Fig. 6: Activation of MET1 by ubiquitinated histone H3.
a Structural comparison of the MET1 RFTS2 domain (left) and the DNMT1 RFTS domain bound to ubiquitinated H3 (right, PDB: 5WVO). Color schemes for the RFTS domains are consistent with those in Fig. 5. In the right panel, the histone H3 tail and ubiquitins are shown as green cartoons and transparent surface models, respectively. b Amino acid sequence of the putative UIM motif in the RFTS2 domain of MET1. c In vitro ubiquitination of C-terminal FLAG tagged H3 tail by VIM1 using wild-type ubiquitin (left) or K/R ubiquitin mutant (right). The ubiquitinated H3 was detected using anti-FLAG antibody. Experiments were independently repeated three times with similar results. d In vitro DNA methylation assay of MET1full in the presence of ubiquitin (ub), histone H3 tail (H3WT), mono-ubiquitinated H3s: H3C14ub (H3-14ub), H3C18ub (H3-18ub), and H3C23ub (H3-23ub), dual mono-ubiquitinated H3s: H3C14ub/18ub (H3-14/18ub2), H3C18ub/C23ub (H3-18/23ub2), and H3C14ub/C23ub (H3-14/23ub2), and triple mono-ubiquitinated H3: H3C14ub/C18ub/C23ub (H3-14/18/23ub3). DNA methylation rates were monitored after 1 h incubation with 10 μM hemimethylated DNA at 30 °C. Data are presented as the mean ± SD for n = 3 independent biological replicates. Statistical significance was assessed using a two-tailed Student’s t-test relative to MET1full. e Interaction between a series of RFTS domains of MET1 and the H3C18ub/C23ub analog (H3ub2S-S) analyzed by EMSA. Experiments were independently repeated three times with similar results.
