Fig. 3

Crosstalk between H2BK120ub and the N-terminal tail of H2A. a In vitro ubiquitylation assays. MNs containing truncated or acetylated versions of H2A were treated with purified human E1, UBE2A, and RNF20/40 ligases. Top: western blot analysis of in vitro ubiquitylation reactions. Bottom: quantification of the immunoblotting data. Normalized H2BK120ub levels are plotted relative to wild-type (wt) MNs (n = 4 for H2AΔ10 and H2AΔ15; n = 3 for all other samples). b As per a but employing 12mer nucleosome arrays possessing the indicated histone PTMs (n = 6). c Top: sequence alignment of the N-terminal tails of human H2A variants. Bottom: superposition of the crystal structures of MNs containing canonical H2A (PDB: 1KX5) and H2A.Z (PDB: 1F66). The secondary structure of H2A is rendered in yellow and H2B is in red. H2AK15/H2A.Z V17 and H2BK120 are shown as sticks. In vitro ubiquitylation assays using MNs (d) or 12mer nucleosome arrays (e) containing H2A/H2A.Z mutants and chimeras (H2A_pQ: H2A K5/9/13/15Q; H2A_TS: N-terminal tail H2A.Z fused to the H2A core; H2A.Z_TS: N-terminal tail H2A fused to the H2A.Z core; for complete amino acid sequences, see Supplementary Fig. 7c). Top: western blot analysis of in vitro ubiquitylation reactions. Bottom: quantification of the immunoblotting data as per panel a (n = 5 for d; n = 6 for e). All data are mean ± SEM (full western blot images are presented in Supplementary Fig. 15)