Cellular and Clinical Impact of Haploinsufficiency for Genes Involved in ATR Signaling
2007; Elsevier BV; Volume: 81; Issue: 1 Linguagem: Inglês
10.1086/518696
ISSN1537-6605
AutoresMark O’Driscoll, William B. Dobyns, Johanna M. van Hagen, Penny A. Jeggo,
Tópico(s)RNA regulation and disease
ResumoAtaxia telangiectasia and Rad3-related (ATR) protein, a kinase that regulates a DNA damage–response pathway, is mutated in ATR-Seckel syndrome (ATR-SS), a disorder characterized by severe microcephaly and growth delay. Impaired ATR signaling is also observed in cell lines from additional disorders characterized by microcephaly and growth delay, including non–ATR-SS, Nijmegen breakage syndrome, and MCPH1 (microcephaly, primary autosomal recessive, 1)–dependent primary microcephaly. Here, we examined ATR-pathway function in cell lines from three haploinsufficient contiguous gene-deletion disorders—a subset of blepharophimosis-ptosis-epicanthus inversus syndrome, Miller-Dieker lissencephaly syndrome, and Williams-Beuren syndrome—in which the deleted region encompasses ATR, RPA1, and RFC2, respectively. These three genes function in ATR signaling. Cell lines from these disorders displayed an impaired ATR-dependent DNA damage response. Thus, we describe ATR signaling as a pathway unusually sensitive to haploinsufficiency and identify three further human disorders displaying a defective ATR-dependent DNA damage response. The striking correlation of ATR-pathway dysfunction with the presence of microcephaly and growth delay strongly suggests a causal relationship. Ataxia telangiectasia and Rad3-related (ATR) protein, a kinase that regulates a DNA damage–response pathway, is mutated in ATR-Seckel syndrome (ATR-SS), a disorder characterized by severe microcephaly and growth delay. Impaired ATR signaling is also observed in cell lines from additional disorders characterized by microcephaly and growth delay, including non–ATR-SS, Nijmegen breakage syndrome, and MCPH1 (microcephaly, primary autosomal recessive, 1)–dependent primary microcephaly. Here, we examined ATR-pathway function in cell lines from three haploinsufficient contiguous gene-deletion disorders—a subset of blepharophimosis-ptosis-epicanthus inversus syndrome, Miller-Dieker lissencephaly syndrome, and Williams-Beuren syndrome—in which the deleted region encompasses ATR, RPA1, and RFC2, respectively. These three genes function in ATR signaling. Cell lines from these disorders displayed an impaired ATR-dependent DNA damage response. Thus, we describe ATR signaling as a pathway unusually sensitive to haploinsufficiency and identify three further human disorders displaying a defective ATR-dependent DNA damage response. The striking correlation of ATR-pathway dysfunction with the presence of microcephaly and growth delay strongly suggests a causal relationship. Ataxia telangiectasia and Rad3-related (ATR) protein is a central component of a DNA damage–response signaling pathway.1Abraham RT Cell cycle checkpoint signaling through the ATM and ATR kinases.Genes Dev. 2001; 15: 2177-2196Crossref PubMed Scopus (1598) Google Scholar, 2Shiloh Y ATM and ATR: networking cellular responses to DNA damage.Curr Opin Genet Dev. 2001; 11: 71-77Crossref PubMed Scopus (502) Google ScholarATR (MIM *601215) is mutated in two families displaying Seckel syndrome (SS) (MIM 210600), a disorder characterized by severe microcephaly, proportionate dwarfism, and dysmorphic facial features.3O'Driscoll M Ruiz-Perez VL Woods CG Jeggo PA Goodship JA A splicing mutation affecting expression of ataxia-telangiectasia and Rad3-related protein (ATR) results in Seckel syndrome.Nat Genet. 2003; 33: 497-501Crossref PubMed Scopus (607) Google Scholar, 4O'Driscoll M Jeggo PA Clinical impact of ATR checkpoint signalling failure in humans.Cell Cycle. 