-30, al-though not required for enzymatic activity, increases the his-tone substrate specificity of the WRAD complex. We also showthat WRAD requires zinc for catalytic activity, displaysMichaelis-Menten kinetics, and is inhibited by S-adenosyl-homocysteine. In addition, we demonstrate that WRAD pref-erentially methylates lysine 4 of histone H3 within the contextof the H3/H4 tetramer but does not methylate nucleosomalhistone H3 on its own. In contrast, we find that MLL1 andWRAD are required for nucleosomal histone H3 methylation,and we provide evidence suggesting that each plays distinctstructural and catalytic roles in the recognition and methyla-tion of a nucleosome substrate. Our results indicate thatWRAD is a new H3K4 methyltransferase with functions thatinclude regulating the substrate and product specificities ofthe MLL1 core complex.

Eukaryotic gene expression programs are established andmaintained in part by enzymes that methylate the epsilonamino group of histone lysine residues. Histone lysine meth-ylation regulates gene expression by recruiting proteins thatstabilize or remodel distinct chromatin states (1–3). Lysineresidues can be mono-, di-, or trimethylated with distinctfunctional consequences, increasing the combinatorial signal-

ing potential of lysine methylation (4, 5). Although it has be-come increasingly clear that regulation of the degree of lysinemethylation plays a functionally significant role in eukaryoticgene regulation, the molecular mechanisms involved are onlybeginning to be understood.Recent data suggest several models for the regulation of the

degree of methylation by histone lysine methyltransferases.One model suggests that multiple methylation is achieved bydistinct histone lysine methyltransferases that have evolved tocatalyze the addition of one, two, or three methyl groups to asingle lysine side chain. In this model, the addition of eachmethyl group is sequentially catalyzed by a distinct enzymeand is supported by the existence of several SET domain en-zymes that differ in their abilities to use mono- or dimethyll-ysine as a substrate for further methylation (6), a phenome-non known as “product specificity” (7). In contrast, analternative model suggests that multiple lysine methylationmay be achieved by allosteric control of a single SET domainenzyme. In the allosteric model, the degree of lysine methyla-tion is controlled by proteins that interact with and alter theconformation of the catalytic SET domain, altering its abilityto catalyze the addition of each methyl group to a lysine sidechain. The regulation of the trimethylation activity of theESET/SETDB1 protein by interaction with the mAM protein(8) is an example of this type of regulation.Another often cited example for allosteric control of multi-

ple lysine methylation is the SET1 family of HMTases. SET1family enzymes assemble into conserved multi-subunit com-plexes that are important for the maintenance of transcrip-tionally accessible forms of chromatin through the regulationof the degree of histone H3 lysine 4 (H3K4)2 methylation (9–21). Evidence supporting the allosteric model for multiplelysine methylation stems from sequence alignments predict-ing that SET1 family SET domains should only monomethyl-ate their substrates (6, 7, 22). However, mono-, di-, and trim-ethylation activities have been attributed to SET1 familycomplexes in vivo and in vitro (4, 9–11, 23). These resultshave led to the suggestion that SET domain-interacting pro-teins alter the conformation of the SET1 family active site,allowing allosteric control of multiple lysine methylation (24,25). However, recent biochemical data suggest that SET1 fam-

* This work was supported, in whole or in part, by National Institutes ofHealth Grant R01CA140522 (to M. S. C.). This work was also supported byResearch Scholar Grant RSG-09-245-01-DMC from the American CancerSociety (to M. S. C.).

1 To whom correspondence should be addressed: 107 College Pl., Syracuse,NY 13244. Tel.: 315-443-2964; Fax: 315-443-2012; E-mail: [email protected].

2 The abbreviations used are: H3K4, H3 lysine 4; [3H]AdoMet, [3H]methylS-adenosylmethionine; AdoHyc, S-adenosyl-homocysteine.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 5, pp. 3359 –3369, February 4, 2011© 2011 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

FEBRUARY 4, 2011 • VOLUME 286 • NUMBER 5 JOURNAL OF BIOLOGICAL CHEMISTRY 3359

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ily complexes instead use a sequential mechanism for multi-ple lysine methylation.The human MLL1 (mixed lineage leukemia protein 1) is a

member of the SET1 family of H3K4 methyltransferases andhas been shown to interact with an evolutionarily conservedgroup of non-SET domain proteins that include WDR5,RbBP5, Ash2L, and DPY-30 (26), components that have previ-ously been shown to play important roles in cellular differen-tiation (27–29), development (30, 31), transcription (32), dos-age compensation (33, 34), and oncogenesis (35, 36). Theseproteins, when in complex with MLL1, form the MLL1 corecomplex (10) and are required for the regulation of HOXgenes during hematopoiesis and development (37–42). Werecently reported that the intrinsic activity of an isolatedMLL1 SET domain is indeed that of an H3K4 monomethyl-transferase (43). Surprisingly, we discovered that WDR5,RbBP5, Ash2L, and DPY-30 form a subcomplex (called“WRAD”) that possesses an H3K4 methyltransferase activitythat is independent of the MLL1 SET domain (26, 43). In ad-dition, contrary to the predictions of the allosteric model formultiple lysine methylation, we demonstrated that WRADcatalyzes H3K4 dimethylation within the context of the MLL1core complex, providing the first example of a sequential mul-tiple lysine methylation system (43). Because the componentsof the WRAD complex lack homology to known S-adenosyl-methionine-dependent methyltransferase folds, relatively lit-tle is understood about how this novel enzyme works. Herewe further characterize the enzymatic activity and substratespecificity of WRAD in isolation and in the presence of MLL1.

