LAUSR

Dear Editor, Histone marks deposited by post-translational modifications (PTMs) frequently occur in interrelated combinational patterns to create a complex and precise control on the chromatin structure and function. One of the landmark findings of histone PTM crosstalk is the trans-histone regulation of histone H3 lysine 79 (H3K79) methylation by the monoubiquitination of histone H2B on lysine 120 (H2BK120). Mono-, di-, and tri-methylation of histone H3K79 serves as a prominent histone mark that participates in transcription regulation and DNA damage response. H2BK120 monoubiquitination (H2BK120ub1) is a prerequisite for the efficient methylation of H3K79 by the unique non-SET domaincontaining histone methyltransferase DOT1L (Disrupter of telomere silencing protein 1-like) in vivo. Incorporation of chemically monoubiquitinated H2B into in vitro reconstituted nucleosome directly stimulates the catalytic activity of DOT1L (Supplementary information, Figs. S1 and S2). It still remains poorly understood how the H3K79 methyl marks are deposited and how the associated PTM crosstalk occurs on nucleosome. Here we present the cryo-electron microscopy (cryo-EM) structure of the catalytic domain of human DOT1L (residues 1–416) in complex with an H2BK120ub1 nucleosome core particle (abbreviated as ubNCP) at an overall resolution of 4.1 Å (Supplementary information, Figs. S3-S5 and Table S1). The structure of DOT1LubNCP reveals that DOT1L extensively interacts with core histones on the disk-face of nucleosome (buried surface area, ~2020 Å), with its C-terminal region (residues 269–331) sandwiched between ubiquitin and the histone H2A-H2B dimer (Fig. 1a). The direct association of DOT1L with the H2BK120-conjugated ubiquitin extends the recognition interface between DOT1L and histone surface, and probably increases the binding affinity of DOT1L toward the H2BK120ub1 nucleosome. We used a DOT1L construct (residues 1–351), which lacks a positively charged region that binds the nucleosomal DNA, to compare its binding affinity to the histone surface of NCP and ubNCP. GST-pull-down assay clearly showed that DOT1L1–351 only interacts with the ubiquitinated nucleosome, indicating that H2BK120ub1 dramatically increases the weak association of DOT1L with the histone surface of nucleosome (Supplementary information, Fig. S6b, lanes 7–9). The increased binding affinity toward the H2BK120ub1 nucleosome results in the enhanced catalytic efficiency of DOT1L and the accumulation of higher levels of H3K79 diand tri-methylation (Supplementary information, Fig. S2). We also determined the cryo-EM structure of DOT1L in complex with an unmodified NCP at an overall resolution of 5.0 Å (Supplementary information, Fig. S7 and Table S1). The density of DOT1L in this 3D reconstruction is not clear enough to build an atomic model. However, docking of the DOT1L-ubNCP complex structure into this map shows a good fit of the DOT1L-ubNCP structure with the DOT1L-NCP density map, indicating that monoubiquitination of H2BK120 does not change the overall location of DOT1L on the histone surface of nucleosome (Supplementary information, Fig. S7g and h). EM density maps of NCP from the two structures reach to a similar range of resolution (3.5–4.5 Å), whereas the density of DOT1L from the unmodified complex is severely dampened and blurred (Supplementary information, Figs. S3 and S7). This observation suggested that the interaction of DOT1L with the histone surface might be very dynamic, whereas ubiquitin attachment to H2BK120 could rigidly constrain the mobility of DOT1L and lead to an unambiguous 3D reconstruction of the DOT1L-ubNCP complex. Ubiquitin is a highly conserved 76-amino-acid protein with several well-characterized surface patches, such as the I44 patch (residues Leu8, Ile44, His68, and Val70) and the I36 patch (residues Ile36, Leu71, and Leu73), which are frequently recognized by ubiquitin-binding proteins (Supplementary information, Fig. S8a). The DOT1L-ubNCP complex structure indicates that the I36 patch is in close proximity to a hydrophobic surface composed of residues Leu284, Ile290, Leu322, Tyr325, Phe326, and Leu329 within the C-terminal region (residues 269–331) of DOT1L (Fig. 1b). Residue Ile36 of ubiquitin stacks on DOT1L Phe326, which serves as an important recognition interface between ubiquitin and DOT1L (Fig. 1b). Substitution of Phe326 with alanine impaired the stimulatory effect of H2BK120ub1 on DOT1L activity (Fig. 1h, lane 5). By contrast, it had no impact on the catalysis of DOT1L toward the unmodified nucleosome (Supplementary information, Fig. S8g, lane 5). Double mutations of F326A and I36A resulted in a further decrease in the activity of DOT1L toward the mutant ubNCP (Fig. 1h, lane 6). Previously, an alanine scan of the ubiquitin surface revealed that H2BK120ub1 with alanine substitutions of the ubiquitin residues Leu71 and Leu73 failed to stimulate the DOT1L activity. This is consistent with our structure that the I36 patch of ubiquitin participates in the recognition of DOT1L. In addition to binding to ubiquitin, the C-terminal region of DOT1L also interacts with the histone H2A-H2B dimer (Fig. 1a). A long loop (DOT1L, residues 269–290) that connects two parallel strands (β5 and β6) of the central β sheet of DOT1L confers specific recognition of the H2A-H2B surface (Fig. 1c, d). DOT1L sits on the C-terminal helix (αC) of histone H2B and is juxtaposed with the C-terminus of ubiquitin on the αC helix (Fig. 1c). At the DOT1L-H2B interface, DOT1L Pro274 constrains the conformation of DOT1L around residue His109 of H2B, and DOT1L Asp286 forms a salt bridge with residue Lys116 of H2B (Fig. 1c). Mutation of Pro274 or Asp286 severely impaired the HMT (histone methyltransferase) activity of DOT1L toward NCP and ubNCP (Fig. 1i). P271L, a melanoma-associated mutation of DOT1L, is located near to the Pro274-His109 interface (Fig. 1c). It decreased the activity of DOT1L on NCP, but had no distinguishable defect in the methylation of ubNCP (Fig. 1i). Therefore, the