2003; 2: 194-195PubMed Google Scholar, 5Seckel HPG Bird-headed dwarfs: studies in developmental anthropology including human proportions. Springer Karger, Basel, Switzerland1960Google Scholar SS is clinically and genetically heterogeneous.6Alderton GK Joenje H Varon R Borglum AD Jeggo PA O'Driscoll M Seckel syndrome exhibits cellular features demonstrating defects in the ATR signalling pathway.Hum Mol Genet. 2004; 13: 3127-3138Crossref PubMed Scopus (135) Google Scholar Significantly, cell lines derived from additional patients with SS, although not harboring mutations in ATR, display ATR-signaling defects.6Alderton GK Joenje H Varon R Borglum AD Jeggo PA O'Driscoll M Seckel syndrome exhibits cellular features demonstrating defects in the ATR signalling pathway.Hum Mol Genet. 2004; 13: 3127-3138Crossref PubMed Scopus (135) Google Scholar Thus, SS can be attributed to defects in ATR signaling, with a subset of patients, designated "ATR-SS," having mutations in ATR itself. Additionally, three other disorders characterized by microcephaly and growth delay—Nijmegen breakage syndrome (MIM 251260), Fanconi anemia (MIM 227650), and MCPH1 (microcephaly, primary autosomal recessive, 1)–defective primary microcephaly (MIM 251200)—display impaired ATR-signaling responses.7Stiff T Reis C Alderton GK Woodbine L O'Driscoll M Jeggo PA Nbs1 is required for ATR-dependent phosphorylation events.EMBO J. 2005; 24: 199-208Crossref PubMed Scopus (153) Google Scholar, 8Alderton GK Galbiati L Griffith E Surinya KH Neitzel H Jackson AP Jeggo PA O'Driscoll M Regulation of mitotic entry by microcephalin and its overlap with ATR signalling.Nat Cell Biol. 2006; 8: 725-733Crossref PubMed Scopus (142) Google Scholar Together, these findings suggest that impaired ATR signaling can impact development, conferring microcephaly and growth delay. ATR is a phosphoinositol 3-kinase–like kinase (PIKK) that is activated by single-stranded (ss) regions of DNA generated after replication-fork stalling or during the repair of bulky lesions.9Zou L Elledge SJ Sensing DNA damage through ATRIP recognition of RPA-ssDNA complexes.Science. 2003; 300: 1542-1548Crossref PubMed Scopus (1904) Google Scholar ATR interacts with ATRIP (ATR-interacting protein) and is recruited to ssDNA regions, in part by ATRIP's ability to bind to replication protein A (RPA), a complex of three subunits, RPA1–3.9Zou L Elledge SJ Sensing DNA damage through ATRIP recognition of RPA-ssDNA complexes.Science. 2003; 300: 1542-1548Crossref PubMed Scopus (1904) Google Scholar, 10Cortez D Guntuku S Qin J Elledge SJ ATR and ATRIP: partners in checkpoint signaling.Science. 2001; 294: 1713-1716Crossref PubMed Scopus (709) Google Scholar, 11Zou L Liu D Elledge SJ Replication protein A-mediated recruitment and activation of Rad17 complexes.Proc Natl Acad Sci USA. 2003; 100: 13827-13832Crossref PubMed Scopus (335) Google Scholar The Rad17/Rfc2–5 complex, together with a complex involving Rad9, Rad1, and Hus1, also functions to enhance ATR signaling, by impacting either ATR recruitment or activation.12Zou L Cortez D Elledge SJ Regulation of ATR substrate selection by Rad17-dependent loading of Rad9 complexes onto chromatin.Genes Dev. 2002; 16: 198-208Crossref PubMed Scopus (421) Google Scholar Thus, these additional proteins are required for the ATR-signaling response and therefore represent potential candidate genetic defects for SS.6Alderton GK Joenje H Varon R Borglum AD Jeggo PA O'Driscoll M Seckel syndrome exhibits cellular features demonstrating defects in the ATR signalling pathway.Hum Mol Genet. 