Gene expression analysis of pluripotent and differentiated genes during EB formation treated with streptozotocin, o-glacnacylation inhibitor

O-linked-N-acetylglucosamine (O-GlcNAc) has emerged as a critical regulator of diverse cellular processes, but its role in embryonic stem cells (ESCs) and pluripotency has not been investigated. Here we show that O-GlcNAcylation directly regulates core components of the pluripotency network. Blocking O-GlcNAcylation disrupts ESC self-renewal and reprogramming of somatic cells to induced pluripotent stem cells. The core reprogramming factors Oct4 and Sox2 are O-GlcNAcylated in ESCs, but the O-GlcNAc modification is rapidly removed upon differentiation. O-GlcNAc modification of Threonine 228 in Oct4 regulates Oct4 transcriptional activity and is important for inducing many pluripotency related genes, including Klf2, Klf5, Nr5a2, Tbx3 and Tcl1. A T228A point mutation that eliminates this O-GlcNAc modification reduces the capacity of Oct4 to maintain ESC self-renewal and reprogram somatic cells. Overall, our study makes a direct connection between O-GlcNAcylation of key regulatory transcription factors and the activity of the pluripotency network. 2 of E14 stem cell, 2 of embryonic body at day 2, 2 of embryonic body at day 2 treated with streptozotocin, 1 of ZHBTc4 stem cell treated with doxicyclin, and 1 of ZHBTc4 stem cell treated with doxicyclin and streptozotocin were analysed