Robust mechanisms are required to protect the genomic integrity of pluripotent embryonic cells that give rise to the entire organism and future generations through the germ line. Consequently, embryonic stem (ES) cells and, by inference, cells of the inner cell mass (ICM) have considerably lower mutation frequencies than somatic cells (Hong et al., 2007
) and are highly sensitive to DNA damage and other genotoxic stresses (de Waard et al., 2003; Roos et al., 2007; Van Sloun et al., 1999
The intolerance of ES cells toward DNA damage is facilitated by elaborate mechanisms compromising the G1 damage checkpoint and abating the opportunity for repair, routing damaged cells to the more stringent, intact G2/M checkpoint and ultimately apoptosis. In self-renewing ES cells, checkpoint proteins such as p53 (encoded by the Trp53
gene) are sequestered almost entirely in the cytosol, thus preventing effective regulation of their nuclear targets in response to DNA damage (Aladjem et al., 1998; Hong and Stambrook, 2004; Lin et al., 2005; Solozobova et al., 2009
). Indeed, ES cells deleted for Trp53
readily undergo apoptosis in response to DNA damage with kinetics similar to wild-type cells (Aladjem et al., 1998; Lin et al., 2005; Solozobova et al., 2009
), despite a failure to induce canonical somatic targets of p53.
As with the response to DNA damage, models of cell cycle control in the epiblast are largely inferred from studies of mouse ES cells (for reviews see (Orford and Scadden, 2008; White and Dalton, 2005
)). The extremely rapid divisions of mouse ES cells are accomplished by the effective elimination of the early G1 phase. Unlike in somatic cells, constitutively high expression of Cyclin E1 stimulates Cdk2 activity throughout the entire ES cell cycle, thereby maintaining Rb in a hyperphosporylated, inactive state and bypassing both the restriction point (R) and the need for mitogen stimulation of Cyclin D-Cdk4/6 activity (Burdon et al., 2002; Savatier et al., 1994; White et al., 2005
). This constitutive inactivation of Rb results in continual activation by E2f1 of its targets, including Cyclin E1
and the replication machinery, thus rapidly driving ES cells from mitosis into S phase regardless of any damage or stress that might have occurred.
Sin3a function is essential for the growth and viability of mouse embryonic fibroblasts (MEFs). Deletion of Sin3a
in MEFs results in a profound growth defect, significant G2/M accumulation, and increased apoptosis in conjunction with de-repression of hundreds of Myc, E2f1, E2f4, and p53 targets that control cell cycle progression, DNA replication and repair, and cell death (Dannenberg et al., 2005
). Interestingly, while the induction of p21
) in response to Sin3a
deletion requires p53, the growth arrest, replication defects, and apoptosis were not alleviated by either ablation or functional inactivation of p53. Furthermore, genes involved in both non-homologous end-joining (NHEJ) and homologous recombination (HR) repair pathways were aberrantly upregulated in MEFs lacking Sin3a
(Dannenberg et al., 2005
), suggesting a novel role in balancing the relative activities of these two double-strand break (DSB) repair strategies in addition to its reported chromatin-modifying functions during NHEJ (Jazayeri et al., 2004
) and DNA replication (Aparicio et al., 2004
) in yeast.
Genetic studies have shown that Sin3a
is required for early mouse embryonic development between E3.5 and gastrulation at E6.5 (Cowley et al., 2005; Dannenberg et al., 2005
). Similarly, knock-down of Sin3a by siRNAs resulted in severely impaired proliferation in ES cells (Fazzio et al., 2008
). However, as outlined above, many of the genes overexpressed in Sin3a
MEFs that led to growth defects and apoptosis are already highly expressed in early embryonic cells, and thus their de-repression in the ICM or ES cells is not necessarily expected to have such adverse effects. Therefore, the mechanisms underlying this embryonic requirement for Sin3a are not at all clear.
To better understand the requirement for Sin3a/HDAC complexes in early mammalian embryogenesis, we examined in detail embryos and ES cells deleted for Sin3a. We found that Sin3a is absolutely required for proliferation and survival of cells in the ICM as embryos implant, but cells in the trophectoderm lineage appeared largely normal in Sin3a
−/− embryos. We identify a number of proteins interacting with Sin3a in ES cells, further implicating the complex in cell cycle control and the DNA damage response. We propose that Sin3a is essential to maintain both the unusual cell cycle of pluripotent embryonic cells and their genomic integrity.