Visualizing Live Chromatin Dynamics

Abstract

In this talk, I will discuss the following two topics: (1) transcription initiation dynamics in yeast mitochondria and (2) chromatin dynamics under replication stress. Using single molecule FRET techniques, we show that the yeast mitochondrial transcriptional complex dynamically transitions among closed, open, and scrunched states throughout the initiation stage. Then abruptly at position +8, a sharp irreversible transition occurs to bring the complex into an unbent conformation along with promoter release. Stalled initiation complexes remain in dynamic scrunching and unscrunching states without dissociating the RNA transcript, suggesting the existence of backtracking transitions that have possible regulatory roles. In the second part of the talk, I will present our recent studies of chromatin dynamics using CRISPR imaging techniques. We developed a CRISPR system integrating split fluorophores and signal-amplifying tags in order to suppress background signals and it allows tracking small repeat or non-repetitive gene loci for an extended time using conventional microscopy. Stochastic optical reconstruction microscopy of a chromosomal domain in living cells reveals fast conformational changes within the domain. Heterochromatin domains exhibit long extensions under replication stress, revealing the implications of active chromatin reorganization in the repair of heterochromatin domains.