title: Stalled replication forks within heterochromatin require ATRX for protection.
abstract: Expansive growth of neural progenitor cells (NPCs) is a prerequisite to the temporal waves of neuronal differentiation that generate the six-layered neocortex, while also placing a heavy burden on proteins that regulate chromatin packaging and genome integrity. This problem is further reflected by the growing number of developmental disorders caused by mutations in chromatin regulators. ATRX gene mutations cause a severe intellectual disability disorder (α-thalassemia mental retardation X-linked (ATRX) syndrome; OMIM no. 301040), characterized by microcephaly, urogenital abnormalities and α-thalassemia. Although the ATRX protein is required for the maintenance of repetitive DNA within heterochromatin, how this translates to disease pathogenesis remain poorly understood and was a focus of this study. We demonstrate that Atrx(FoxG1Cre) forebrain-specific conditional knockout mice display poly(ADP-ribose) polymerase-1 (Parp-1) hyperactivation during neurogenesis and generate fewer late-born Cux1- and Brn2-positive neurons that accounts for the reduced cortical size. Moreover, DNA damage, induced Parp-1 and Atm activation is elevated in progenitor cells and contributes to their increased level of cell death. ATRX-null HeLa cells are similarly sensitive to hydroxyurea-induced replication stress, accumulate DNA damage and proliferate poorly. Impaired BRCA1-RAD51 colocalization and PARP-1 hyperactivation indicated that stalled replication forks are not efficiently protected. DNA fiber assays confirmed that MRE11 degradation of stalled replication forks was rampant in the absence of ATRX or DAXX. Indeed, fork degradation in ATRX-null cells could be attenuated by treatment with the MRE11 inhibitor mirin, or exacerbated by inhibiting PARP-1 activity. Taken together, these results suggest that ATRX is required to limit replication stress during cellular proliferation, whereas upregulation of PARP-1 activity functions as a compensatory mechanism to protect stalled forks, limiting genomic damage, and facilitating late-born neuron production.
authors.affiliation: Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, ON K1H 8L6, Canada.
authors.forename: M S
authors.initials: MS
authors.lastname: Huh
authors.affiliation: Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, ON K1H 8L6, Canada.|Department of Biochemistry, Microbiology, and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, ON K1H 8M5, Canada.
authors.forename: D
authors.initials: D
authors.lastname: Ivanochko
authors.affiliation: Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, ON K1H 8L6, Canada.|Department of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, Ottawa, ON K1H 8M5, Canada.
authors.forename: L E
authors.initials: LE
authors.lastname: Hashem
authors.affiliation: Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, ON K1H 8L6, Canada.|Department of Biochemistry, Microbiology, and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, ON K1H 8M5, Canada.
authors.forename: M
authors.initials: M
authors.lastname: Curtin
authors.affiliation: Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, ON K1H 8L6, Canada.|Department of Biochemistry, Microbiology, and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, ON K1H 8M5, Canada.
authors.forename: M
authors.initials: M
authors.lastname: Delorme
authors.affiliation: Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, ON K1H 8L6, Canada.|Department of Biochemistry, Microbiology, and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, ON K1H 8M5, Canada.
authors.forename: E
authors.initials: E
authors.lastname: Goodall
authors.affiliation: Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, ON K1H 8L6, Canada.
authors.forename: K
authors.initials: K
authors.lastname: Yan
authors.affiliation: Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, ON K1H 8L6, Canada.|Department of Biochemistry, Microbiology, and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, ON K1H 8M5, Canada.|Department of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, Ottawa, ON K1H 8M5, Canada.
authors.forename: D J
authors.initials: DJ
authors.lastname: Picketts
mesh_terms: D000818:Animals; D064007:Ataxia Telangiectasia Mutated Proteins / Q000235:genetics / Q000378:metabolism; D019313:BRCA1 Protein; D002352:Carrier Proteins / Q000235:genetics; D049109:Cell Proliferation / Q000187:drug effects; D056970:Co-Repressor Proteins; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004249:DNA Damage; D004265:DNA Helicases / Q000172:deficiency / Q000235:genetics*; D045643:DNA Repair Enzymes / Q000235:genetics / Q000378:metabolism; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D006367:HeLa Cells; D006570:Heterochromatin / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D006918:Hydroxyurea / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins / Q000172:deficiency / Q000235:genetics; D000076228:MRE11 Homologue Protein; D051379:Mice; D018345:Mice, Knockout; D018832:Molecular Chaperones; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism; D055495:Neurogenesis / Q000235:genetics; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D009687:Nuclear Proteins / Q000172:deficiency / Q000235:genetics* / Q000378:metabolism; D050809:POU Domain Factors / Q000235:genetics / Q000378:metabolism; D000071137:Poly (ADP-Ribose) Polymerase-1 / Q000235:genetics* / Q000378:metabolism; D016548:Prosencephalon / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D051135:Rad51 Recombinase / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism; D000075924:X-linked Nuclear Protein
chemical_list: D019313:BRCA1 Protein; C000605931:Brca1 protein, mouse; D002352:Carrier Proteins; D056970:Co-Repressor Proteins; C000624101:Cux1 protein, mouse; D004268:DNA-Binding Proteins; C491473:Daxx protein, mouse; D006570:Heterochromatin; D018398:Homeodomain Proteins; D047908:Intracellular Signaling Peptides and Proteins; D018832:Molecular Chaperones; C512689:Mre11a protein, mouse; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D050809:POU Domain Factors; D012097:Repressor Proteins; D025521:Tumor Suppressor Proteins; C074066:Pou3f2 protein, mouse; D004247:DNA; C499321:Parp1 protein, mouse; D000071137:Poly (ADP-Ribose) Polymerase-1; D064007:Ataxia Telangiectasia Mutated Proteins; C576325:Atm protein, mouse; D051135:Rad51 Recombinase; C495154:Rad51 protein, mouse; D000076228:MRE11 Homologue Protein; D004265:DNA Helicases; C490462:Atrx protein, mouse; D000075924:X-linked Nuclear Protein; D045643:DNA Repair Enzymes; D006918:Hydroxyurea