2004; 13: 3127-3138Crossref PubMed Scopus (135) Google Scholar Hence, defects in these genes may confer "Seckel-like" clinical features. High-resolution genetic mapping studies have led to the characterization of contiguous-gene-deletion disorders caused by heterozygous microdeletions that result in haploinsufficiency for a single or, more usually, several genes.13Lupski JR Genomic disorders: structural features of the genome can lead to DNA rearrangements and human disease traits.Trends Genet. 1998; 14: 417-422Abstract Full Text Full Text PDF PubMed Scopus (658) Google Scholar, 14Shaw-Smith C Redon R Rickman L Rio M Willatt L Fiegler H Firth H Sanlaville D Winter R Colleaux L et al.Microarray based comparative genomic hybridisation (array-CGH) detects submicroscopic chromosomal deletions and duplications in patients with learning disability/mental retardation and dysmorphic features.J Med Genet. 2004; 41: 241-248Crossref PubMed Scopus (419) Google Scholar It is likely that the clinical manifestations of these disorders arise from the combined impact of haploinsufficiency for multiple genes.15Page GP George V Go RC Page PZ Allison DB "Are we there yet?": Deciding when one has demonstrated specific genetic causation in complex diseases and quantitative traits.Am J Hum Genet. 2003; 73: 711-719Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar It is also possible that there are critical genes or pathways sensitive to haploinsufficiency, either alone or when combined with haploinsufficiency for other genes. While investigating disorders exhibiting microcephaly and growth delay, we were struck by the observation that the microdeletion in three such disorders involved haploinsufficiency for genes involved in ATR-pathway function. Blepharophimosis-ptosis-epicanthus inversus syndrome (BPES [MIM 110100]), which is characterized by small eye sockets (blepharophimosis), drooping eyelids (ptosis), and upward-folding inner eyelids (epicanthus inversus), is an autosomal dominant disorder caused by mutation of the putative forkhead transcription factor FOXL2 (MIM *605597).16Crisponi L Manila D Loi A Chiappe F Uda M Amati P Bisceglia L Zelante L Nagaraja R Porcu S et al.The putative forkhead transcription factor FOXL2 is mutated in blepharophimosis/ptosis/epicanthus inversus syndrome.Nat Genet. 2001; 27: 159-166Crossref PubMed Scopus (764) Google Scholar, 17De Baere E Dixon MJ Small KW Jabs EW Leroy BP Devriendt KG Gillerot Y Mortier G Meire F Van Maldergem L et al.Spectrum of FOXL2 gene mutations in blepharophimosis-ptosis-epicanthus inversus (BPES) families demonstrates a genotype-phenotype correlation.Hum Mol Genet. 2001; 10: 1591-1600Crossref PubMed Scopus (219) Google Scholar Seventeen cases of BPES with heterozygous interstitial deletions of various sizes on chromosome 3q leading to loss of FOXL2 have been documented (reviewed by de Rue et al.18de Ru MH Gille JJ Nieuwint AW Bijlsma JJ van der Blij JB van Hagen JM Interstitial deletion in 3q in a patient with blepharophimosis-ptosis-epicanthus inversus syndrome (BPES) and microcephaly, mild mental retardation and growth delay: clinical report and review of the literature.Am J Med Genetic A. 2005; 137: 81-87Crossref PubMed Scopus (30) Google Scholar). Of these, 13 patients were also reported to exhibit microcephaly and growth retardation, clinical features not normally associated with BPES. Recently, the microdeletion in one such patient was carefully mapped and was shown to encompass ATR, which localizes to the same region.18de Ru MH Gille JJ Nieuwint AW Bijlsma JJ van der Blij JB van Hagen JM Interstitial deletion in 3q in a patient with blepharophimosis-ptosis-epicanthus inversus syndrome (BPES) and microcephaly, mild mental retardation and growth delay: clinical report and review of the literature.Am J Med Genetic A. 2005; 137: 81-87Crossref PubMed Scopus (30) Google Scholar It was therefore proposed that the non-BPES features observed