Critical role for P53 in regulating the cell cycle of ground state embryonic stem cells [RNA-seq]. Critical role for P53 in regulating the cell cycle of ground state embryonic stem cells [RNA-seq]

Mouse Embryonic Stem Cells (ESCs) grown in serum-supplemented conditions are characterized by an extremely short G1-phase due to the lack of G1-phase control. Concordantly, the G1-phase-specific P53-P21 pathway is compromised in serum ESCs. Here we provide evidence that P53 is activated upon transition of serum ESCs to their pluripotent ground state using serum-free 2i conditions and modulates G1-phase progression. Our data shows that the elongated G1-phase characteristic of ground state ESCs is dependent on P53. RNA-seq and ChIP-seq analyses reveal that P53 directly regulates the expression of the Retinoblastoma (RB) protein and that the hypo-phosphorylated, active RB protein plays a key role in G1-phase control. Our findings suggest that the P53-P21 pathway is active in ground state 2i ESCs and that its role in the G1-checkpoint is abolished in serum ESCs. Taken together, the data reveals a mechanism by which inactivation of P53 can lead to loss of RB and uncontrolled cell proliferation. Overall design: FUCCI reporter constructed R1 mESCs were cultured in either serum or 2i conditions. G1-phase cells were sorted and collected using the BD FACS Aria. RNA were extracted from G1 cells and library prepared

小鼠胚胎干细胞（Mouse Embryonic Stem Cells, ESCs）在血清补充培养基中培养时，因缺乏G1期调控而呈现G1期极短的特征。与此一致的是，血清培养的ESCs中G1期特异性的P53-P21通路功能受损。本研究证实，当血清培养的ESCs在无血清2i条件下转换至多能基态时，P53被激活并调控G1期进程。本研究数据显示，基态ESCs所特有的延长G1期依赖于P53。RNA测序（RNA-seq）与染色质免疫沉淀测序（Chromatin Immunoprecipitation Sequencing, ChIP-seq）分析表明，P53直接调控成视网膜细胞瘤（Retinoblastoma, RB）蛋白的表达，而去磷酸化的活性RB蛋白在G1期调控中发挥关键作用。本研究结果提示，P53-P21通路在基态2i ESCs中具有活性，但其在G1检验点的功能在血清培养的ESCs中丧失。综上，本研究数据揭示了P53失活可通过何种机制导致RB表达缺失及细胞增殖失控。实验设计：将构建了FUCCI报告基因的R1小鼠ESCs分别在血清或2i条件下培养。采用BD FACS Aria流式细胞仪分选并收集G1期细胞，从G1期细胞中提取RNA并构建测序文库。