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.
Authors
L. Ashley Watson, Lauren A. Solomon, Jennifer Ruizhe Li, Yan Jiang, Matthew Edwards, Kazuo Shin-ya, Frank Beier, Nathalie G. Bérubé
(A) AtrxloxP/Y MEFs were untransduced or transduced with adenovirus expressing Cre recombinase fused to GFP (Ad-CreGFP) or Ad-GFP and subsequently treated with HU for 24 hours or γ-irradiated at the indicated doses. Cell viability was measured at 24 hours after HU treatment (n = 4) and at 6 hours after irradiation (n = 3) via trypan blue dye exclusion. (B) Control and cKO NPCs were treated with HU or MMC for 24 hours or γ-irradiated at the indicated doses. Cell viability was measured at 24 hours after HU and MMC treatment and at 6 hours after irradiation (n = 3). (C) Co-immunofluorescence detection of PCNA, a marker of replication foci, and γH2AX in control and cKO E13.5 cortical cryosections. Results were quantified by measuring the ratio of γH2AX staining that localized to late-replicating PCNA foci to total γH2AX staining per cell, to account for overall lower levels of γH2AX signal in control cells (300 nuclei counted, n = 3). Scale bar: 12 μm. (D) Control and cKO NPCs were treated with 20 μm TMS for 2 hours, and γH2AX signal was imaged 6 hours after treatment. Scale bar: 70 μm. (E) Control and cKO NPCs were treated with TMS for 24 hours, and cell viability was measured 24 hours after treatment (n = 3). Original magnification, ×600 (C); ×100 (D). *P < 0.05.