in such patients might be due to haploinsufficiency for ATR.18de Ru MH Gille JJ Nieuwint AW Bijlsma JJ van der Blij JB van Hagen JM Interstitial deletion in 3q in a patient with blepharophimosis-ptosis-epicanthus inversus syndrome (BPES) and microcephaly, mild mental retardation and growth delay: clinical report and review of the literature.Am J Med Genetic A. 2005; 137: 81-87Crossref PubMed Scopus (30) Google Scholar Hemizygous deletions on chromosome 17p also confer microcephaly and growth delay.19Cardoso C Leventer RJ Ward HL Toyo-Oka K Chung J Gross A Martin CL Allanson J Pilz DT Olney AH et al.Refinement of a 400-kb critical region allows genotypic differentiation between isolated lissencephaly, Miller-Dieker syndrome, and other phenotypes secondary to deletions of 17p13.3.Am J Hum Genet. 2003; 72: 918-930Abstract Full Text Full Text PDF PubMed Scopus (184) Google ScholarPAHFAH1B1/Lis1 (MIM *601545) encodes a protein, lissencephaly 1 (Lis1) that functions in neuronal migration.20Leventer RJ Cardoso C Ledbetter DH Dobyns WB LIS1: from cortical malformation to essential protein of cellular dynamics.Trends Neurosci. 2001; 24: 489-492Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar Mutations in or heterozygous deletions of PAHFAH1B1/Lis1 alone cause isolated lissencephaly sequence (ILS), a disorder typified by reduced neuronal migration resulting in a "smooth brain" (lissencephaly).21Cardoso C Leventer RJ Dowling JJ Ward HL Chung J Petras KS Roseberry JA Weiss AM Das S Martin CL et al.Clinical and molecular basis of classical lissencephaly: mutations in the LIS1 gene (PAFAH1B1).Hum Mutat. 2002; 19: 4-15Crossref PubMed Scopus (87) Google Scholar Larger deletions identified in some patients with ILS confer a more severe grade of lissencephaly associated with craniofacial abnormalities (ILS+). Even larger deletions extending from the PAHFAH1B1/Lis1 gene to the telomere are observed in Miller-Dieker lissencephaly syndrome (MDLS [MIM 247200]), a disorder characterized by the most severe grade of lissencephaly, with craniofacial abnormalities, microcephaly, and growth retardation. RPA1 (MIM *179835), the largest subunit of RPA, is heterozygously deleted in patients with MDLS and ILS+ but not ILS.19Cardoso C Leventer RJ Ward HL Toyo-Oka K Chung J Gross A Martin CL Allanson J Pilz DT Olney AH et al.Refinement of a 400-kb critical region allows genotypic differentiation between isolated lissencephaly, Miller-Dieker syndrome, and other phenotypes secondary to deletions of 17p13.3.Am J Hum Genet. 2003; 72: 918-930Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar Finally, Williams-Beuren syndrome (WBS [MIM 194050]) is caused by hemizygous deletion of chromosome 7q11.23, which results in the haploinsufficiency of multiple genes.22Tassabehji M Williams-Beuren syndrome: a challenge for genotype-phenotype correlations.Hum Mol Genet. 2003; 12: R229-R237Crossref PubMed Scopus (141) Google Scholar WBS is characterized by facial dysmorphia, microcephaly, growth retardation, and supravalvular aortic stenosis (SVAS [MIM 185500]). Hemizygous deletion or mutations in ELN (MIM *130160) alone, the gene encoding elastin, a structural component of arteries, cause SVAS.23Tassabehji M Metcalfe K Donnai D Hurst J Reardon W Burch M Read AP Elastin: genomic structure and point mutations in patients with supravalvular aortic stenosis.Hum Mol Genet. 1997; 6: 1029-1036Crossref PubMed Scopus (120) Google Scholar Patients with WBS, in contrast, have larger deletions encompassing replication factor C2 (RFC2 [MIM *600404]), a subunit of replication factor C (RF-C).24Wu YQ Sutton VR Nickerson E Lupski JR Potocki L Korenberg JR Greenberg F Tassabehji M Shaffer LG Delineation of the common critical region in Williams syndrome and clinical correlation of growth, heart defects, ethnicity, and parental origin.Am J Med Genet. 