O-GlcNAc transferase associates with the MCM2–7 complex and its silencing destabilizes MCM–MCM interactions

Abstract

O-GlcNAcylation of proteins is governed by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). The homeostasis of O-GlcNAc cycling is regulated during cell cycle progression and is essential for proper cellular division. We previously reported the O-GlcNAcylation of the minichromosome maintenance proteins MCM2, MCM3, MCM6 and MCM7. These proteins belong to the MCM2–7 complex which is crucial for the initiation of DNA replication through its DNA helicase activity. Here we show that the six subunits of MCM2–7 are O-GlcNAcylated and that O-GlcNAcylation of MCM proteins mainly occurs in the chromatin-bound fraction of synchronized human cells. Moreover, we identify stable interaction between OGT and several MCM subunits. We also show that down-regulation of OGT decreases the chromatin binding of MCM2, MCM6 and MCM7 without affecting their steady-state level. Finally, OGT silencing or OGA inhibition destabilizes MCM2/6 and MCM4/7 interactions in the chromatin-enriched fraction. In conclusion, OGT is a new partner of the MCM2–7 complex and O-GlcNAcylation homeostasis might regulate MCM2–7 complex by regulating the chromatin loading of MCM6 and MCM7 and stabilizing MCM/MCM interactions.Electronic supplementary materialThe online version of this article (10.1007/s00018-018-2874-0) contains supplementary material, which is available to authorized users.

Cyclin D1 Stability Is Partly Controlled by O-GlcNAcylation

“…The various treatments with the inhibitors were done as indicated in the text and figure legends. Reverse transfection of siRNA was performed in complete medium (2 × 10 5 or 3.75 × 10 5 cells in 6-well plates for HEK 293T and MCF7 cells respectively; 6 × 10 5 or 1 × 10 6 in 100-mm dishes for HEK 293T and MCF7 cells, respectively), as previously described (41). Transfected cells were harvested 72 h later.…”

Exploring the Potential of β-Catenin O-GlcNAcylation by Using Fluorescence-Based Engineering and Imaging

“…Briefly, HeLa cells were transfected with the different GFP-β-catenin constructs ( Figure 2 a) and treated with 200 µM of Ac 4 GalNAz for 24 h. Then, 50 µg of the labeled proteins were resuspended in 20 mM HEPES and 1% ( w / v ) SDS, pH 7.9, and incubated for 1 h at room temperature with a 4.4 kDa DBCO-PEG (polyethylene glycol) at a final concentration of 10 mM in DMSO. DBCO-PEG was synthesized, as previously described [ 35 ]. One volume of PEGylated proteins was precipitated using the mix of chloroform:methanol:water (3:0.75:2) in order to remove the excess of DBCO-PEG.…”

Post-Translational Modifications of the Mini-Chromosome Maintenance Proteins in DNA Replication

“…MCM2–7 proteins (particularly present in the chromatin-bound fraction) are all substrates of O -GlcNAcylation in human cells [146,147,148]. We explored the YinOYang1.2 server and found some predicted MCM O -GlcNAcylation sites (Table 6) [149].…”

“…We explored the YinOYang1.2 server and found some predicted MCM O -GlcNAcylation sites (Table 6) [149]. Moreover, OGT directly interacts with MCM3, MCM6, and MCM7; OGT depletion decreases the interactions between MCM subunits, which subsequently impairs chromatin loading with no impact on replication rate [148]. This finding indicates that O -GlcNAcylation likely predominantly occurs on the dormant MCM complex.…”

“…This finding indicates that O -GlcNAcylation likely predominantly occurs on the dormant MCM complex. Given that OGT can be recruited to DNA damage sites [150] and that the dormant MCM complex is required to maintain genome stability [16], MCM O -GlcNAcylation may help ensure a rapid response to DNA damage and replication stress [148].…”