Journal: Journal of Biochemistry
Article Title: DAXX promotes SUMOylation of chromatin-trapped DNMT1
doi: 10.1093/jb/mvag011
Figure Lengend Snippet: DAXX interacts with SUMOylated DNMT1 on chromatin through SIMs. (A) The multiple sequence alignment of DAXX from different indicated species. DAXX contains SUMO-interacting motifs (SIMs), four-helix bundle (4HB), histone binding domain (HBD) and acidic residue-rich domain ( ad ). (B) Xenopus sperm nuclei were incubated in egg extracts supplemented with buffer, 5-aza-dCTP or 5-aza-dCTP and SUMO inhibitor ML-792. Isolated chromatin was analyzed by immunoblotting with indicated antibodies. (C) Xenopus sperm nuclei were incubated in mock-depleted or DNMT1-depleted extracts in the presence of buffer or 5-aza-dCTP. Isolated chromatin was analyzed by immunoblotting with indicated antibodies. (D) 3× FLAG-tagged xDAXX WT, 3× FLAG-tagged xDAXX SIM1 mutants (I7AI8A point mutant or 1–14 deletion mutant) or 3× FLAG-tagged xDAXX SIM2 mutants (V911A/I912A point mutant or 909–916 deletion mutant) were added to DAXX-depleted extracts. (E) Xenopus sperm nuclei were incubated in mock-depleted or DAXX-depleted extracts supplemented with xDAXX WT, xDAXX SIM1 mutants or xDAXX SIM2 mutants in the presence of buffer or 5-aza-dCTP.
Article Snippet: For DNMT1 inhibition, egg extracts were incubated with 50 μM 5-aza-dCTP (Jena Bioscience) or with 100 μM GSK-3484862 (MedChemExpress).
Techniques: Sequencing, Binding Assay, Residue, Incubation, Isolation, Western Blot, Mutagenesis