Evidence that ubiquitylated H2B corrals hDot1L on the nucleosomal surface to induce H3K79 methylation

Ubiquitylation of histone H2B at lysine 120 (H2B-Ub), a post-translational modification first discovered in 1980, plays a critical role in diverse nuclear processes including the regulation of transcription and DNA damage repair. Herein, we use a suite of protein chemistry methods to explore how H2B-Ub stimulates hDot1L-mediated methylation of histone H3 on lysine 79 (H3K79me). By using semisynthetic ‘designer' chromatin containing H2B-Ub bearing a site-specifically installed photocrosslinker, here we report an interaction between a functional hotspot on ubiquitin and the N-terminus of histone H2A. Our biochemical studies indicate that this interaction is required for stimulation of hDot1L activity and leads to a repositioning of hDot1L on the nucleosomal surface, which likely places the active site of the enzyme proximal to H3K79. Collectively, our data converge on a possible mechanism for hDot1L stimulation in which H2B-Ub physically ‘corrals' the enzyme into a productive binding orientation.

ThermoFisher) and Sequest HT (Thermo Fisher Scientific) search engine nodes were employed to match MS/MS spectra against a database consisting of H2A and Ub, allowing for a parent ion mass window of ±6 ppm, ≤3 missed trypsin cleavages, and methionine oxidation as a variable modification. 74.62% of H2A sequence was covered by identified peptides and 88.16% of Ub 9 sequence. Peptide identifications at high confidence are highlighted in green, medium confidence in yellow, low confidence in red. (b) Raw spectrum of crosslinked peptide, H2A(7-10)-Ub(55-74).
The most probable crosslinked peptide, H2A(7-10)-Ub(55-74), was identified by Stavrox 1 (v.3.4.12) and manually inspected using Xcalibur (v. 2.2, ThermoFisher). Left, high-resolution MS spectrum of the parent ion. Right, fragment ion mass spectrum between Ub(55-74) and H2A(1-10). The peptide sequence (with glutamate analogue indicated as 'e' and photo-Leu indicated as 'Z') is annotated to indicate the detected b n and y n ions. Ions of the crosslinked α-peptide are represented in red (b n ) and blue (y n ), while ions of the β-peptide are represented in pink (b n ) and green (y n ).

Preparation of ubiquitin thioester
All constructs (e.g. Ub

Synthesis of H2BssUb constructs by asymmetric disulfide formation
H2BssUb was prepared according to published protocols. 6 The purest HPLC fractions from HPLC purification were pooled and analyzed by ESI-MS (Supplementary Figure   13).

Generation of H2BssUb conjugate with photoLeu at position 73 of ubiquitin.
This construct was prepared in a similar manner to that of H2B-ubiquitin conjugate 1, as outlined in Figure 1b. The only difference was use of a synthetic C-terminal ubiquitin 22 fragment in which photoLeu was incorporated at position 73 to give Ub(64-76)E64C/L73L*. Otherwise, the synthesis was identical to that of 1.

Preparation of H2B-Ub conjugates by expressed protein ligation
H2B-Ub was prepared using an established sequential expressed protein ligation procedure. 7 Note, the ubiquitin in these semisynthetic constructs harbored a G76A mutation. The purest fractions from HPLC purification were pooled and analyzed by ESI-MS (Supplementary Figure 13).

DNA preparation
Unlabeled and biotinylated 601-147-1 was prepared by PCR as previously described 8 using unlabeled or biotinylated primers, respectively. For large scale mononucleosome formation, a 153bp segment containing the 601 DNA sequence was prepared as previously described 2 . For array formation, a plasmid containing 4 copies of a 177 base pair repeat of the 601 nucleosome positioning sequence (4-177-601) flanked by EcoRV sites was purified from a 6 L culture of DH5alpha cells as previously described. 8