EXPERIMENTAL PROCEDURES

Protein Expression and Purification—A construct of humanMLL1 consisting of amino acid residues 3745–3969(MLL3745) as well as full-length human proteins WDR5,RbBP5, Ash2L, and DPY30 were individually expressed inEscherichia coli and purified as described previously (43, 44).The WRAD or MWRAD complexes were created by mixingequimolar amounts of each recombinant component. Sedi-mentation velocity analytical ultracentrifugation was used toassess complex formation as described below. Histones wereexpressed, purified, refolded, and reconstituted into nucleo-somes using the small scale reconstitution procedure as de-scribed by Dyer et al. (45) using the 255-base pair 54A54 dou-ble-stranded DNA fragment from the murine mammarytumor virus 3�-LTR promoter and flanking sequences as de-scribed by Flause et al. (46).Analytical Ultracentrifugation—Analytical ultracentrifuga-

tion experiments were carried out using a Beckman CoulterProteomeLabTM XL-A analytical ultracentrifuge equippedwith absorbance optics and an eight-hole An-50 Ti analyticalrotor. Sedimentation velocity experiments were carried out at10 °C and 50,000 rpm (200,000 � g) using 3-mm two-sectorcharcoal-filled Epon centerpieces with quartz windows. Foreach sample, 300 scans were collected with the time intervalbetween scans set to 0. Protein samples in 20 mM Tris-Cl, pH7.5, 300 mM NaCl, 1 mM TCEP, and 1 �M ZnCl2 were run atvarious concentrations as described under “Results.” Sedi-mentation boundaries were analyzed by the continuous distri-

bution (c(s)) method using the program SEDFIT (47). Theprogram SEDNTERP version 1.09 (48) was used to correct theexperimental S value (s) to standard conditions at 20 °C inwater (S20,w) and to calculate the partial specific volume ofeach protein.[3H]Methyltransferase Assays—Histone methyltransferase

activity assays were carried out by combining 5 �g of chickencore histones (Millipore) and 1 �Ci of [3H]methyl S-adenosyl-methionine ([3H]AdoMet; MP Biomedicals) in the presenceof the WRA(D) enzyme at a final concentration of 4.3 �M.The reaction buffer contained 50 mM Tris, pH 8.5, 200 mM

NaCl, 3 mM DTT, 5 mM MgCl2, and 5% glycerol. The reac-tions were incubated at 15 °C for 8 h and quenched by dilu-tion with 5� SDS loading buffer. The reactions were sepa-rated by 18% Tris-glycine PAGE or 4–12% Bis-Tris PAGE(Invitrogen). The gels were soaked in autoradiography en-hancer solution (ENLIGHTNING; PerkinElmer Life Science)for 30 min, dried, and exposed to film at �80 °C overnight orfor up to 4 days. For assays comparing the enzymatic activityof WRAD with full-length unmodified or monomethylatedrecombinant Xenopus laevis histone H3 (Active Motif�), 4 �gof histone H3 was incubated with 1 �Ci of [3H]AdoMet in thepresence of the WRA(D) enzyme at a final concentration of4.3 �M. The reactions were processed as described above.Enzyme Kinetics—Enzymatic reactions (20 �l) were initi-

ated by the addition of enzyme (1–17 �M) to reaction mix-tures containing a fixed concentration of histone H3 peptide(residues 1–20) and variable concentrations of [3H]AdoMet(PerkinElmer Life Sciences; specific activity, 12 �Ci/mM) orfixed [3H]AdoMet concentrations and variable concentra-tions of histone H3 peptide in assay buffer (50 mM Tris, pH8.5, 200 mM NaCl, 3 mM DTT, 5 mM MgCl2, and 5% glyc-erol). The reactions were incubated at 15 °C for 4 h andthen stopped by the addition of 5 �l of 5� SDS loadingbuffer. Control time course experiments showed that reac-tions were still linear after 4 h with various substrate andcoenzyme combinations. Quenched reactions were separatedby 4–12% Bis-Tris PAGE and stained with Coomassie Bril-liant Blue R. Peptide bands were excised from the gel and dis-solved in 750 �l of SolvableTM (PerkinElmer Life Sciences) at50 °C for 3 h. The extracted peptide solution was mixedwith 5 ml of scintillation mixture (ULTIMA GOLD XR;PerkinElmer Life Sciences) and counted using a BeckmanCoulter LS6500 scintillation counter. Initial rates were plottedas a function of the variable substrate and fitted to theMichaelis-Menten equation (Equation 1) using the nonlinearleast squares regression analysis in SigmaPlot. To determinethe apparent inhibition constant (Ki) for S-adenosyl-homo-cysteine (AdoHyc), enzymatic assays were conducted by incu-bating fixed concentrations of [3H]AdoMet (25 �M), histoneH3 peptide (500 �M), and WRAD (4.3 �M) with variable con-centrations of AdoHyc (0–250 �M). The reactions were al-lowed to proceed for 4 h, quenched with SDS loading buffer,and separated by PAGE. Peptide bands were excised, dis-solved with Solvable, and counted by LSC as described above.The inhibition curve was fitted to Equation 2 using SigmaPlotsoftware.

WRAD, a Novel Multi-subunit Histone Methyltransferase

3360 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 286 • NUMBER 5 • FEBRUARY 4, 2011

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�0 � Vmax�S�/�Km � �S�� (Eq. 1)

�0 � Vmax�S�/�Km�1 � �I�/Ki� � �S�� (Eq. 2)

Western Blotting—Enzymatic assays with reconstituted nu-cleosomes were carried out as described above. The reactionswere quenched by the addition of SDS loading buffer after 8 hand resolved by 18% Tris-glycine PAGE. The proteins wereblotted onto nitrocellulose membranes and probed with anti-bodies specific for H3K4 monomethyllysine (1:500 dilution;Abcam catalogue number ab8895), dimethyllysine (1:1000dilution, Abcam catalogue number ab32356), or trimethyll-ysine (1:500 dilution, Active Motif catalogue number 39160).

