We previously discovered that ATM in addition to serving man

We previously discovered that ATM, in addition to serving many roles in cell-cycle regulation and DNA damage response, also functions as a key epigenetic regulator in neurons, likely underlying the ataxic symptoms as well as neurodegeneration. ATM indirectly regulates nuclear-cytoplasmic shuttling of HDAC4 (Li et al., 2012), and it directly phosphorylates EZH2, the enzymatic component of the PRC2 complex (Li et al., 2013). Each of these mechanisms contributes to ataxia symptoms in knockout mouse models, because reversal restores behavioral function. ATM also phosphorylates TET1, leading to conversion of 5mC to 5hmC marks, thought to be a key step in DNA demethylation (Jiang et al., 2015). Others have reported an effect on the DNMT1 methyltransferase via ATM phosphorylation of the Rb protein (Shamma et al., 2013). Any or all of these mechanisms could be affected or modulated by domains within a truncated ATM protein. Although others have reported A-T iPSC (Nayler et al., 2012; Lee et al., 2013; Fukawatase et al., 2014), there has been no systematic preparation of a range of ATM mutation sites and no broad assessment of genome stability in iPSC cultures. It is expected that mutations occur spontaneously in tissue culture and presumably even in vivo. We examined several A-T iPSC lines and sublines derived from individually picked colonies for evidence of such reversion. We found that iPSC made from one subject yielded phenotypic and genotypic differences among sublines. Analysis of the resulting isogenic pair of cell lines greatly focuses the identification of cellular mechanisms, such as gene expression differences with or without ATM.
Results Blood samples were obtained from six subjects harboring previously identified ATM mutations (Figure 1A): two from subjects diagnosed with A-T (labeled as “Q”) and four from carrier parents (labeled as “CAR”). iPSC were prepared from four of these subjects (asterisks in Figure 1A) using enriched, activated T cells and non-integrating Sendai viral vectors to deliver reprogramming factors (Moore et al., 2012). Sample iPSC colonies from each subject had standard morphology and stained positive for Oct4 and TRA-1-60, as shown in Figure 1B. For each subject, several sublines were picked from single colonies, expanded, and stored as frozen stocks. A subset of these was tested for gene expression patterns consistent with pluripotency. As shown in Figure 1C, three iPSC sublines (Q1SA, Q3SA, and Q3SC) all clustered with unrelated iPSC prepared from a non-A-T subject (“iPSC”), but they clustered separately from H1 human embryonic stem cell (hESC)-derived neural stem find protocol (NSC) at day 0 (NSC0) or NSC following 5 days of differentiation (NSC5) (Sauvageau et al., 2013), as well as dopaminergic neurons (DAN) differentiated from iPSC using the dual-SMAD protocol (Kriks et al., 2011). Furthermore, analysis of gene expression patterns using the PluriTest algorithm (Müller et al., 2011) identifies each iPSC line as being consistent with pluripotency (pluri-raw values ≥ 27, novelty ≤ 1.8). These results demonstrate successful iPSC reprogramming. To confirm the presence of ATM gene mutations, we assessed ATM protein expression by western blot (Figures 1D, S1A, and S1B). A band corresponding to full-length ATM protein was clearly visible in the CAR3 and Q1SA iPSC lines, as expected (Figure S1A). Q1SA encodes a missense mutation (c.7181 C > T; encoding S2394L, Figure 1A) that is predicted to affect ATM activity but not translation of full-length protein. However, independent sublines prepared from subject JHU_Q3 expressed unexpected variation in ATM expression (Figures 1D and S1B). Some sublines had no ATM protein, as expected (sublines Q3SA, Q3SE, and Q3SI), but others expressed full-length protein (Q3SC and Q3SG). Furthermore, different passage numbers from the same subline exhibited varying quantities of ATM protein, peaking at P7 and then diminishing but not disappearing in later passages (Q3SC; Figures 1D and S1B). We selected two sublines, Q3SA and Q3SC, as contrasting examples without or with ATM expression, respectively. ATM activity in sample iPSC lines was confirmed by the X-irradiation (XR)-induced phosphorylation of CHK2 as detected by western blots (Figure 1E). The pCHK2 band was enhanced by XR (8 Gy) and diminished by pre-treatment with the ATM-specific inhibitor KU-55933 (KU) in unrelated control iPSC (SC1) and in Q3SC. Little pCHK2 was detected in Q1SA. Results were confirmed by observing the presence of XR-induced γH2A.X nuclear puncta with the same cell lines exhibiting positive (CAR3 and Q3SC) or negative (Q1SA and Q3SA) results (Figures 1F and S1C). Therefore, Q1SA expressed full-length ATM protein but had little or no kinase activity, presumably because of the presence of a previously unreported C > T variant at position c.7181, producing a S2394L that apparently affects activation. However, results indicate both the presence of ATM protein and its function in the Q3SC subline where none was expected on the basis of the diagnostic genotype provided with the subject cells. Results from Q3SA matched those expectations, as no ATM protein or activity was observed, with one allele terminating shortly after a frameshift mutation (c.217_218 delGA) and the other allele terminating at a point mutation (c.7792 C > T; R2598X), each truncating translation prior to the kinase domain.