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1.
Genet Med ; 25(7): 100861, 2023 Jul.
Article in English | MEDLINE | ID: mdl-37087635

ABSTRACT

PURPOSE: This study aimed to establish variants in CBX1, encoding heterochromatin protein 1ß (HP1ß), as a cause of a novel syndromic neurodevelopmental disorder. METHODS: Patients with CBX1 variants were identified, and clinician researchers were connected using GeneMatcher and physician referrals. Clinical histories were collected from each patient. To investigate the pathogenicity of identified variants, we performed in vitro cellular assays and neurobehavioral and cytological analyses of neuronal cells obtained from newly generated Cbx1 mutant mouse lines. RESULTS: In 3 unrelated individuals with developmental delay, hypotonia, and autistic features, we identified heterozygous de novo variants in CBX1. The identified variants were in the chromodomain, the functional domain of HP1ß, which mediates interactions with chromatin. Cbx1 chromodomain mutant mice displayed increased latency-to-peak response, suggesting the possibility of synaptic delay or myelination deficits. Cytological and chromatin immunoprecipitation experiments confirmed the reduction of mutant HP1ß binding to heterochromatin, whereas HP1ß interactome analysis demonstrated that the majority of HP1ß-interacting proteins remained unchanged between the wild-type and mutant HP1ß. CONCLUSION: These collective findings confirm the role of CBX1 in developmental disabilities through the disruption of HP1ß chromatin binding during neurocognitive development. Because HP1ß forms homodimers and heterodimers, mutant HP1ß likely sequesters wild-type HP1ß and other HP1 proteins, exerting dominant-negative effects.


Subject(s)
Chromobox Protein Homolog 5 , Heterochromatin , Animals , Mice , Chromatin/genetics , Chromosomal Proteins, Non-Histone/genetics , Histones/genetics , Histones/metabolism
2.
Cell Rep ; 36(2): 109383, 2021 07 13.
Article in English | MEDLINE | ID: mdl-34260925

ABSTRACT

DNA double-strand breaks (DSBs) are repaired mainly by non-homologous end joining (NHEJ) or homologous recombination (HR). RIF1 negatively regulates resection through the effector Shieldin, which associates with a short 3' single-stranded DNA (ssDNA) overhang by the MRN (MRE11-RAD50-NBS1) complex, to prevent further resection and HR repair. In this study, we show that RIF1, but not Shieldin, inhibits the accumulation of CtIP at DSB sites immediately after damage, suggesting that RIF1 has another effector besides Shieldin. We find that protein phosphatase 1 (PP1), a known RIF1 effector in replication, localizes at damage sites dependent on RIF1, where it suppresses downstream CtIP accumulation and limits the resection by the MRN complex. PP1 therefore acts as a RIF1 effector distinct from Shieldin. Furthermore, PP1 deficiency in the context of Shieldin depletion elevates HR immediately after irradiation. We conclude that PP1 inhibits resection before the action of Shieldin to prevent precocious HR in the early phase of the damage response.


Subject(s)
Cell Cycle Proteins/metabolism , DNA Breaks, Double-Stranded , DNA-Binding Proteins/metabolism , Protein Phosphatase 1/metabolism , Telomere-Binding Proteins/metabolism , BRCA1 Protein/metabolism , Base Sequence , DNA Breaks, Double-Stranded/drug effects , Endodeoxyribonucleases/metabolism , HeLa Cells , Homologous Recombination/drug effects , Humans , Multiprotein Complexes/metabolism , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Protein Binding/drug effects
3.
Cell Rep ; 20(2): 297-307, 2017 07 11.
Article in English | MEDLINE | ID: mdl-28700933

ABSTRACT

DNA double-strand breaks (DSBs) are repaired by either the homology-directed repair (HDR) or the non-homologous end-joining (NHEJ) pathway. RIF1 (RAP1-interacting factor homolog) was recently shown to stimulate NHEJ through an interaction with 53BP1 (p53-binding protein 1) phosphorylated at S/TQ sites, but the molecular mechanism underlying pathway choice remains unclear. Here, we show that SCAI (suppressor of cancer cell invasion) binds to 53BP1 phosphorylated at S/TP sites and facilitates HDR. Upon DNA damage, RIF1 immediately accumulates at damage sites and then gradually dissociates from 53BP1 and is subsequently replaced with SCAI. Depletion of SCAI reduces both the accumulation of HDR factors, including BRCA1 (breast cancer susceptibility gene 1), at damage sites and the efficiency of HDR, as detected by a reporter assay system. These data suggest that SCAI inhibits RIF1 function to allow BRCA1-mediated repair, which possibly includes alt-NHEJ and resection-dependent NHEJ in G1, as well as HDR in S/G2.


Subject(s)
BRCA1 Protein/metabolism , DNA Repair/physiology , Telomere-Binding Proteins/metabolism , Transcription Factors/metabolism , BRCA1 Protein/genetics , DNA Breaks, Double-Stranded , DNA Damage/genetics , DNA Damage/physiology , DNA Repair/genetics , Humans , Kinetics , Phosphorylation/genetics , Phosphorylation/physiology , Protein Binding/genetics , Protein Binding/physiology , Telomere-Binding Proteins/genetics , Transcription Factors/genetics , Tumor Suppressor p53-Binding Protein 1/genetics , Tumor Suppressor p53-Binding Protein 1/metabolism
4.
Cell Rep ; 18(2): 520-532, 2017 01 10.
Article in English | MEDLINE | ID: mdl-28076794

ABSTRACT

BRCA1 promotes homologous recombination (HR) by activating DNA-end resection. By contrast, 53BP1 forms a barrier that inhibits DNA-end resection. Here, we show that BRCA1 promotes DNA-end resection by relieving the 53BP1-dependent barrier. We show that 53BP1 is phosphorylated by ATM in S/G2 phase, promoting RIF1 recruitment, which inhibits resection. 53BP1 is promptly dephosphorylated and RIF1 released, despite remaining unrepaired DNA double-strand breaks (DSBs). When resection is impaired by CtIP/MRE11 endonuclease inhibition, 53BP1 phosphorylation and RIF1 are sustained due to ongoing ATM signaling. BRCA1 depletion also sustains 53BP1 phosphorylation and RIF1 recruitment. We identify the phosphatase PP4C as having a major role in 53BP1 dephosphorylation and RIF1 release. BRCA1 or PP4C depletion impairs 53BP1 repositioning, EXO1 recruitment, and HR progression. 53BP1 or RIF1 depletion restores resection, RAD51 loading, and HR in PP4C-depleted cells. Our findings suggest that BRCA1 promotes PP4C-dependent 53BP1 dephosphorylation and RIF1 release, directing repair toward HR.


Subject(s)
BRCA1 Protein/metabolism , DNA Repair , Homologous Recombination , Signal Transduction , Tumor Suppressor p53-Binding Protein 1/metabolism , Ataxia Telangiectasia Mutated Proteins/metabolism , Carrier Proteins/metabolism , DNA Breaks, Double-Stranded , DNA Repair Enzymes/metabolism , Endodeoxyribonucleases , Exodeoxyribonucleases/metabolism , G2 Phase , Humans , MRE11 Homologue Protein/metabolism , Nuclear Proteins/metabolism , Phosphoprotein Phosphatases/metabolism , Phosphorylation , S Phase , Telomere-Binding Proteins/metabolism
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