RESULTS

WRAD Is a Histone H3 Lysine 4-specific Methyltransferase—We previously demonstrated that the isolated humanWRADcomplex monomethylates but does not dimethylate histoneH3 peptides (residues 1–20) at lysine 4 (43). In contrast, wedemonstrated that WRAD acquires the ability to monom-ethylate histone peptides that are unmethylated or mono-methylated at lysine 4 when assembled with a catalyticallyinactive variant of the MLL1 SET domain (43). These resultssuggest that WRAD is a one-carbon methyl transfer enzymethat is capable of transferring one methyl group to histone H3on its own or to a previously monomethylated H3K4 substratebut only when in complex with MLL1.To better understand the substrate specificity of the iso-

lated WRAD complex under our standard in vitro assayconditions, we characterized the methylation activity ofrecombinant human WRAD with different histone sub-strates. When purified, chicken core histones and[3H]AdoMet are incubated in the presence of WRAD,[3H]methyl is incorporated into histone H3 but not his-tones H2A, H2B, H4, or H1 (Fig. 1A). To identify lysineresidues methylated by WRAD, we compared activityamong recombinant full-length X. laevis histone H3 pro-teins that were unmethylated or chemically modified tointroduce a monomethyllysine analogue at lysine 4 (ActiveMotif�). The results show that unmethylated histone H3 isa substrate for methylation by WRAD (Fig. 1B, lane 1) butnot histone H3 previously monomethylated at lysine 4 (Fig.1B, lane 2). To determine whether the chemically modifiedform of histone H3 can be dimethylated, we added toWRAD the wild-type MLL1 SET domain construct (resi-dues 3745–3969 (MLL3745)) to form the fully assembledMLL1 core complex, which we previously demonstrated tobe required for dimethylation of H3K4 (43). In these assays,methylation of monomethylated histone H3 is readily ob-served (Fig. 1C, lane 2), indicating that the monomethyllysineanalogue is an adequate mimic of the H3K4 monomethylatedstate.These results indicate that histone H3 is the preferred sub-

strate for isolated humanWRAD, which, in the absence ofMLL1, preferentially monomethylates lysine 4. These resultsare in agreement with our previous demonstration that theisolated WRAD complex monomethylates H3 peptides in anH3K4-dependent manner (43).

WRAD Preferentially Methylates the Histone H3/H4 Tet-ramer but Does Not Methylate Nucleosomal Histone H3—Wenext compared the enzymatic activity of WRAD using as sub-strates recombinant X. laevis histone H3 in the context of freehistone H3, the dialyzed histone octamer (which under theassayed conditions dissociates into an H3/H4 tetramer andtwo H2B/H2A dimers (49)), and a reconstituted nucleosome.The results show that free recombinant H3 and the H3/H4tetramer are methylated by WRAD (Fig. 1D, lanes 1 and 2),with the H3/H4 tetramer showing greater methylation. Incontrast, reconstituted nucleosomes are not a substrate forWRAD (Fig. 1D, lane 3). It should be noted that the lack ofactivity with nucleosomes does not appear to be due to inhibi-tion by free DNA, because the addition of a stoichiometricequivalent of free DNA to assays with the histone octamerdoes not significantly affect methylation by WRAD (data notshown). These results indicate that histone H3 within the

FIGURE 1. WRAD is a histone H3 lysine 4-specific monomethyltrans-ferase. A, enzymatic assays showing the methyltransferase activity ofWRAD after a period of 8 h using [3H]methyl-S-adenosylmethionine andpurified chicken core histones as substrates. Enzymatic reactions were sepa-rated by 18% Tris-glycine SDS-PAGE and visualized with Coomassie BrilliantBlue (left panel, lanes 1 and 2) or fluorography (right panel, lanes 3 and 4).B, enzymatic activity of WRAD using 4 �g of full-length histone H3 proteins(Active Motif�) that were either unmodified (H3, lane 1) or previouslymonomethylated at lysine 4 (H3K4me1, lane 2). The reactions were sepa-rated by 18% Tris-glycine PAGE and visualized with Coomassie Brilliant Blue(upper panel) and fluorography (3H-Methyl, lower panel). C, enzymatic activ-ity of MLL1 core complex after a period of 8 h using 4 �g of full-length his-tone H3 proteins that were either unmodified (H3, lane 1) or previouslymonomethylated at lysine 4 (H3K4me1, lane 2). The reaction products wereseparated by 4 –12% Bis-Tris PAGE and visualized as described in B. D, com-parison of the enzymatic activity of WRAD using as substrates recombinanthistone H3 (1.5 �g, lane 1), dialyzed histone octamer (6 �g, lane 2), or anequivalent amount of reconstituted nucleosomes (lane 3). The reactionswere separated by 18% Tris-glycine SDS-PAGE and visualized as describedin B above. Octamer* denotes that the histone octamer dissociates into oneH3/H4 tetramer and two H2A/H2B dimers upon dialysis into assay buffer(see text).



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context of the histone H3/H4 tetramer is the preferred sub-strate for WRAD when recombinant histones are used as asubstrate.WRA Is the Minimal Complex Required for Enzymatic

Activity—To identify the minimal complex required for meth-ylation by WRAD, we compared activity among the individualcomponents and all possible binary and ternary complexesusing purified chicken core histones as a substrate. As shownin Fig. 2A, no activity is observed when the assays are con-ducted with the individual components of the WRAD com-plex (lanes 1–4) or with any of the binary complexes (lanes5–7 and 13–15), similar to that observed in the absence ofenzyme (lane 16). Enzymatic activity is observed only withWRA andWRAD complexes (lanes 8 and 9, respectively) butnot with any of the other ternary complexes (lanes 10–12).WRAD activity depends on the dose of the enzyme, as indi-

cated by the nearly linear dependence of activity on enzymeconcentration (Fig. 2B). The addition of DPY-30 to the WRAsubcomplex results in a 38% increase in the slope of the doseresponse curve (Fig. 2B), suggesting that DPY-30 may func-tion to enhance the activity of WRAD. The enhanced activity

in the presence of DPY-30 may be due in part to stabilizationof the complex, because sedimentation velocity analytical ul-tracentrifugation experiments show that the WRAD complexis slightly more stable upon dilution than the WRA complex(Fig. 2C). For example, the WRAD complex represents 71%(4.1 s), 69% (4.0 s), and 63% (3.9 s) of all species in the samplerun at concentrations of 2.2, 1.1, and 0.6 �M, respectively (Fig.2C, upper panel). In contrast, in the absence of DPY-30, theWRA subcomplex represents 67% (4.0 s), 65% (3.8 s), and 53%(3.5 s) of all species at the same concentrations, respectively(Fig. 2C, lower panel). DPY-30 promotes complex formationat each protein concentration. Taken together, these resultsindicate that WDR5, RbBP5, and Ash2L form the minimalcomplex required for methylation of histone H3 and thatDPY-30 functions to enhance complex stability and enzy-matic activity.DPY-30 Increases the Histone Substrate Specificity of the

WRAD Complex—To determine apparent steady-state kineticparameters for WRA andWRAD complexes, we performedenzymatic reactions with variable amounts of AdoMet at aconstant concentration of histone H3 peptide (residues 1–20).

