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?5A). Furthermore, the fraction of 4?C cells entering mitosis, as assessed by the mitotic marker phospho-histone H3 (Ser10), was significantly (p?UNC2881 with fluorescently labelled Annexin V, which binds to phosphatidyl serine on the cell membrane of apoptotic cells. By 36?h after tamoxifen addition, approximately 68% of ES cells deleted for Sin3a undergo apoptosis, as compared with approximately 12% of untreated cells ( Fig.?5C). Taken together, these results indicate that cells lacking Sin3a arrest at the G2/M checkpoint and die by apoptosis. Given the lack of a functional G1/S damage checkpoint in ES cells, and prior gene expression evidence implicating Sin3a in DSB repair pathways in somatic cells, we hypothesised that the observed G2 arrest in ES cells upon loss of Sin3a could arise from failure to resolve DSBs. Indeed, probing whole-cell lysates from untreated and tamoxifen-treated this website ES cells revealed a rapid increase in ATM/ATR phosphorylation targets ��H2AX (S139), phospho-53BP1 (S166), and phospho-SMC1 (S966), coincident with the loss of Sin3a (Fig.?5D). Whereas H2AX and 53BP1 are hyperphosphorylated in response to a variety of DSB types, SMC1 is typically phosphorylated by ATM as part of the S-phase damage checkpoint (Kim et al., 2002?and?Yazdi et al., 2002). An increase in the abundance of these markers is consistent with DSBs arising during SRT1720 DNA replication in the absence of Sin3a. From this, we conclude that Sin3a is required to prevent or repair DSBs that arise in ES cells, possibly during DNA replication. To test whether failure to resolve replicative damage could account for the apparent p53-independent apoptosis seen in mutant ICMs, we next quantified levels of ��H2AX in Sin3a?/? blastocysts by immunofluorescence. Intensity of anti-��H2AX staining was measured in individual ICM and trophectoderm (TE) nuclei and normalised to anti-Oct4 staining intensity in the same cells. Plotting the resulting distributions reveals that both Sin3a?/? ICM and TE cells indeed have significantly increased ?H2AX staining as compared to wild-type or heterozygous littermates ( Fig.?6). It is important to note, however, that increased apoptosis was observed only in ICM cells of Sin3a?/? embryos ( Fig.?3), highlighting the distinct repair pathways in pluripotent vs. trophectoderm cells. Thus both the ex vivo and in vivo results are consistent with unresolved DNA damage leading to apoptosis of pluripotent cells in the absence of Sin3a. Sin3a has been reported to interact with key cell cycle regulatory proteins such as p53 and Rb, but these proteins play minor roles, if any, in self-renewal of pluripotent cells.