Figure 4: MPP8 recognizes methylated Dnmt3a.

(a) ITC measurement of binding of the MPP8 chromodomain to mDnmt3a peptide with (top) and without (bottom) K44me2 methylation. (b) The chromodomain of MPP8 is sufficient to interact with hDNMT3A. Various domains of MPP8 are indicated at left of the panels with a schematic presentation of the Flag-MPP8 constructs. Different MPP8 deletions were coexpressed with full-length Myc-hDNMT3A in 293T cells for immunoprecipitation–western analysis using various antibodies. (c) The N-terminal domain of hDNMT3A associates with endogenous MPP8. Schematic representation shows Myc-hDNMT3A deletion proteins with numbers indicate amino-acid positions (left). The 293T cells transiently transfected with plasmids encoding various Myc-hDNMT3A proteins. Cell lysates were immunoprecipitated with anti-Myc antibody. and western blot was carried out using anti-Myc or MPP8 antibodies (right). (d) K47 methylation stimulates MPP8-hDNMT3A interaction in cells. Myc-hDNMT3A (WT or K47R mutant) was coexpressed with HA–GLP in GLP stable knockdown 293T cells. After Myc-IP, western blot using indicated antibodies analysed methylation of hDNMT3A and its interaction with co-precipitated endogenous MPP8. In, 1% input. (e) Myc-hDNMT3A (WT or K47R mutant) was coexpressed with Flag-G9a in GLP stable knockdown 293T cells (In, 1% input). (f) Monomer structure of MPP8 chromodomain (in surface representation displayed in blue for positive, red for negative and white for neutral) bound to a peptide of hDNMT3AK47me2 (in stick model coloured green). (g) K47me2 of hDNMT3A binds in a partial hydrophobic cage of MPP8 formed by three aromatic residues (Phe59, Trp80 and Tyr83) and one aspartate (Asp87). One of its terminal N-CH₃ groups projects towards the three aromatic rings, whereas the second methyl group points towards Asp87, a typical arrangement of methyl–lysine binding cage⁵². (h) Substitution of the cage-forming residue (W80A) impairs MPP8 interactions with hDNMT3A. Flag-MPP8-WT or W80A mutant was coexpressed with Myc-hDNMT3A in MPP8 stably knockdown 293T cells. Immunoprecipitation–western analyses were carried out using the antibodies indicated. Cell lysates were briefly sonicated followed by micrococcal nuclease treatments before immunoprecipitation to release chromatin-bound proteins. In, 2% input.