FIGURE 2. WDR5, RbBP5, and Ash2L are required for the H3K4 methylation activity of WRAD. A, comparison of enzymatic activity of individual WRADcomponents and all possible binary, ternary, and quaternary complexes. W, WDR5; R, RbBP5; A, Ash2L; D, DPY-30. Histone methyltransferase assays wereconducted for a period of 8 h using [3H]AdoMet and chicken core histones as the substrate. Quenched reactions were separated by 18% Tris-glycine SDS-PAGE and visualized with Coomassie Brilliant Blue (upper panels) and fluorography (lower panels). A no-enzyme control is shown in lane 16. B, WRAD andWRA histone methyltransferase activity as a function of enzyme concentration. Methyltransferase activity assays were conducted with 25 �M [3H]AdoMetand 500 �M histone H3 peptide (residues 1–20) with varying concentrations of WRAD (open triangles) or WRA (open circles). Each point corresponds to theaverage of duplicate measurements with the error bars indicating the standard error of measurement. Linear regression fitting of the data gave slopes of0.0016 and 0.001, and R2 values of 0.99 and 0.97 for WRAD and WRA complexes, respectively. C, diffusion-free sedimentation coefficient distributions (c(s))derived from sedimentation velocity analytical ultracentrifugation of WRAD (upper panel) and WRA (lower panel) complexes at concentrations of 2.2 �M

(solid line), 1.1 �M (dashed line), and 0.55 �M (dotted line).



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Michaelis-Menten kinetics were observed for both WRA andWRAD complexes (Fig. 3). Nonlinear least square regressionfitting of the data to the Michaelis-Menten equation showsthat AdoMet binds to WRAD andWRA in enzymatic reac-tions with relatively similar apparent Km values of 8 and 14�M, respectively (Fig. 3A and Table 1). In contrast, when thehistone H3 peptide is the variable substrate, the apparent Kmvalue for histone H3 decreases �7-fold from 2.4 to 0.34 mM

when DPY-30 is added to the complex (Fig. 3B and Table 1).These data suggest that DPY-30 functions to increase thespecificity of WRAD for histone H3, as reflected by thegreater increase in the apparent specificity constant (kcat/Km)for histone H3 (3-fold) compared with that of AdoMet (1.6-fold) when DPY-30 is added to the complex (Table 1).WRAD Is Inhibited by S-Adenosyl-homocysteine—Because

the co-factor product AdoHyc is often a potent competitive

inhibitor of AdoMet dependent enzymes (50), we comparedthe enzymatic activity of WRAD with increasing concentra-tions of AdoHyc. As shown in Fig. 3C, WRAD activity is in-hibited in a saturable manner with increasing concentrationsof AdoHyc. Fitting the data to equation 2 (materials andmethods) reveals an apparent Ki value for AdoHyc of �9.0 2.0 �M, which is comparable with that of other AdoMet-de-pendent methyltransferases (50).WRAD Requires Zinc for Catalytic Activity—To determine

whether the activity of WRAD is metal ion-dependent, wecompared methylation of histone H3 peptides by WRAD inthe presence of increasing concentrations of chelating agentsEDTA or 1,10-phenanthroline. As shown in Fig. 4 (A and B),the enzymatic activity of WRAD is highly sensitive to bothchelating agents. In the presence of 1 mM EDTA, the enzy-matic activity of WRAD is inhibited by 80% (Fig. 4A). In con-

FIGURE 3. Steady-state kinetics and inhibition analysis of WRA(D). A, comparison of WRA (open circle) and WRAD (open triangle) kinetics with AdoMet asthe variable substrate (ranging from 0.5 to 25 �M) and fixed concentrations (1 mM) of histone H3 peptide (residues 1–20). Rates of methylation are themeans of duplicate measurements standard error of measurement. Apparent kinetic parameters were determined by fitting the data to the Michaelis-Menten equation (Equation 1, “Experimental Procedures”). B, comparison of WRA (open circles) and WRAD (open triangles) kinetics with histone H3 peptideas the variable substrate (ranging from 25 to 5000 �M). The data are represented and fitted as described for A. For clarity, the values for the concentrationrange from 0 to 1000 �M are shown. C, comparison of the enzymatic activity of WRAD (4.3 �M) with increasing concentrations of AdoHyc (1–250 �M). Activ-ity assays were conducted with fixed concentrations of AdoMet (25 �M) and histone H3 peptide (500 �M). Each point represents the means S.E. of mea-surement from duplicate measurements. The data were fit to Equation 2 (“Experimental Procedures”).

FIGURE 4. Zinc is required for the H3K4 methyltransferase activity of WRAD. A, WRAD enzymatic activity (mean S.E.) with increasing concentrationsof EDTA. B, WRAD enzymatic activity (mean S.E.) with increasing concentrations of 1,10-phenanthroline. C, relative WRAD activity after preincubationwith 1 mM EDTA and the addition of different divalent cations: zinc, cobalt, magnesium, or calcium. The data were normalized relative to the untreated con-trol (0 mM EDTA) and represent the means the standard error of measurement from two independent experiments.