1998; 78: 82-89Crossref PubMed Scopus (87) Google Scholar RF-C loads proliferating cell nuclear antigen (PCNA) onto chromatin during DNA replication, and four of its five subunits, Rfc2–5, form a complex with Rad17 that functions in ATR signaling.11Zou L Liu D Elledge SJ Replication protein A-mediated recruitment and activation of Rad17 complexes.Proc Natl Acad Sci USA. 2003; 100: 13827-13832Crossref PubMed Scopus (335) Google Scholar, 25Yao NY Johnson A Bowman GD Kuriyan J O'Donnell M Mechanism of proliferating cell nuclear antigen clamp opening by replication factor C.J Biol Chem. 2006; 281: 17528-17539Crossref PubMed Scopus (52) Google Scholar, 26Johnson A Yao NY Bowman GD Kuriyan J O'Donnell M The replication factor C clamp loader requires arginine finger sensors to drive DNA binding and proliferating cell nuclear antigen loading.J Biol Chem. 2006; 281: 35531-35543Crossref PubMed Scopus (48) Google Scholar, 27Ellison V Stillman B Biochemical characterization of DNA damage checkpoint complexes: clamp loader and clamp complexes with specificity for 5′ recessed DNA.PLoS Biol. 2003; 1: E33Crossref PubMed Scopus (270) Google Scholar The association of severe microcephaly and growth retardation with heterozygous loss of genes encoding proteins involved in the ATR-signaling pathway prompted us to examine whether haploinsufficiency for these genes impacts the ATR-signaling response. We therefore examined cell lines obtained from these contiguous gene disorders for their ability to effect the ATR-dependent DNA-damage response. We employed sensitive assays capable of detecting defective ATR-pathway function that we had established elsewhere to examine SS cell lines. Strikingly, we observed defects in ATR signaling in all three disorders, demonstrating that ATR signaling is sensitive to haploinsufficiency. Lymphoblastoid cell lines (LBLs) were cultured in RPMI 1640 with 15% fetal calf serum. GM02188 (wild type [WT]) and DK0064 (ATR-SS) have been described elsewhere.6Alderton GK Joenje H Varon R Borglum AD Jeggo PA O'Driscoll M Seckel syndrome exhibits cellular features demonstrating defects in the ATR signalling pathway.Hum Mol Genet. 2004; 13: 3127-3138Crossref PubMed Scopus (135) Google Scholar VD9396 (BPES-ATR+/−) LBLs were obtained from J.M.v.H.18de Ru MH Gille JJ Nieuwint AW Bijlsma JJ van der Blij JB van Hagen JM Interstitial deletion in 3q in a patient with blepharophimosis-ptosis-epicanthus inversus syndrome (BPES) and microcephaly, mild mental retardation and growth delay: clinical report and review of the literature.Am J Med Genetic A. 2005; 137: 81-87Crossref PubMed Scopus (30) Google Scholar ILS, ILS+, and MDLS LBLs—DR00-063a1 (Con-MR), LP99-017 (ILS A), LP94-013 (ILS B), LP90-017 (ILS+ A), LP99-086 (ILS+ B), LP91-026 (ILS+ C), L95-059 (MDLS-A), LP92-005 (MDLS-B), LP90-006 (MDLS-C), and LP88-002 (MDLS-D)—have been described elsewhere and were provided by W.B.D.19Cardoso C Leventer RJ Ward HL Toyo-Oka K Chung J Gross A Martin CL Allanson J Pilz DT Olney AH et al.Refinement of a 400-kb critical region allows genotypic differentiation between isolated lissencephaly, Miller-Dieker syndrome, and other phenotypes secondary to deletions of 17p13.3.Am J Hum Genet. 