Effect of MOF mediated UHRF1 acetylation on global DNA methyaltion maintenance in mouse embryonic stem cells.

The multi-domain protein UHRF1 (ubiquitin-like, containing PHD and RING finger domains, 1) recruits DNMT1 for DNA methylation maintenance during DNA replication. Here, we show that MOF (Males absent On the First) is an acetyltransferase of UHRF1 to acetylate UHRF1 at Lys670 in the pre-RING linker region whereas HDAC1 is a deacetylase of UHRF1 at the same site. The MOF/HDAC1-mediated acetylation in UHRF1 is cell-cycle regulated and peaks at G1/S phase, in line with the function of UHRF1 in recruiting DNMT1 to maintain DNA methylation. In addition, UHRF1 acetylation significantly enhances its E3 ligase activity and elimination of UHRF1 acetylation at these sites attenuates UHRF1-mediated H3 ubiquitination, which in turn impairs the DNMT1 recruitment and DNA methylation. Taken together, these findings not only identify MOF as a new acetyltransferase for UHRF1 but also reveal a novel mechanism underlying the regulation of DNA methylation maintenance through MOF-mediated UHRF1 acetylation. Mouse WT ESCs, Uhrf1-/- ESCs were cultured on 0.1% gelatin (Sigma) coated dishes in DMEM (high glucose, Gibco, 11965), supplemented with 15% ES-fetal bovine serum (Gibco, 16141-079), 100 units/mL Leukemia Inhibitory Factor (Millipore, ESG1107), GlutaMAX Supplement (Life Technologies, 35050), Sodium Pyruvate (Life Technologies, 11360), and 2-Mercaptoethanol (Life Technologies, 31350), maintained in a 37°C incubator with 5% CO2, and passaged every two days. Plasmids containing flag-myc-tagged human wild-type UHRF1, various domain deleted UHRF1, or different point mutated UHRF1 were transfected to Uhrf1 knockout ES cells using Lipofectamine 3000 (Invitrogen, L3000015) according to the manufacturer’s protocol. Cells were selected by Hygromycin and single colonies were picked to obtain cells with homogeneous gene expression levels. Total genomic DNA from different mESCs was extracted using TIANamp Genomic DNA Kit (TIANGEN, DP304). The double-enzyme RRBS experiments were performed according to a previously published protocol (PMC3570491).