TABLE 1Summary of apparent kinetic parameters for WRA and WRAD complexes

Enzyme KmAdoMet Km

H3 peptide KcatAdoMet Kcat

H3 peptide Kcat/KmAdoMet Kcat/Km

H3 peptide

�M �M �10�4 h�1 �10�4 h�1 �10�4 �M�1 h�1 �10�4 �M�1 h�1

WRA 13.7 3.7 2382 269 20 70 1.5 0.03WRAD 7.9 1.7 338 54 18 30 2.3 0.09



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trast, the enzymatic activity of WRAD is inhibited 95% inthe presence of 1 mM 1,10-phenanthroline (Fig. 4B). To deter-mine which metal ion is required for activity, we preincubatedWRAD with 1 mM EDTA and then compared enzymatic ac-tivity after the addition of excess amounts of divalent cationsof zinc, cobalt, magnesium, or calcium. As shown in Fig. 4C,when 1 mM ZnCl2 is added to EDTA-treated WRAD, most ofthe catalytic activity is regained. In contrast, the addition of astoichiometric equivalent of CoCl2, MgCl2, or CaCl2 resultedin relatively small increases in enzymatic activity. These re-sults suggest that WRAD requires zinc for catalytic activity.WRAD Is Required for Methylation of Nucleosomal Histone

H3 by the MLL1 Core Complex—To further characterize therole of WRA(D) in the substrate specificity of the MLL1 corecomplex, we compared the enzymatic activity of WRA(D) inthe presence and absence of an MLL1 SET domain constructMLL3745 (amino acid residues 3745–3969) using recombinanthistone octamers or reconstituted nucleosomes as substrates(Fig. 5). We previously demonstrated that MLL3745 is theminimal MLL1 construct required for the assembly and enzy-matic activity of the MLL1 core complex in vitro (43, 44). Inthis investigation, when the histone octamer is used as a sub-strate, a significantly greater amount of enzymatic activity wasobserved by the minimal MLL1 core complex (MWRA) (Fig.5A, lane 2) compared with that of MLL1 alone (Fig. 5A, lane1) or that of the WRA or WRAD complexes alone (Fig. 5A,lanes 3 and 4, respectively). These results suggest that the his-tone H3/H4 tetramer is the preferred substrate for the MLL1core complex. In contrast, when reconstituted nucleosomesare used as a substrate, little activity could be observed whenthe assays were conducted with the isolated MLL1 SET do-main construct (Fig. 5A, lane 5) or the isolated WRA (lane 7)or WRAD (lane 8) complexes. Significant methylation of anucleosomal substrate was only observed when the assayswere conducted with a fully assembled complex containing

MLL3745 and WRA (MWRA; Fig. 5A, lane 6) or MLL3745 andWRAD (MWRAD; Fig. 6A, lane 5). These results indicate thata fully assembled MLL1 core complex is required for methyla-tion of nucleosomal histone H3.To determine the product specificity of nucleosome meth-

ylation by the MLL1 core complex, we compared relativeamounts of mono-, di-, and trimethylation of nucleosomescatalyzed by MLL3745, WRAD, and the fully assembled MLL1core complex by Western blotting using methylation state-specific antibodies (Fig. 5B). The results show that the fullyassembled MLL1 core complex (MWRAD) catalyzes signifi-cant mono- and dimethylation of histone H3 when the assaysare conducted with reconstituted nucleosomes (Fig. 5B, lane1). In contrast, dimethylation of H3 is absent and H3K4monomethylation is significantly reduced when the assays areconducted with isolated MLL3745 (lane 2) or the isolatedWRAD complex (lane 3). These results demonstrate that thefully assembled MLL1 core complex is required to catalyzemono- and dimethylation of histone H3 within the context ofa nucleosomal substrate.MLL1 and WRAD Each Play Distinct Catalytic and Struc-

tural Roles in the Methylation of the Nucleosome by the MLL1Core Complex—To begin to understand why MLL1 andWRAD are required for methylation of nucleosomal histoneH3, we compared nucleosome methylation among MLL1 SETdomain variants that are either catalytically inactive or mu-tated to catalyze mono-, di-, and trimethylation of histone H3.We previously showed that the N3906A substitution in theMLL1 SET domain abolishes enzymatic activity using histonepeptides as a substrate (43). In this investigation, althoughlittle nucleosome histone H3 methylation could be observedwhen the assays were conducted with the isolated WRADenzyme (Fig. 5B, lane 3), an increase in the monomethylationof nucleosomal histone H3 was observed when WRAD wasassembled with the inactive N3906A variant of MLL1 (Fig. 5B,

FIGURE 5. MLL1 and WRAD are each required for methylation of nucleosomal histone H3 by the MLL1 core complex. A, comparison of the enzymaticactivity of the MLL1 SET domain (M), WRA(D), or the fully assembled MLL1 core complex (MWRA) using the dialyzed histone octamer or reconstituted nu-cleosomes as substrates. Histone methyltransferase assays were conducted for a period of 8 h. Quenched reactions were separated by 18% Tris-glycineSDS-PAGE and visualized with Coomassie Brilliant Blue (upper panels) and fluorography (lower panels, overnight (O/N) and 4-day exposures). B, mono-, di-,and trimethylation of reconstituted nucleosomes were compared using wild-type MLL3745 (M(wild-type)), WRAD, or the fully assembled MLL1 core complex(MWRAD). In addition, nucleosome methylation activities were compared among MLL1 core complexes assembled with a loss-of-function variant of MLL1(M(N3906A)) or a gain-of-function variant of MLL1 (M(Y3942F)). Western blotting with antibodies specific for the H3K4 mono-, di-, or trimethylated forms of his-tone H3 was used to detect nucleosome methylation, as described under “Experimental Procedures.”



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lane 4). These results indicate that MLL1 facilitates nucleo-some monomethylation by WRAD, even when MLL1 is cata-lytically inactive.To further test this hypothesis, we compared nucleosome

methylation by the gain-of-function Y3942F variant of MLL1in the presence and absence of WRAD. We previouslyshowed that replacement of Tyr-3942 with phenylalanineconverts the MLL1 SET domain, in the absence of WRAD,into a processive mono-, di-, and trimethyltransferase whenusing histone peptides as a substrate (43). However, in thisinvestigation, when nucleosomes are used as the substratewith the isolated Y3942F MLL1 SET domain enzyme,monomethylation is reduced, and di- and trimethylation ofhistone H3 are not observed (Fig. 5B, lane 5). In contrast,when WRAD is added to the Y3942F MLL1 SET domain,mono-, di-, and trimethylation of histone H3 are significantlyincreased (Fig. 5B, lane 6). Even though the Y3942F MLL1SET domain has the ability to mono-, di-, and trimethylatehistone H3 peptides on its own, it cannot fully methylate nu-cleosomal histone H3 without WRAD. Taken together, theseresults suggest that MLL1 and WRAD, in addition to beingrequired for the mono- and dimethylation activity of the wild-type MLL1 core complex, each play a structural role that al-lows the complex to use nucleosomes as a substrate.Arginine 3765 of MLL1 Is Required for the Interaction be-

