Competitive rDNA binding by dCas9 induces outside-in disassembly of the nucleolus

The inside-out assembly and outside-in disassembly of the nucleolus are well-accepted models, yet direct in-cell evidence remains elusive. Here, we employed a dCas9-based competitive binding system to specifically target the rDNA promoter within the nucleolus, effectively inhibiting rDNA transcription. This transcriptional blockade induced a stepwise, outside-in disassembly of the nucleolus. NPM1 was the first to disappear from the nucleolus, followed by a progressive reduction in the fluorescence intensities of FBL and UBF. Additionally, UBF relocated from the nucleolar core to the periphery. These findings provide the first direct evidence in cells supporting the outside-in disassembly of the nucleolus. Furthermore, our results suggest that the dynamic inside-out assembly and outside-in disassembly of the nucleolus.