Go to JCI Insight
  • About
  • Editors
  • Consulting Editors
  • For authors
  • Publication ethics
  • Publication alerts by email
  • Advertising
  • Job board
  • Contact
  • Clinical Research and Public Health
  • Current issue
  • Past issues
  • By specialty
    • COVID-19
    • Cardiology
    • Gastroenterology
    • Immunology
    • Metabolism
    • Nephrology
    • Neuroscience
    • Oncology
    • Pulmonology
    • Vascular biology
    • All ...
  • Videos
    • Conversations with Giants in Medicine
    • Video Abstracts
  • Reviews
    • View all reviews ...
    • Complement Biology and Therapeutics (May 2025)
    • Evolving insights into MASLD and MASH pathogenesis and treatment (Apr 2025)
    • Microbiome in Health and Disease (Feb 2025)
    • Substance Use Disorders (Oct 2024)
    • Clonal Hematopoiesis (Oct 2024)
    • Sex Differences in Medicine (Sep 2024)
    • Vascular Malformations (Apr 2024)
    • View all review series ...
  • Viewpoint
  • Collections
    • In-Press Preview
    • Clinical Research and Public Health
    • Research Letters
    • Letters to the Editor
    • Editorials
    • Commentaries
    • Editor's notes
    • Reviews
    • Viewpoints
    • 100th anniversary
    • Top read articles

  • Current issue
  • Past issues
  • Specialties
  • Reviews
  • Review series
  • Conversations with Giants in Medicine
  • Video Abstracts
  • In-Press Preview
  • Clinical Research and Public Health
  • Research Letters
  • Letters to the Editor
  • Editorials
  • Commentaries
  • Editor's notes
  • Reviews
  • Viewpoints
  • 100th anniversary
  • Top read articles
  • About
  • Editors
  • Consulting Editors
  • For authors
  • Publication ethics
  • Publication alerts by email
  • Advertising
  • Job board
  • Contact
CDCA7 and HELLS mutations undermine nonhomologous end joining in centromeric instability syndrome
Motoko Unoki, … , Claire Francastel, Hiroyuki Sasaki
Motoko Unoki, … , Claire Francastel, Hiroyuki Sasaki
Published October 11, 2018
Citation Information: J Clin Invest. 2019;129(1):78-92. https://doi.org/10.1172/JCI99751.
View: Text | PDF
Research Article Cell biology Genetics

CDCA7 and HELLS mutations undermine nonhomologous end joining in centromeric instability syndrome

  • Text
  • PDF
Abstract

Mutations in CDCA7 and HELLS that respectively encode a CXXC-type zinc finger protein and an SNF2 family chromatin remodeler cause immunodeficiency, centromeric instability, and facial anomalies (ICF) syndrome types 3 and 4. Here, we demonstrate that the classical nonhomologous end joining (C-NHEJ) proteins Ku80 and Ku70, as well as HELLS, coimmunoprecipitated with CDCA7. The coimmunoprecipitation of the repair proteins was sensitive to nuclease treatment and an ICF3 mutation in CDCA7 that impairs its chromatin binding. The functional importance of these interactions was strongly suggested by the compromised C-NHEJ activity and significant delay in Ku80 accumulation at DNA damage sites in CDCA7- and HELLS-deficient HEK293 cells. Consistent with the repair defect, these cells displayed increased apoptosis, abnormal chromosome segregation, aneuploidy, centrosome amplification, and significant accumulation of γH2AX signals. Although less prominent, cells with mutations in the other ICF genes DNMT3B and ZBTB24 (responsible for ICF types 1 and 2, respectively) showed similar defects. Importantly, lymphoblastoid cells from ICF patients shared the same changes detected in the mutant HEK293 cells to varying degrees. Although the C-NHEJ defect alone did not cause CG hypomethylation, CDCA7 and HELLS are involved in maintaining CG methylation at centromeric and pericentromeric repeats. The defect in C-NHEJ may account for some common features of ICF cells, including centromeric instability, abnormal chromosome segregation, and apoptosis.

Authors

Motoko Unoki, Hironori Funabiki, Guillaume Velasco, Claire Francastel, Hiroyuki Sasaki

×

Figure 4

ICF mutant cells display enlarged nuclei, centrosome amplification, and abnormal chromosome segregation.

Options: View larger image (or click on image) Download as PowerPoint
ICF mutant cells display enlarged nuclei, centrosome amplification, and ...
(A) Representative images from biological triplicate showing immunostaining of centrosomes with anti-pericentrin antibody (green) in WT and mutant cells. Nuclei of CDCA7, HELLS, and Ku80 mutant cells were significantly enlarged (DAPI staining, blue). Arrows and arrowheads indicate giant aggregated centrosomes and metaphase chromosome spreads with an aberrant number of centrosomes, respectively. Scale bars: 20 μm. The right panel shows the distribution of areas (μm2) of individual nuclei measured using ZEN 2012 software. At least 30 nuclei (the exact number is shown in parentheses) were measured for each clone. Each box indicates 25th to 75th percentile, and a bar in the box indicates the median. X indicates the mean. *P < 0.0033 and **P < 0.0007 (Mann-Whitney U test) were considered statistically significant at the 5% and 1% levels, respectively, after Bonferroni correction. The exact P value, which was significant (P < 0.01) before the correction, is shown for reference. (B) Enlarged images of representative metaphase chromosome spreads and centrosomes from A. The right panel shows the percentage composition of metaphase cells with indicated centrosome numbers. At least 40 metaphase cells (the exact number is shown in parentheses) were analyzed for each clone. Scale bars: 5 μm. (C) Representative images from biological triplicate showing anaphase chromosomes stained with DAPI. At least 50 anaphase-nuclei (the exact number is shown in parentheses) were observed for each clone (mean ± SEM). The right panel shows the percentage anaphase images with bridges. *P < 0.0033 (Student’s 2-tailed t test) was considered statistically significant at the 5% level after Bonferroni correction. The exact P values, which were significant (P < 0.05) before the correction, are shown for reference. Scale bars: 5 μm.

Copyright © 2025 American Society for Clinical Investigation
ISSN: 0021-9738 (print), 1558-8238 (online)

Sign up for email alerts