tween MLL1 andWRAD and for NucleosomeMethylation—The results presented above suggest that factors controllingthe assembly of the MLL1 core complex may be important for

the regulation of nucleosomal histone H3 methylation. Wepreviously demonstrated that the interaction between MLL1and the WRA complex is dependent on WDR5 recognition ofarginine 3765 in MLL1 (44, 51). To test the hypothesis that afully assembled MLL1 core complex is required for methyla-tion of a nucleosomal substrate, we compared the enzymaticactivity of the wild-type and R3765A MLL1 SET domains inthe presence and absence of WRAD using recombinant his-tone octamers or nucleosomes as substrates. As shown in Fig.6A, like that of wild-type MLL1, the R3765A variant of MLL1is capable of methylating the histone octamer (Fig. 6A, lane 1)but not a nucleosomal substrate (Fig. 6A, lane 6). In contrast,althoughWRAD in the presence of wild-typeMLL1 activatesnucleosomemethylation (Fig. 6A, lane 5), WRAD activation ofnucleosomal methylation is mostly abolished when Arg-3765 ofMLL1 is replaced with alanine (Fig. 6A, lane 7). Sedimentationvelocity analytical ultracentrifugation experiments confirm thatreplacement of Arg-3765 with alanine disrupts the formation oftheMLL1 core complex (5.4 s, Fig. 6B, upper panel), resulting intwo largely noninteracting species corresponding to freeMLL1at 1.7 s andmostly freeWRAD at 4.5 s (Fig. 6B, lower panel).These results demonstrate that nucleosomal histone H3methyl-ation by theMLL1 core complex is dependent on the interactionbetweenMLL1 andWRAD.

DISCUSSION

Most histone lysine methyltransferases characterized todate possess the evolutionarily conserved SET domain. The

FIGURE 6. Arginine 3765 of MLL1 is required for the interaction between MLL1 and WRAD and for nucleosomal histone H3 methylation. A, compari-son of the enzymatic activity of wild-type (M) and R3765A (MR3765A) MLL1 SET domains in the presence and absence of WRAD using dialyzed histone oc-tamers or reconstituted nucleosomes as substrates. Quenched reactions were separated by 18% Tris-glycine SDS-PAGE and visualized with Coomassie Bril-liant Blue (upper panel) and fluorography (3H-Methyl (lower panels), overnight (O/N) and 4-day exposures)). Lanes 1 and 2 are assays conducted with dialyzedhistone octamers; lanes 3–7 are assays conducted with a reconstituted nucleosome substrate. B, diffusion-free sedimentation coefficient distribution (c(s))derived from sedimentation velocity analytical ultracentrifugation of the MLL1 core complex assembled with wild type (upper panel) or the R3765A variantof MLL1 (lower panel). The experimental sedimentation coefficients (s) are indicated.



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SET domain is a �130-amino acid motif that possesses aunique �-fold knot-like structure that functions to align themethyl donor S-adenosylmethionine and the peptide methylacceptor lysine (52). The only other non-SET domainHKMTase identified to date is the Dot1 protein, whichmethyl-ates lysine 79 in the globular domain of histone H3 within thecontext of the nucleosome (53–55). The three-dimensionalstructure of Dot1 shows that it possesses the more classical�/� methyltransferase fold using AdoMet as the methyl do-nor (56). In this investigation, we describe the characteriza-tion of a new histone lysine methyltransferase activity in amulti-subunit complex lacking homology to that of the SETor Dot1 family of histone methyltransferases. This enzyme iscomposed minimally of three proteins including WDR5 andRbBP5, both possessing WD-40 repeat domains; and Ash2L,which contains a conserved PHD finger and a SPRY domain.To our knowledge, these domains have not previously beenshown to be directly involved in S-adenosylmethionine-de-pendent methyltransferase reactions.The evidence we present strongly supports the existence of

this previously unrecognized enzymatic activity. Using a truebiochemical reconstitution system, where each human com-ponent is individually expressed and purified to homogeneityfrom E. coli, we demonstrate that the activity of WRA(D) islinearly dependent on the dose of the enzyme and that it dis-plays Michealis-Menten kinetics with respect to both histonepeptides and co-factor AdoMet. We also show that as withother AdoMet-dependent enzymes, the co-factor productS-adenosyl-homocysteine is a potent inhibitor of WRADmethyltransferase activity. Furthermore, we provide evidencesuggesting that zinc is required for WRAD activity. It is notclear at present whether zinc plays a direct role in catalysis orwhether it is required for subunit association.We also show that WDR5, RbBP5, and Ash2L are the mini-

mal components required for activity. Interestingly, DPY-30appears to function by modulating the histone substrate spec-ificity of WRAD. Because the individual components lack cat-alytic activity on their own, contamination with a bacterialmethyltransferase can be ruled out. Whether the active site isentirely contained within one subunit or whether it is sharedamong subunits is currently unknown.The complex requirement for the enzymatic activity of

WRAD may explain why this activity has not been observedpreviously. For example, in Saccharomyces cerevisiae, theSET1 protein assembles into a complex homologous to that ofthe MLL1 core complex, called COMPASS (15–17, 57), andregulates lysine 4 methylation, gene silencing, and gene ex-pression (58–60). Deletion of the SET1 gene in budding yeastgreatly reduces lysine 4 methylation (59, 61), which has led tothe suggestion that the SET1 protein is the only lysine 4methyltransferase in yeast, despite possessing homologues ofthe proteins that make up WRAD: Ash2L (Bre2/CPS60),RbBP5 (Swd1/CPS50), WDR5 (Swd3/CPS30), and DPY-30(Sdc1/CPS25). However, unlike WRAD in mammalian cells,which exists as an independent complex in the absence ofMLL1 (9, 10), the yeast homologue of WRAD apparently doesnot exist in a complex in the absence of the SET1 protein,because deletion of SET1 prevents co-immunoprecipitation of

the Bre2-Sdc1 subcomplex with the Swd1-Swd3 subcomplex(16, 62). It may be that in budding yeast, the SET1 protein isrequired to stabilize formation of the Bre2-Sdc1-Swd1-Swd3(WRAD) active site, which is required for enzymatic activity.We previously demonstrated that WRAD functions within

