Human ATRX mutations are associated with cognitive deficits, developmental abnormalities, and cancer. We show that the Atrx-null embryonic mouse brain accumulates replicative damage at telomeres and pericentromeric heterochromatin, which is exacerbated by loss of p53 and linked to ATM activation. ATRX-deficient neuroprogenitors exhibited higher incidence of telomere fusions and increased sensitivity to replication stress–inducing drugs. Treatment of Atrx-null neuroprogenitors with the G-quadruplex (G4) ligand telomestatin increased DNA damage, indicating that ATRX likely aids in the replication of telomeric G4-DNA structures. Unexpectedly, mutant mice displayed reduced growth, shortened life span, lordokyphosis, cataracts, heart enlargement, and hypoglycemia, as well as reduction of mineral bone density, trabecular bone content, and subcutaneous fat. We show that a subset of these defects can be attributed to loss of ATRX in the embryonic anterior pituitary that resulted in low circulating levels of thyroxine and IGF-1. Our findings suggest that loss of ATRX increases DNA damage locally in the forebrain and anterior pituitary and causes tissue attrition and other systemic defects similar to those seen in aging.
L. Ashley Watson, Lauren A. Solomon, Jennifer Ruizhe Li, Yan Jiang, Matthew Edwards, Kazuo Shin-ya, Frank Beier, Nathalie G. Bérubé
Increased DNA damage leads to ATM activation and p53-dependent apoptosis in the Atrx-null embryonic brain.
(A) Immunostaining for γH2AX in E13.5 control (Ctrl), cKO, and cKO;p53–/– compound mutant cortical cryosections. Scale bar: 100 μm. DAPI staining of E13.5 forebrain highlights in green the hippocampal hem (H), cortex (Ctx), and basal ganglia (BG) regions where γH2AX foci per unit area were scored. Control, cKO, and cKO;p53–/– (n = 3); p53–/– (n = 2). (B) γH2AX staining in P0.5 control, cKO, and cKO;p53–/– cortical cryosections. Scale bar: 200 μm. DAPI staining of P0.5 forebrain highlights in green the hippocampus (H) and cortex (Ctx) regions where γH2AX foci per unit area were scored. Control and cKO (n = 3); cKO;p53–/– and p53–/– (n = 2). (C) Co-immunofluorescence detection of γH2AX (red) and activated caspase-3 (AC3; green) in E13.5 cortical cryosections. Scale bar: 30 μm. AC3+ cells were scored for the presence (AC3 + γH2AX) or absence (AC3 – γH2AX) of DNA damage (n = 3). (D) Western blot analysis of nuclear protein extracts obtained from E13.5 telencephalon (n = 3). While levels of ATR and phospho-ATR were not increased (left panels), phospho-ATM was noticeably increased in the cKO extracts compared with controls (indicated by an asterisk). Original magnification, ×100 (A and B); ×200 (C). *P < 0.05.