Cell Cycle and Developmental Regulations of Replication Factors in Mouse Embryonic Stem Cells

Hiroko Fujii-Yamamoto,Hisao Masai,Jung Min Kim,Ken-Ichi Arai

doi:10.1074/jbc.m412224200

Abstract

Embryonic stem (ES) cells can grow rapidly and permanently while maintaining their differentiation capacity. To gain insight into how the cell cycle progression of undifferentiated murine ES cells is regulated, we have examined the expression patterns of various replication and cell cycle regulators. Most factors including cyclins, Cdc6, and geminin are rather constitutively expressed during the cell cycle of ES cells. Furthermore, the transcript levels of almost all the cell cycle regulators we investigated except for p21 and p27 are higher in undifferentiated ES cells than in murine embryonic fibroblasts (MEFs), and the increased stability of mRNA in ES cells may be partially responsible for this at least with some of the factors. More strikingly, the transcriptional levels of these factors are strongly correlated with the acetylated state of histone H3 at their promoter regions. However, the methylation state of histone or CpG methylation of the promoter region is not generally correlated significantly with the expression pattern of these factors in both cell types. On the protein level, Cdc6, ASK, cyclin A2, and cyclin B1 are extremely abundant in ES cells compared with MEFs. Furthermore, they are rapidly down-regulated upon induction of differentiation of ES cells. The significance of these findings is discussed in relation to the unusual proliferative properties of ES cells in an undifferentiated state.

Highlights

Embryonic stem (ES) cells can grow rapidly and permanently while maintaining their differentiation capacity
P21 and p27 were significantly up-regulated during the same treatment. These results indicate that expression of Cdc6, ASK, cyclin A2, and cyclin B1 proteins is under the regulation of the differentiation state of the cells, and their overexpression is correlated with the undifferentiated state of ES cells
The results revealed no significant difference in the stability of Mcm2, ASK, cyclin A2, and cyclin B1 proteins between ES cells and murine embryonic fibroblasts (MEFs) (Fig. 6B)

Summary

EXPERIMENTAL PROCEDURES

Cell Culture and Synchronization—ES cell lines, CCE28 and D3, were cultured on gelatin-coated culture dishes without feeder layer. For synchronous release from M phase arrest, ES cells were first treated with 2.5 mM thymidine (Sigma) for 12 h, washed twice with prewarmed PBS and incubated in the presence of 150 ng/ml of TN-16 (Biomol) for 5 h. These cells were washed with PBS and released into prewarmed medium for the indicated time. For release from G1/S phase, cells were first treated with 2.5 mM thymidine (Sigma) for 12 h, washed twice with prewarmed PBS and released into prewarmed medium for 6 h. The primer sets used in this study were the same as those used in the ChIP assay (Table SII) or otherwise shown in Table SIII (Supplemental Materials)

RESULTS

Cell Cycle Regulators in Mouse Embryonic Stem Cells

DISCUSSION