Live-Cell Imaging of Chromatin Condensation Dynamics by CRISPR

• Main Authors: Yuan Xue, Murat Acar
• Format: Article
• Language: English
• Published: Elsevier 2018-06-01
• Series: iScience
• Online Access: http://www.sciencedirect.com/science/article/pii/S2589004218300774

Summary: The spatiotemporal organization of chromatin plays central roles in cellular function. The ribosomal DNA (rDNA) chromatin undergoes dynamic structural changes during mitosis and stress. Here, we developed a CRISPR-based imaging system and tracked the condensation dynamics of rDNA chromatin in live yeast cells under glucose starvation. We found that acute glucose starvation triggers rapid condensation of rDNA. Time-lapse microscopy revealed two stages for rDNA condensation: a “primary stage,” when relaxed rDNA chromatin forms higher order loops or rings, and a “secondary stage,” when the rDNA rings further condense into compact clusters. Twisting of rDNA rings accompanies the secondary stage. The condensin complex, but not the cohesin complex, is required for efficient rDNA condensation in response to glucose starvation. Furthermore, we found that the DNA helicase Sgs1 is essential for the survival of cells expressing rDNA-bound dCas9, suggesting a role for helicases in facilitating DNA replication at dCas9-binding sites. : Genetics; Techniques in Genetics; Molecular Biology; Chromosome Organization; Live Imaging Subject Areas: Genetics, Techniques in Genetics, Molecular Biology, Chromosome Organization, Live Imaging