2003; 72: 918-930Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar WBS and respective parental LBLs—GM14183 (WT-I), GM14182 (WBS-I), GM14295 (WT-II), and GM14297 (WBS-II)—were obtained from Coriell Cell Repository. Antibodies were obtained as follows. α-ATR (N19), α-Lis1 (H-300), and α-β-tubulin (H235) were obtained from Autogen Bioclear; α-RPA1 (Ab-1), α-H2AX (DR-1016), and α-NBS1 (Ab-1) were obtained from Merck; α–γ-H2AX, α-pS10 Histone H3, and α-ATRIP were obtained from Upstate Technology; and α-pSer317-Chk1 was obtained from New England Biolabs. Western blotting was performed as described elsewhere.8Alderton GK Galbiati L Griffith E Surinya KH Neitzel H Jackson AP Jeggo PA O'Driscoll M Regulation of mitotic entry by microcephalin and its overlap with ATR signalling.Nat Cell Biol. 2006; 8: 725-733Crossref PubMed Scopus (142) Google Scholar For γ-H2AX analysis, a chromatin extraction step was included. In brief, 1×107 cells were washed once in PBS and were resuspended in 100 μl hypotonic buffer (10 mM HEPES [pH 7.5], 5 mM KCl, 1.5 mM MgCl2, 1 mM dithiothreitol, 10 mM NaF, 1 mM Na2VO3, 10 mM β-glycerolphosphate, 0.5% IPEGAL/Nonident P-40, and Protease Inhibitor Cocktail from Sigma). Lysates were incubated on ice for 15 min, were pelleted, and were washed twice (200 μl each) in hypotonic buffer. The pellet was treated with hypertonic buffer (hypotonic buffer with 0.5 M NaCl) and was incubated on ice for 15 min. After washing in hypertonic buffer, the chromatin pellets were resuspended in 100 μl of SDS-PAGE loading buffer (with 5% SDS, 10% β-mercaptoethanol) and were sonicated. Ten microliters of the chromatin fraction was separated on 17% SDS-PAGE. Cells were irradiated with 5 J/m2 UV-C, were immediately seeded into complete medium with 1.5 μM nocodazole, and were incubated for 24 h before being cytospun onto poly-D-lysine–coated slides and processed for immunofluorescence with α-pS10-Histone H3 and for counterstaining with 4',6-diamidino-2-phenylindole. Cells were treated with 5 mM hydroxyurea (HU) in the presence of 1.5 μM nocodazole for 24 h and were processed as described elsewhere.6Alderton GK Joenje H Varon R Borglum AD Jeggo PA O'Driscoll M Seckel syndrome exhibits cellular features demonstrating defects in the ATR signalling pathway.Hum Mol Genet. 2004; 13: 3127-3138Crossref PubMed Scopus (135) Google Scholar With use of Genejuice (Novagen), 3×105 cells/ml, in 3-ml quantities, were transiently transfected with 2 μg of pcDNA3-ATR, pcDNA3.1-RPA1, or pcDNA3.1-RFC2, according to the manufacturer's instructions, and were incubated for 24 h before NF processing. For complementation of the G2/M checkpoint, cells were incubated for 24 h and were retransfected for a further 24 h before processing. The control WT LBL GM02188 was used for small-interfering RNA (siRNA) experiments. Cells were transfected once with 10 nM of respective siRNA duplex (GFPi, ATRi, Lis1i, or RPA1i), with use of SiPort NeoFX transfection reagent according to the manufacturer's instructions (Ambion). The oligonucleotides (sense) used were ATR 5′-AAC CUC CGU GAU GUU GCU UGA-3′, RPA1 5′-AAA CCA UCC ACG AAG CUU AUA GGC C-3′, Lis1 5′-UUG AUU UGG CCG UAC CAU ACG UAC C-3′, and a control oligonucleotide directed to GFP 5′-AAC ACU UGU CAC UAC UUU CTC. A previous study reported heterozygosity for ATR due to an interstitial deletion on 3q in a boy with BPES who displayed non-BPES clinical features that included mental retardation (MR), microcephaly, and growth delay.18de Ru MH Gille JJ Nieuwint AW Bijlsma JJ van der Blij JB van Hagen JM Interstitial deletion in 3q in a patient with blepharophimosis-ptosis-epicanthus inversus syndrome (BPES) and microcephaly, mild mental retardation and growth delay: clinical report and review of the literature.Am J Med Genetic A. 2005; 137: 81-87Crossref PubMed Scopus (30) Google Scholar A representation of the deletion is shown in figure 1A. We obtained an LBL derived from this patient (BPES-ATR+/−) and, by immunoblotting, observed approximately twofold reduced ATR protein levels compared with those of a control LBL (fig. 1B). The level of ATRIP was also reduced approximately twofold, consistent with reports that ATR and ATRIP are coregulated.10Cortez D Guntuku S Qin J Elledge SJ ATR and ATRIP: partners in checkpoint signaling.Science. 