Figure 6: MPP8 binds self-methylated GLP.

(a) GLP self-methylates lysine residue at K205. Wild-type (WT) or K205R mutated GST–GLP–N terminal fragment (residues 191–280) was incubated with (lanes 3–4) or without (lanes 1–2) His–GLP–SET domain (residues 955–1,298) for methylation assays. Auto-radiogram (right) shows methylation. (b) MPP8 binds to methylated GLP at K205 in vitro. GST–GLP–N–WT or its K205R mutant in the context of fragment 191–280 was incubated with His–GLP–SET domain for in vitro methylation. After being purified and immobilized on glutathione beads, GST–GLP–N terminal fragment was incubated with in vitro translated FL-³⁵S–MPP8–WT (top panel) or its W80A mutant (bottom panel) for GST pull-down. Label 'B' indicates the bound fraction and 'Ft' the flow through. In, 5% input. (c) MPP8–GLP interaction is mediated through GLP–K205 in cells. Flag-MPP8-WT was coexpressed with HA-GLP–WT, K199R, K205R or K209R mutant in 293T cells. Cell lysates were immunoprecipitated with anti-HA (top panel) or Flag (bottom panel) antibody. Co-immunoprecipitated Flag-MPP8 or HA–GLP were analysed by western blot using indicated antibodies. In, 5% input. (d) MPP8–GLP interaction requires intact chromodomain of MPP8. HA-GLP–WT was coexpressed with Flag-MPP8-WT or its W80A mutant in MPP8 stably knockdown 293T cells. Immunoprecipitation–western analyses were carried out using indicated antibodies. Methyl–lysine binding deficient MPP8 mutant W80A abrogates MPP8–GLP interaction in cells (comparing lanes 3 and 6). In, 5% input. (e) A hypothetical model of G9a/GLP–Dnmt3a-MPP8 complex mediated by generating and reading of methyl–lysine. A cartoon illustration of the proposed model of signalling cross-talk between the K44 methylated Dnmt3a, by the GLP component of the GLP/G9a heterodimer⁶, and recognized by the MPP8, resulting in a repressive chromatin with H3K9 methylation and DNA CpG methylation on chromatin templates.