Here we show that CtIP a bona fide
Here we show that CtIP, a bona fide regulator of DNA end resection (Huertas and Jackson, 2009; Huertas et al., 2008; Sartori et al., 2007), is upregulated during cell reprogramming, and that it is essential for this process. Similarly, CHK1 has been suggested to be pertinent to generating iPSCs (Ruiz et al., 2015). CtIP is known to be a key player in maintaining genomic stability, and we reasoned that CtIP could be appropriately activated to repair DSBs generated by the presence of reprogramming factors. Selective CtIP depletion during mouse and human cell reprogramming interferes with iPSC generation and triggers apoptosis. In agreement with these data, BRCA1, which accelerates DNA resection through its interaction with CtIP (Cruz-Garcia et al., 2014), is also required for successful reprogramming (Gonzalez et al., 2013). In addition, other proteins linked to HR repair, such as BRCA2 and RAD51, are critical for cell reprogramming, at least in mice (Gonzalez et al., 2013). We also observed that CtIP depletion during cell reprogramming in mouse and human EHT 1864 hampers the growth and maintenance of their derived iPSCs. As a matter of fact, the human C1 clone obtained from cells bearing an inducible shRNA in the presence of IPTG showed the same long-term problems in viability regardless of the restoration of CtIP expression (data not shown, see Figure 3G for CtIP expression recovery). Likewise, Brca1-deficient MEFs have problems for cell reprogramming, and the derived iPSCs are unable to establish colonies (Gonzalez et al., 2013). This clearly differentiates proteins involved in early steps of HR, such as CtIP and BRCA1 in resection, from those affecting later steps, such as BRCA2 and RAD51, which are in fact dispensable for iPSC colony expansion (Gonzalez et al., 2013). We reasoned that iPSCs are defective for DNA end resection, which is critical for choosing between HR and NHEJ (Gomez-Cabello et al., 2013). As mentioned before, we suggest that both pluripotent and differentiated cells can likely repair their DNA by both NHEJ and HR, such that eliminating one of them only has a mild effect on cell viability unless an exogenous source of damage is present. However, during cell reprogramming, recombination is the only mechanism able to deal with the endogenous damage. We postulate that this is due to the nature of the DNA lesion, as replication stress will readily cause the appearance of one-ended DNA DSBs and will require recombination to restore replication. In the absence of CtIP, those breaks would be erroneously repaired, inducing the observed chromosomal abnormalities and CNV differences (Figures 3H and 5F). Indeed, this seems to be a recurrent mechanism that causes cells undergoing reprogramming to acquire a high degree of chromosomal instability. Even by analyzing cells from a single clone that differentiated in the absence of CtIP (the C1 clone), we can observe that each metaphase has a different number of chromosomes (Figure 3H). Thus, in the absence of CtIP, DSBs created by replication stress would be repaired through more mutagenic repair pathways, thereby increasing mutagenesis and chromosomal rearrangements (Bunting et al., 2010). This is consistent with an increase in DNA damage and genetic instability and would ultimately lead to apoptosis during reprogramming. We could not expand the only clone we were able to obtain from CtIP-depleted hiPSCs (C1), but did establish a few clones from CtIP-depleted miPSCs, albeit with low efficiency. These clones showed high levels of alterations in chromosome number and CNV with respect to iPSCs generated under normal CtIP levels. These data are consistent with early embryonic lethality (E4.0) observed for CtIP knockout mice, which shows a slightly elevated apoptosis (Chen et al., 2005). Although this lethality has been previously associated with the retinoblastoma protein, our data support the idea that the resection function of CtIP is required for embryonic viability (Polato et al., 2014). Curiously, we found that established pluripotent cells, namely miPSCs and ES-D3 cells, did not require CtIP protein (at least not in the absence of an exogenous source of DNA damage). Cell reprogramming seems to be one such source of internal stress, so it is possible that other situations arise during normal embryogenesis in which CtIP, and specifically its resection activity, are essential.