2001; 294: 1713-1716Crossref PubMed Scopus (709) Google Scholar NBS1 served as a loading control and was expressed at similar levels in both lines (fig. 1B). An early step in the DNA damage response regulated by ATR is phosphorylation of the histone H2A variant, H2AX, on serine 139 (termed "γ-H2AX"), which can be detected by the immunoblotting of chromatin-bound proteins with α–γ-H2AX antibodies.28Fernandez-Capetillo O Lee A Nussenzweig M Nussenzweig A H2AX: the histone guardian of the genome.DNA Repair. 2004; 3: 959-967Crossref PubMed Scopus (761) Google Scholar After exposure to HU, an agent that causes replication-fork stalling, a strong γ-H2AX signal was observed in control LBLs, indicating activation of ATR-dependent damage-response signaling. However, in marked contrast, detectable γ-H2AX was not observed in the ATR-SS LBLs, nor in LBLs derived from the patient with BPES-ATR+/− (fig. 1C). Chk1 represents an important phosphorylation target of ATR that is required for the stability of arrested replication forks and cell-cycle arrest.29Chen Y Sanchez Y Chk1 in the DNA damage response: conserved roles from yeasts to mammals.DNA Repair. 2004; 3: 1025-1032Crossref PubMed Scopus (150) Google Scholar, 30Liu Q Guntuku S Cui XS Matsuoka S Cortez D Tamai K Luo G Carattini-Rivera S DeMayo F Bradley A et al.Chk1 is an essential kinase that is regulated by Atr and required for the G(2)/M DNA damage checkpoint.Genes Dev. 2000; 14: 1448-1459Crossref PubMed Scopus (185) Google Scholar, 31Carr AM Moudjou M Bentley NJ Hagan IM The chk1 pathway is required to prevent mitosis following cell-cycle arrest at "start".Curr Biol. 1995; 5: 1179-1190Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 32Petermann E Caldecott KW Evidence that the ATR/Chk1 pathway maintains normal replication fork progression during unperturbed S phase.Cell Cycle. 2006; 5: 2203-2209Crossref PubMed Scopus (100) Google Scholar, 33Zachos G Rainey M Gillespie D Chk1-deficient tumour cells are viable but exhibit multiple checkpoint and survival defects.EMBO J. 2003; 22: 713-723Crossref PubMed Scopus (220) Google Scholar HU-induced phosphorylation of Chk1 on serine 317, a known ATR target site, was also significantly diminished in LBLs from the patient with BPES-ATR+/−, similar to ATR-SS LBLs (fig. 1D).6Alderton GK Joenje H Varon R Borglum AD Jeggo PA O'Driscoll M Seckel syndrome exhibits cellular features demonstrating defects in the ATR signalling pathway.Hum Mol Genet. 2004; 13: 3127-3138Crossref PubMed Scopus (135) Google Scholar An important end point of ATR activation regulated by Chk1 is onset of G2/M checkpoint arrest, which serves to prevent cells harboring DNA damage from entering mitosis.6Alderton GK Joenje H Varon R Borglum AD Jeggo PA O'Driscoll M Seckel syndrome exhibits cellular features demonstrating defects in the ATR signalling pathway.Hum Mol Genet. 2004; 13: 3127-3138Crossref PubMed Scopus (135) Google Scholar To monitor G2/M checkpoint arrest, the percentage of mitotic cells was examined in untreated cells or 24 h postirradiation with UV (5 Jm−2). Control LBLs showed a marked reduction in mitotic cells due to arrest at the G2/M checkpoint, whereas ATR-SS and BPES-ATR+/− cells showed a mitotic index (MI) similar to that observed in the absence of UV treatment (fig. 1E). A further hallmark of impaired ATR signaling is the presence of cells with NF after treatment with HU.6Alderton GK Joenje H Varon R Borglum AD Jeggo PA O'Driscoll M Seckel syndrome exhibits cellular features demonstrating defects in the ATR signalling pathway.Hum Mol Genet. 