Developmental Activation of the Rb–E2F Pathway and Establishment of Cell Cycle-regulated Cyclin-dependent Kinase Activity during Embryonic Stem Cell Differentiation

Abstract

To understand cell cycle control mechanisms in early development and how they change during differentiation, we used embryonic stem cells to model embryonic events. Our results demonstrate that as pluripotent cells differentiate, the length of G 1 phase increases substantially. At the molecular level, this is associated with a significant change in the size of active cyclin-dependent kinase (Cdk) complexes, the establishment of cell cycle-regulated Cdk2 activity and the activation of a functional Rb-E2F pathway. The switch from constitutive to cell cycle-dependent Cdk2 activity coincides with temporal changes in cyclin A2 and E1 protein levels during the cell cycle. Transcriptional mechanisms underpin the downregulation of cyclin levels and the establishment of their periodicity during differentiation. As pluripotent cells differentiate and pRb/p107 kinase activities become cell cycle dependent, the E2F-pRb pathway is activated and imposes cell cycle-regulated transcriptional control on E2F target genes, such as cyclin E1. These results suggest the existence of a feedback loop where Cdk2 controls its own activity through regulation of cyclin E1 transcription. Changes in rates of cell division, cell cycle structure and the establishment of cell cycle-regulated Cdk2 activity can therefore be explained by activation of the E2F-pRb pathway. INTRODUCTIONCell proliferation is coordinated by the activity of cyclindependent kinase (Cdk) activities (Nigg, 1995). This family of kinases functions by regulating the activity of proteins required for progression through the different cell cycle phases and hence must themselves be tightly cell cycle regulated. At one level, this is achieved through the cell cycleregulated synthesis of cyclin regulatory subunits, which bind and activate their catalytic Cdk partner. Inactivation of Cdk activity from one cell cycle phase is required for transition into the next by a mechanism involving cyclin destruction (Tyers and Jorgensen, 2000;Breeden, 2003). Hence, the ordered synthesis and destruction of phase-specific cyclin regulatory subunits are essential elements of normal cell cycle progression. Cyclin expression levels are controlled, in part at least, through transcriptional regulation. Cell cycleregulated cyclin E1 and A2 transcription is controlled by E2F transcription factors that are subject to repression through recruitment of "pocket proteins," such as pRb and p107, to the promoter regions of these genes. Repression of E2F-dependent transcription is lifted through phosphorylation of pocket proteins by Cdk activities that become active during G 1 phase (Harbour et al., 1999;Zhang et al., 2000), allowing for the transcriptional activation of genes required for the G 1 -S transition, such as cyclin E1.Pluripotent cells of late preimplantation and early postimplantation embryos exhibit two remarkable features. First, their cell division rates are very short, and their division is controlled through unusual mechanisms of Cdk regulation. Second, they have extraordinary developmental...

Human Embryonic Stem Cells Are Capable of Executing G1/S Checkpoint Activation

“…In somatic cells, the transition from G1 to S-phase, which is followed by DNA synthesis, is initiated by CDK4 and CDK6 in conjunction with D-type cyclins, and is further propelled mainly by CDK2 complexed with cyclin E and cyclin A, with the key target of these CDKs being retinoblastoma protein (RB)/E2F-mediated transcriptional regulation [21]. In mouse ESCs, the RB/E2F pathway is constitutively activated, thus eliminating effective regulation of the G1/S transition [22][23][24]. In contrast, several lines of evidence published by four independent laboratories and unpublished data from our laboratory indicate that hESCs can modify CDK activity to promote and/or inhibit cell cycle progression.…”

A New Coactivator Function for Zac1's C2H2 Zinc Finger DNA-Binding Domain in Selectively Controlling PCAF Activity

“…Pluripotent cells of embryonic origin exhibit a short G 1 phase that is driven by precocious and unrestrained cyclin expression (12,26,34). Although differentiation is coupled with the upregulation of p21 Cip1 and p27 Kip1 (40), many of the mechanisms and factors underlying cell cycle maturation in early neurons are largely unknown. While a role for p53 has been proposed in these processes (26), our data show that Lif withdrawal results in a strong downregulation of p53 mRNA and protein expression.…”

Screening of human pluripotent stem cells using CGH and FISH reveals low-grade mosaic aneuploidy and a recurrent amplification of chromosome 1q

“…As it turns out, in early development, the strict requirements for cell growth are suspended to allow rapid cell proliferation 38,39 and is activated again when ESCs are induced to differentiate into embryoid bodies. 40,41 A recent study, conducted by Mayshar et al 34 , identified a substantial number of hiPS cell lines carrying full and partial chromosomal aberrations. To conclude, both hESCs and hiPSCs should be tightly monitored regarding their genomic stability.…”