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1 Figure S1 Schematic of expressed protein ligation (EPL). Protein-αthioester is generated by thiolysis of the corresponding recombinant intein fusion protein. A native chemical ligation reaction is performed between this thioester and a synthetic peptide bearing an N-terminal cysteine. Transthioesterification, followed by an S-N acyl shift yields a native amide bond between the polypeptides. 1

2 Figure S2 Synthesis of H2B-C. a, Synthetic scheme showing synthesis of peptide 1. Resin 8 was prepared using solid phase peptide synthesis with an Fmoc protection strategy. v, K120 Mtt removal with TFA:TIS:DCM (1:1:98), was followed by coupling of bromoacetic acid to the exposed ε-nh 2, yielding peptidyl resin 9. vi, alkylation of resin 9 with S-(t-butyl) protected ligation auxiliary, followed by cleavage from the resin, afforded peptide 10. vii, TCEP treatment of peptide 10 immediately prior to ligation, yielded peptide 1. b, Analytical RP- HPLC analysis of peptide 10. c, ESI-MS characterization of peptide 10. Two charge states of the peptide are labelled. Peak at results from MS fragmentation of the ligation auxiliary attached to peptide

3 Figure S3 Characterization of ubiquitin(1-75) and H2B(1-116)-α-thioesters. a, Analytical RP-HPLC analysis, top panel, and ESI-MS analysis, bottom panel, of ubiquitin(1-75)-mes, 2. b, Analytical RP-HPLC analysis, top panel, and ESI- MS analysis, bottom panel, of H2B(1-116)-MES,

4 Figure S4 Characterization of semisynthesis intermediates. ESI-MS characterization of (a), ligation product 3; (b), photolysis product 4; and (c), ligation product

5 Figure S5 Characterization of recombinant Xenopus histone proteins by ESI-MS. ESI-MS characterization of (a), recombinant H2A; (b), H2B; (c), H3; (d), H4; and (e), H3(K79R). 5

6 Figure S6 Analysis of purified hdot1l proteins. a, Purified FLAG-hDot1L was analyzed by 8% SDS-PAGE and Coomassie staining. b, Purified hdot1l(1-416) was analyzed by 15% SDS-PAGE and Coomassie staining. Arrows mark expected sizes of the respective proteins. 6

7 Figure S7 hdot1l methyltransferase assay on unmodified substrates. a, A 3 H-SAM methyltransferase assay was performed with recombinant H3, recombinant octamers, a chromatinized plasmid with recombinant histones, and (H3/H4) 2 tetramers. Coomassie staining, bottom panel, was followed by fluorography, top panel. b, A 3 H-SAM methyltransferase assay was performed with unmodified, wild-type octamers, and nucleosomes, and ubiquitylated nucleosomes. Loading was verified by Coomassie staining, top panel, and methyltransferase activity was quantitated by scintillation counting. 7

8 Figure S8 hdot1l methyltransferase assay on chromatinized plasmids containing wild-type H2B and uh2b. a, Micrococcal nuclease digestion of chromatinized plasmids containing H2B, left, and uh2b, right to verify chromatin assembly. b, 3 H-SAM methyltransferase assay with 350 ng of chromatinized template containing H2B, left, and uh2b, right, and 15 ng FLAG-hDot1L. Assay samples were separated on 15% SDS-PAGE. Fluorography is shown. Arrow indicates projected position of H3 and the asterisk indicates H3 proteolysis product. 8

9 Figure S9 Ligation of 5 and 3 unmodified nucleosomes. A ligation was performed using 5 and 3 nucleosomes assembled with unmodified histones. Ligation products were separated on a 5% acrylamide TBE native gel and stained with ethidium bromide. MN = mononucleosome; DN = dinucleosome. 9

10 Figure S10 Methyltransferase assay with ubiquitin competition. a, A 3 H- SAM methyltransferase assay performed with mononucleosomes and FLAGhDot1L. 10 µm and 1 mm ubiquitin were added as indicated. Assay samples were separated on a 5% acrylamide TBE gel, stained with ethidium bromide, bottom panel of each pair, followed by fluorography, top panel. b, Filter-binding and scintillation counting were used to quantify methyltransferase activity in the presence and absence of 1 mm ubiquitin. Error bars indicate s.e.m. Asterisk indicates p < 0.05 using a two-tailed unpaired Student s t-test. (t-score = 2.28; 10 degrees of freedom; n = 6) 10

11 Figure S11 Gel-shift of radiolabeled nucleosomes with competing cold nucleosomes. 32 P labelled nucleosomes with and without uh2b were combined with five molar equivalents of hdot1l(1-416) and increasing molar equivalents of cold unmodified nucleosomes to compete the gel-shift of the radiolabeled nucleosomes. Samples were separated on a 5% TBE gel and imaged with a phosphorimager. All nucleosomes bear the H3(K79R) mutant to prevent methylation. The asterisk indicates 32 P labelling. 11