the context of the MLL1 core complex to sequentially methyl-ate H3 substrates previously monomethylated at H3K4 (43).The enzymatic activity of WRAD may also play a functionallysignificant role independent of MLL1 or other SET1 familyenzymes in the cell. Evidence supporting this hypothesiscomes from size exclusion chromatography of mammaliannuclear extracts, demonstrating that WDR5, RbBP5, andAsh2L, in addition to co-eluting with MLL1, also co-elutes asa distinct �150-kDa complex lacking MLL1 (9). The apparentmolecular mass of this complex determined by gel filtration iscomparable with the theoretical molecular mass for theWDR5, RbBP5, and Ash2L complex (156 kDa) with 1:1:1 stoi-chiometry and is consistent with our sedimentation velocityanalytical ultracentrifugation characterization of WRA(D).WRA(D) has also been identified in other complexes that ap-pear to lack SET1 family enzymes, such as the CHD8 andNIF1 complexes (63–65). Whether WRAD brings a histonemethyltransferase activity to these complexes remains to bedetermined. Intriguingly, enzymatic assays with an immuno-precipitated NIF1 complex shows histone H3 methylationactivity, albeit in a manner that is largely independent ofH3K4 (65). It is therefore likely that the majority of enzymaticactivity of the NIF1 complex is due to co-immunopurificationof other histone lysine or arginine methyltransferases. How-ever, it is also possible that the substrate specificity of WRADis altered within the context of the NIF1 complex. Furtherexperimentation will be required to distinguish thesehypotheses.A surprising finding from this investigation is the demon-

stration that WRAD lacks catalytic activity with a reconsti-tuted nucleosome substrate, suggesting that isolated WRADmay play a role prior to or during nucleosome assembly. Asimilar lack of activity on nucleosomes is observed when theassays are conducted with a purified MLL1 SET domain con-struct containing residues 3745–3969, likewise suggestingthat MLL1 in the absence of other components does not sig-nificantly methylate nucleosomal histone H3. Nucleosomalhistone H3 methylation is observed only when the assays areconducted with a fully assembled MLL1 core complex con-taining a stoichiometric equivalent of MLL1 and WRAD thatcatalyzes H3K4 mono- and dimethylation under our assayconditions. Why the fully assembled MLL1 core complex isrequired for nucleosome methylation is currently unknown.One possibility is that the histone H3 N-terminal tail mayinteract with nucleosomal DNA in a way that requires thefully assembled MLL1 core complex to make it available as asubstrate for methylation. This mechanism is consistent withthe demonstration that histone acetylation facilitates the as-sociation of the MLL1 SET domain with nucleosomes in vitro(66). However, a purely charged based mechanism predictsthat a sufficient amount of free DNA should also inhibit his-tone peptide or full-length H3 methylation. The addition ofdouble-stranded DNA to methylation reactions with histone



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peptides or full-length histone H3 does not significantly in-hibit methylation by the isolated MLL3745 or WRAD enzymes(not shown), suggesting that there is something unique aboutthe structure of the nucleosome that is recognized only by thefully assembled MLL1 core complex.Several lines of evidence suggest that the globular domains

of histones within the context of the nucleosome are impor-tant for H3K4 methylation by the MLL1 core complex. Theglobular domains of histones H2A, H2B, H3, and H4 are par-tially exposed on the face of the nucleosome (67), providing apotential interaction surface that may function to anchor theMLL1 core complex and correctly position the active site formethylation of the histone H3 tail. Consistent with this hy-pothesis, mutagenesis experiments in yeast revealed a clusterof histone H2A and H2B amino acids required for H3K4methylation (Fig. 7A) (68, 69). This cluster includes histoneH2B residue Lys-123 (Lys-117 in the X. laevis nucleosomestructure (70)) that is targeted for ubiquitination (71, 72). It ishas been suggested that ubiquitination of histone H2B is re-quired for H3K4 methylation in a potential histone “cross-talk” mechanism (73). However, recent data suggest that theH2B C-terminal helix is recognized by COMPASS (68) andthat ubiquitination of H2B K123 stabilizes H2A/H2B dimerassociation with the nucleosome, perhaps making it a bettersubstrate for lysine 4 methylation by COMPASS (74). What-ever the mechanism, these data are consistent with a model inwhich the MLL1 core complex recognizes more than just theN-terminal tails of histones and that the structured globularcore domain of the nucleosome plays an important role in theregulation of nucleosomal H3K4 methylation.

Our demonstration that the fully assembled MLL1 corecomplex is required for nucleosome methylation suggests anovel mechanism for controlling nucleosomal H3K4 methyla-tion. We suggest that nucleosomal H3K4 methylation may becontrolled by factors that regulate the interaction betweenMLL1 and WRAD. In support of this hypothesis, we demon-strate that nucleosomal methylation by the MLL1 core com-plex requires Arg-3765 of MLL1, which we have previouslyshown to be required for the assembly and the H3K4 dim-ethylation activity of the MLL1 core complex (44, 51). In addi-tion, genome-wide chromatin immunoprecipitation studiesshow that although Menin, MLL1, and RbBP5 localize to thepromoters of thousands of genes in different mammalian celllines, they are not always found together (75). This result sug-gests that mechanisms exist in vivo to regulate the assembly ofthe MLL1 core complex in different genomic contexts andpresumably methylation of nucleosomal histone H3.Based on these observations, we propose the following

