The BAF A12T mutation disrupts lamin A/C interaction, impairing robust repair of nuclear envelope ruptures in Nestor–Guillermo progeria syndrome cells

Abstract

Nestor–Guillermo progeria syndrome (NGPS) is caused by a homozygous alanine-to-threonine mutation at position 12 (A12T) in barrier-to-autointegration factor (BAF). It is characterized by accelerated aging with severe skeletal abnormalities. BAF is an essential protein binding to DNA and nuclear envelope (NE) proteins, involved in NE rupture repair. Here, we assessed the impact of BAF A12T on NE integrity using NGPS-derived patient fibroblasts. We observed a strong defect in lamin A/C accumulation to NE ruptures in NGPS cells, restored upon homozygous reversion of the pathogenic BAF A12T mutation with CRISPR/Cas9. By combining in vitro and cellular assays, we demonstrated that while the A12T mutation does not affect BAF 3D structure and phosphorylation by VRK1, it specifically decreases the interaction between BAF and lamin A/C. Finally, we revealed that the disrupted interaction does not prevent repair of NE ruptures but instead generates weak points in the NE that lead to a higher frequency of NE re-rupturing in NGPS cells. We propose that this NE fragility could directly contribute to the premature aging phenotype in patients.

A multiparametric anti-aging CRISPR screen uncovers a role for BAF in protein translation

“…2E and Fig. S2C), further characterized in our recent study ( 14 ). NE ruptures are identified on fixed cells by the presence of nuclear blebs (observed using LAP2 staining), the first step in the NE rupturing process ( 28–30 ) (Fig.…”

“…While a targeted (320 genes) multiparametric siRNA screen has been carried out previously in a cellular model of HGPS ( 39 ), our screening approach has interrogated the entire human protein coding genome (19,200 genes) and used NGPS patient cells. By using both WT fibroblasts from an unaffected individual, as well as NGPS fibroblasts in which we reversed the BAF A12T homozygous mutation using CRISPR/Cas9 ( 14 ), we were able to identify a reliable combination of four phenotypes that were both specific to the BAF A12T mutation, and quantifiable by high throughput microscopy. This approach allowed us to reduce the number of hits to ∼0.2% of the whole genome library, and to identify genes showing the best “rescue” across several phenotypes.…”

“…Cells were grown at 37°C in a 5% CO 2 incubator in Dulbecco’s modified Eagle’s medium containing 4.5 g/L glucose (Sigma) supplemented with 2 mM L-glutamine (Sigma), 10% foetal bovine serum, 100 units/mL penicillin and 0.1 g/L streptomycin (complete medium). WT cells expressing Flag-BAF or Flag-A12T BAF were described before ( 14 ) and maintained in complete medium containing 100 μg/mL hygromycin B (Toku-E, #H007, made up to 100 mg/mL in PBS). Cells were passaged twice a week and used within 12 passages.…”

“…After 20 minutes of incubation at room temperature, the transfection mixes were distributed in 384-well imaging plates (Greiner #781182) and overlaid with 1200 NGPS2-Cas9 cells/well in 30 μL of complete DMEM medium. WT cells and NGPS2 WT cells (from the 2 clones in which the BAF A12T mutation was reversed using CRISPR Cas9 ( 14 ) – see Fig. S3A) were added in columns 1 and 2 of each plate (that do not contain transfection complexes).…”

“…The first major step relied on the identification of a combination of phenotypes, specific to the BAF A12T mutation, significantly different in NGPS cells compared to WT cells, and amenable to high throughput screening. To this aim, we took advantage of our recently established NGPS2 isogenic cell lines, in which we reversed the homozygous BAF A12T mutation using CRISPR/Cas9 (NGPS2 corrected) ( 14 ). The screen set-up also required the design of specific pipelines to reliably identify and quantify these phenotypes.…”