2004; 13: 3127-3138Crossref PubMed Scopus (135) Google Scholar ATR-SS and BPES-ATR+/− LBLs showed markedly elevated levels of cells displaying NF after HU treatment, in contrast to those of control LBLs (fig. 1F). To verify that the failure to effect G2/M checkpoint arrest and that the damage-induced NF phenotype of BPES-ATR+/− cells were directly attributable to an impaired ATR response, the cells were transfected with ATR cDNA and were reexamined for these phenotypes. Significant UV-induced G2/M arrest (fig. 1G) and reduced NF (fig. 1H) were observed after transfection with ATR cDNA. Collectively, these results provide strong evidence that haploinsufficiency for ATR in the BPES-ATR+/− cell line confers an impaired response to DNA damage that is similar in magnitude to that observed in an ATR-SS cell line. A representation of the deletion on chromosome 17p observed in MDLS is shown in figure 2A. The size of the heterozygous deletions on chromosome 17p in a panel of LBLs derived from patients with ILS, ILS+, and MDLS and from a control patient (Con-MR) are shown in figure 2B. ILS A and ILS B were derived from patients with mild ILS due to microdeletions involving Lis1 only. ILS+ A, ILS+ B, and ILS+ C were obtained from patients with more-severe ILS and craniofacial abnormalities. MDLS-A, -B, -C, and -D, which have deletions extending from Lis1 to the telomere, were derived from MDLS-affected patients with the severest grade of lissencephaly together with microcephaly and growth retardation. Con-MR, which has a heterozygous telomeric deletion that does not involve either RPA1 or Lis1, was derived from a patient with mild MR (fig. 2B). This patient does not exhibit MDLS, lissencephaly, microcephaly, or growth delay. The location of RPA1 is shown in figure 2B, demonstrating that this gene is deleted in some but not all LBLs in the panel. To examine whether haploinsufficiency for RPA1 correlated with impaired ATR-dependent damage-response signaling, we examined the response to DNA damage in this panel of LBLs. First, we examined RPA1 expression in whole-cell extracts (WCEs) derived from Con-MR, ILS A, and ILS B, compared with MDLS-A, -B, and -C. All three MDLS LBLs displayed reduced levels of RPA1 compared with those of Con-MR, ILS A, and ILS B LBLs, which is consistent with the deletion mapping (fig. 2B). Next, we examined HU-induced γ-H2AX formation in representative LBLs, either haploinsufficient for RPA1 or with both copies of the gene. Impaired HU-induced γ-H2AX was observed in the ATR-SS, ILS+ A, and MDLS A LBLs, the latter two of which exhibit haploinsufficiency for RPA1 (fig. 2B and 2D). Normal HU-induced γ-H2AX was observed in WT control LBLs and in those cell lines in which the deletion did not encompass RPA1 (Con-MR, ILS A, and ILS B) (fig. 2B and 2D). Similarly, MDLS-A LBLs also exhibited impaired HU-induced Chk1-pSer317 formation, unlike Con-MR and ILS A LBLs (fig. 2E). We also examined the ability to activate UV-induced G2/M checkpoint arrest and observed impaired arrest specifically in the seven LBLs with haploinsufficiency for RPA1 (figs. 2B and 3A). Furthermore, we examined HU-induced NF and observed elevated levels specifically in those LBLs from patients haploinsufficient for RPA1 (figs. 2B and 3B).Figure 3.Haploinsufficiency of RPA1, which specifically segregates with a defective ATR-dependent DNA damage response. A, Defective ATR-dependent G2/M checkpoint arrest, which segregates with RPA1 haploinsufficiency. Con-MR, ILS A, and ILS B cells showing a reduction in MI (percentage of mitosis) at 24 h after UV irradiation (5 J/m2), indicating G2/M checkpoint arrest. ILS+ A, ILS+ B, ILS+ C, and MDLS-A, -B, -C, and -D cell lines failed to show a decrease in MI after UV treatment. B, Increased HU-induced NF segregating with RPA1 haploinsufficiency. No increase in HU-induced NF is seen in Con-MR,
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