model (Fig. 7): MLL1 localizes to the promoters of targetgenes through the DNA-binding domains of MLL1 located inthe N-terminal half-of MLL1 (MLL-N, which includes DNAbinding AT hooks and CXXC domain (76)) and by interactionwith other transcription factors and co-activators (i.e.Menin(20, 39) or CBP (77)) but is inactive in the methylation of pro-moter-associated nucleosomes in the absence of WRAD (Fig.7B). Lack of promoter nucleosome methylation by MLL1 pre-vents the recruitment of other factors required for transcrip-tion (i.e. TFIID complex (78) or ATP-dependent nucleosomeremodeling complexes (1, 3, 79, 80)). WRAD binding to thenucleosome allows formation of the holo-MLL1 core com-plex, leading to liberation of the H3 N-terminal tail andmono-, di-, and presumably trimethylation of H3K4 and sub-sequently gene activation (Fig. 7C). The interaction of WRADwith the MLL1 core complex may be regulated by sequence-specific transcription factors that have been shown to interactwith Ash2L, such as Mef2d (81), Ap2� (32), or Tbx1 (82). Inaddition, based on previous results demonstrating that argi-nine methylation modulates the peptidyl arginine bindingactivity of WDR5 (83), we suggest that methylation of Arg-3765 of MLL1 may be a mechanism to control the assembly ofthe MLL1 core complex. Confirmation of this hypothesis willdepend on the identification of enzymes that methylate Arg-3765 of MLL1 in vivo.In summary, in this investigation we have characterized the

enzymatic activity ofWRAD, a newmulti-subunit histonemeth-yltransferase lacking a conserved SET domain.We also demon-strate thatWRAD, in addition to being required for sequentialH3K4methylation, is also required for methylation of nucleoso-mal histone H3 by theMLL1 core complex. Identification ofamino acid residues involved in the catalytic mechanism of thisenzyme will facilitate experiments designed for a greater under-standing of the roleWRAD plays in vivo.

Acknowledgments—We thank Kyle Fahey, Jeremy French-Lawyer, andMelody Sanders for a critical reading of this manuscript. We thankTim Richmond for histone plasmids and Andrew Flaus and TomOwen-Hughes for the murine mammary tumor virus LTR DNA.

FIGURE 7. Proposed model for the regulation of nucleosome methyla-tion by the MLL1 core complex. A, surface model of the X. laevis nucleo-some core particle (drawn with Protein Data Bank coordinate 1KX5 (70)).Highlighted are histone H2A and H2B residues required for H3K4 methyla-tion in budding yeast (69). Histone H3K4 is highlighted in yellow, H2A resi-dues are shown in dark blue, and H2B residues are green. B, MLL1 interactswith DNA through AT hooks and CXXC domains located in the MLL-N sub-unit of the MLL1 core complex. In the absence of WRAD, the histone H3N-terminal tail interacts with linker DNA and is not a substrate for methyla-tion. Consequently, transcription of MLL1 target genes is repressed. W,WDR5; R, RbBP5; A, Ash2L; and D, DPY-30. MLL-N is the 300-kDa N-terminalportion of MLL1, and MLL-C is the 180-kDa C-terminal portion of MLL1 con-taining the SET domain. C, the WDR5 component of WRAD interacts withArg-3765 of MLL1 resulting in the assembly of the MLL1 core complex. TheMLL1 complex interacts extensively with the nucleosome core and liberatesthe histone H3 N-terminal tail, which becomes a substrate for mono- anddimethylation at lysine 4, a critical step in the pathway that is required fortranscription of MLL1-dependent genes (indicated by the dashed arrow).



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CosgroveAnamika Patel, Valarie E. Vought, Venkatasubramanian Dharmarajan and Michael S.

Protein-1 (MLL1) Core ComplexSequential Nucleosomal Histone H3 Methylation by the Mixed Lineage Leukemia

A Novel Non-SET Domain Multi-subunit Methyltransferase Required for

doi: 10.1074/jbc.M110.174524 originally published online November 24, 20102011, 286:3359-3369.J. Biol. Chem.

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VOLUME 286 (2011) PAGES 1260 –1268DOI 10.1074/jbc.A110.183483

Nature of the ferryl heme in Compounds I and II.Andrea Gumiero, Clive L. Metcalfe, Arwen R. Pearson, Emma Lloyd Raven,and Peter C. E. Moody

PAGE 1263:

The legend to Fig. 1 should read as follows.FIGURE 1. Stereo images of the crystal structures of the ferryl hemeintermediates ofCcPandAPX.A–E, CcPCompound I (A), APXCom-pound II (B), APX Compound III (ferrous-oxy) (C), APX Compound I(D), and CcP Compound II (E), showing electron density maps calcu-latedwith coefficients 2Fo � Fc (contoured at 2�, shown in blue) and theFo � Fc map (contoured at 4�, shown in green) calculated after refine-ment omitting the oxygen. Oxygen atoms are shown as red spheres, theheme is in red, and the iron is shown as an orange sphere. Key residuesare labeled.

VOLUME 286 (2011) PAGES 3359 –3369DOI 10.1074/jbc.A110.174524

A novel non-SET domain multi-subunit methyltransferaserequired for sequential nucleosomal histone H3methylation by the mixed lineage leukemia protein-1(MLL1) core complex.Anamika Patel, Valarie E. Vought, Venkatasubramanian Dharmarajan,and Michael S. Cosgrove

PAGE 3363:

In Table 1, a factor of 10�4 was incorrectly inserted in front of theunits h�1 and �M�1 h�1 for the apparent Kcat and Kcat/Km values. As aresult, the reported values in Table 1 underrepresent the apparent Kcat

values by a factor of 104. Our error does not affect the conclusions of thearticle.We apologize for any confusion that this error may have caused.The corrected Table 1 is shown below.

VOLUME 285 (2010) PAGES 35967–35978DOI 10.1074/jbc.A110.155770

Forcing switch from short- to intermediate- and long-lived states of the �A domain generates LFA-1/ICAM-1catch bonds.Wei Chen, Jizhong Lou, and Cheng Zhu

PAGE 35972:

The y axis values of Fig. 2H were miscalculated by multiplying(instead of dividing) the values of Fig. 2G by the zero-force lifetime.

TABLE 1Summary of apparent kinetic parameters for WRA and WRADcomplexes

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 20, p. 18344, May 20, 2011© 2011 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

18344 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 286 • NUMBER 20 • MAY 20, 2011

ADDITIONS AND CORRECTIONS This paper is available online at www.jbc.org

We suggest that subscribers photocopy these corrections and insert the photocopies in the original publication at the location of the origi-nal article. Authors are urged to introduce these corrections into any reprints they distribute. Secondary (abstract) services are urged tocarry notice of these corrections as prominently as they carried the original abstracts.

anovelnon-setdomainmulti-subunitmethyltransferase ... · tions were incubated at 15 °c for 8 h and...

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