RESUMEN
Ribosomal DNA (rDNA) encodes the ribosomal RNA genes and represents an intrinsically unstable genomic region. However, the underlying mechanisms and implications for genome integrity remain elusive. Here, we use Bloom syndrome (BS), a rare genetic disease characterized by DNA repair defects and hyper-unstable rDNA, as a model to investigate the mechanisms leading to rDNA instability. We find that in Bloom helicase (BLM) proficient cells, the homologous recombination (HR) pathway in rDNA resembles that in nuclear chromatin; it is initiated by resection, replication protein A (RPA) loading and BRCA2-dependent RAD51 filament formation. However, BLM deficiency compromises RPA-loading and BRCA1/2 recruitment to rDNA, but not RAD51 accumulation. RAD51 accumulates at rDNA despite depletion of long-range resection nucleases and rDNA damage results in micronuclei when BLM is absent. In summary, our findings indicate that rDNA is permissive to RAD51 accumulation in the absence of BLM, leading to micronucleation and potentially global genomic instability.
Asunto(s)
ADN Ribosómico , Inestabilidad Genómica , Recombinasa Rad51 , RecQ Helicasas , Recombinasa Rad51/metabolismo , Recombinasa Rad51/genética , ADN Ribosómico/genética , ADN Ribosómico/metabolismo , Humanos , RecQ Helicasas/metabolismo , RecQ Helicasas/genética , Proteína de Replicación A/metabolismo , Proteína de Replicación A/genética , Recombinación Homóloga , Síndrome de Bloom/genética , Síndrome de Bloom/metabolismo , Proteína BRCA2/metabolismo , Proteína BRCA2/genética , Proteína BRCA1/metabolismo , Proteína BRCA1/genética , Reparación del ADNRESUMEN
Cell shape dynamics during development is tightly regulated and coordinated with cell fate determination. Triggered by an interplay between biochemical and mechanical signals, epithelia form complex tissues by undergoing coordinated cell shape changes, but how such spatiotemporal coordination is controlled remains an open question. To dissect biochemical signaling from purely mechanical cues, we developed a microfluidic system that experimentally triggers epithelial folding to recapitulate stereotypic deformations observed in vivo. Using this system, we observe that the apical or basal direction of folding results in strikingly different mechanical states at the fold boundary, where the balance between tissue tension and torque (arising from the imposed curvature) controls the spread of folding-induced calcium waves at a short timescale and induces spatial patterns of gene expression at longer timescales. Our work uncovers that folding-associated gradients of cell shape and their resulting mechanical stresses direct spatially distinct biochemical responses within the monolayer.