A proper response to DNA damage is critical for the maintenance of genome stability, and it serves as a key barrier to the prevention of cancer. The screen described in this study has led to the identification of several hundred genes that when lost, induce the phosphorylation of H2AX, a robust and reliable marker for DNA damage. We expect there is a strong likelihood of identifying genes with functions in the DNA damage response pathway amongst these hits, as both CMT and splicing genes exhibited either DNA repair or checkpoint defects. This is the first DNA damage response screen of its kind that has been reported in higher eukaryotes, and the results provide new insight into processes that prevent the formation and accumulation of DNA damage. Indeed, many of the genes and processes identified have not been previously linked to the formation of DNA damage, suggesting the events that contribute to genome instability may be more widespread than previously realized.
A number of the genes we identified exhibited a relatively high level of H2AX phosphorylation when knocked down, particularly those known to be involved in DNA replication and DNA damage responses. The genes involved in RNA splicing also caused a high level of H2AX phosphorylation. However, several hundred genes consistently led to low, but reproducible and significant levels of phosphorylation when targeted. Thus, while the high effectors are of obvious importance, those causing low levels of H2AX phosphorylation may also be of interest. Indeed, loss of function of these genes may be tolerated by the cell/organism and could drive genome instability and transformation, while those causing high levels of γH2AX seem more likely to cause cell death or senescence. In this respect, it is interesting that the level of genome instability linked to the CMT genes is relatively low.
For a majority of the genes identified in our screen, it seems likely the increase in γH2AX observed is due to increased spontaneous DNA damage. However, spontaneous or unrepaired DNA damage may not be the only reason for increased γH2AX. For example, H2AX phosphorylation could result from loss of the phosphatases that dephosphorylate γH2AX. In fact, we did identify subunits of the PP2A and PP4 phosphatase complexes that are involved in dephosphorylating γH2AX (Chowdhury et al., 2005
; Chowdhury et al., 2008
; Nakada et al., 2008
). Some of the genes identified may also cause an increase in γH2AX via apoptosis; however, this category was largely eliminated by removing genes that caused overt and widespread cell death, as well as by setting nuclear area parameters to eliminate the identification of nuclear fragments.
Other screens assessing different aspects of the DNA damage response have been carried out in various organisms. For example, the formation of Rad52 foci was examined in Saccharomyces cerevisiae
(Alvaro et al., 2007
), and several screens were also carried out in Caenorhabditis elegans
to identify genes affecting radiation sensitivity (van Haaften et al., 2006
; van Haaften et al., 2004
). Of the genes identified in these screens, many were also found in our data set suggesting that some of the properties measured by previous screens may be linked to increased γH2AX (Table S11
). A proteomic analysis designed to identify the targets of the DNA damage protein kinases has also been carried out using mammalian cells (Matsuoka et al., 2007
). Although this approach is orthogonal to ours, we found significant overlap in the genes and pathways identified by this method and our dataset (110 genes, p = 1.5 × 10-2
) (Table S11
). Interestingly, beyond specific gene overlap between screens, greater commonality was observed between the biological processes and pathways found, suggesting that while individual hits may vary from screen to screen, the enriched pathways observed may provide greater biological insight. For example, mRNA processing genes were also enriched in this proteomic analysis. Nevertheless, the majority of genes found in all studies were not found in the other, and we identified many additional genes and pathways of diverse function not previously linked to the DNA damage response. This indicates that our knowledge of this process is still incomplete and that the screens are not yet saturating. Further, it suggests that a systems biology approach utilizing many genomic datasets could ultimately prove useful in understanding the mechanisms underlying genomic stability.
Altogether, the results of our study indicate the pathways and processes affecting genome stability are much broader than anticipated, and our data provide new unexpected links between the maintenance of genome stability and the kinetochore, the nuclear pore, mRNA processing and Charcot-Marie-Tooth disease. We expect there will be important roles for these genes and pathways in the DNA damage response, cancer, neurodegeneration, aging, and other human diseases, and the nature of these links will be of great interest for future study.