Although our results suggest that Ronin

Although our results suggest that Ronin directly impacts DNA repair through the transcriptional regulation of DNA repair genes, we further sought to determine how much the canonical pluripotency factors might contribute to the transcriptional regulation of DNA repair genes. Oct4 and Sox2 are known to bind upstream of the Gtf2h4 and Rad18 genes (Marson et al., 2008), yet the levels of Oct4 and Sox2 were not affected by Ronin knockout on transcriptional level in our microarray analysis. This observation applies to other pluripotency related factors as well (Supplemental Table S2; Li and Belmonte, 2017). Additionally, Nanog, Suz12 and Tcf3 are also known not to bind to Gtf2h4 or Rad18 (Dejosez et al., 2010). While our results do not formally exclude the involvement of these or other factors, they support the notion that Ronin plays a decisive role in the transcriptional regulations of Rad18 and Gtf2h4. We previously proposed a model in which Ronin regulates its target genes by recruiting histone-modifying enzymes via an interaction with Hcf-1 (Dejosez et al., 2010, 2008). Because Gtf2h4 and Rad18 are bound by Ronin and downregulated upon Ronin knockout, transcriptional regulation of Gtf2h4 and Rad18 could be mediated in part through this mechanism. Stalled replication forks resulting from UV-C damage and other genotoxins are known to activate a DNA damage checkpoint that involves Atr and Chk1 (Heffernan et al., 2002; Liu et al., 2000). The increase in phospho-Chk1 in Ronin-knockout ABT888 after UV-C damage may be explained by an increase in unrepaired damage and/or the inability of DNA replication to proceed past UV-C damage (Coin et al., 2007; Marinoni et al., 1997; Tateishi et al., 2003). Furthermore It is known that knockout of genes in ESCs that are involved in nucleotide excision repair (e.g. Xpc) leads to slowed S phase progression and G2/M arrest after UV-C damage (de Waard et al., 2008). Those observations are in line with our results in Ronin - knockout cells, suggesting that nucleotide excision repair defects contributed to the increased Chk1 phosphorylation and G2/M accumulation observed in our present study. Ronin belongs to a unique protein family characterized by a highly conserved THAP DNA binding domain (Roussigne et al., 2003). The Thap family proteins arose through a process called “molecular domestication,” beginning from an ancient DNA transposon whose modern-day descendent is the P-element transposase (Hammer et al., 2005). As transposition of such elements involves DNA repair, we cannot exclude the possibility that Ronin in addition to its function as a transcriptional regulator (Dejosez et al., 2010; Sabogal et al., 2010) may play a more direct role in the DNA damage response (Weinert et al., 2005). Additionally, other Thap family members, including Thap5, have been suggested to play pro-apoptotic roles in the responses to UV-C irradiation and other sources of stress (Balakrishnan et al., 2011, 2009) and Thap9 was directly shown to have DNA nuclease activity, making this possibility even more likely (Majumdar et al., 2013).
Conclusion In summary, we show Ronin-knockout ESCs to exhibit reduced expression of factors that respond to UV-C damage, as well as increased phospho-Chk1 and G2/M arrest. Future work is warranted to examine whether Ronin performs comparable functions in other cell types. Given the significant differences between ESCs and somatic cells in terms of their DNA repair capacities, spontaneous mutation rates, and cell cycle structures, the insights gained here may not apply universally (Tichy, 2011). However, we think that these findings could be relevant to other highly proliferative cell types, such as tumor cells.
Author contributions
Conflict of interest
Acknowledgements This work was supported by the Huffington Foundation grant PD14-03316, and National Institutes of Health grant R01 GM077442.