Single-molecule imaging of chromatin remodelers reveals role of ATPase in promoting fast kinetics of target search and dissociation from chromatin

Abstract

Conserved ATP-dependent chromatin remodelers establish and maintain genome-wide chromatin architectures of regulatory DNA during cellular lifespan, but the temporal interactions between remodelers and chromatin targets have been obscure. We performed live-cell single-molecule tracking for RSC, SWI/SNF, CHD1, ISW1, ISW2, and INO80 remodeling complexes in budding yeast and detected hyperkinetic behaviors for chromatin-bound molecules that frequently transition to the free state for all complexes. Chromatin-bound remodelers display notably higher diffusion than nucleosomal histones, and strikingly fast dissociation kinetics with 4-7 s mean residence times. These enhanced dynamics require ATP binding or hydrolysis by the catalytic ATPase, uncovering an additional function to its established role in nucleosome remodeling. Kinetic simulations show that multiple remodelers can repeatedly occupy the same promoter region on a timescale of minutes, implicating an unending ‘tug-of-war’ that controls a temporally shifting window of accessibility for the transcription initiation machinery.

FACT is recruited to the +1 nucleosome of transcribed genes and spreads in a Chd1-dependent manner

“…Remarkably, the dissociation kinetics of Spt16 are highly similar to those observed for RNAPII in live cells (RNAPII t sb = 20-23 s) (Nguyen et al, 2021). This characteristic is unique among all the factors we have tested, which includes the entire PIC (Nguyen et al, 2021) and six chromatin-remodeling complexes (Kim et al, 2021). These results suggest a functional connection between FACT and RNAPII on chromatin in vivo.…”

“…In this model, Chd1 therefore promotes the ''processivity'' of FACT during transcription. Interestingly, although FACT (this study) and RNAPII (Nguyen et al, 2021) have a dwell time consistent with both factors persisting on an average gene from start to finish ($22 s), Chd1 resides on chromatin for much shorter time (7 s) (Kim et al, 2021). These observations suggest that different molecules of Chd1 may take turns in promoting the progression of a single FACT complex.…”

“…First, the residence time of FACT and RNAPII are very similar (20-25 s on average) and consistent with both RNAPII and FACT persisting on genes from start to finish, considering the speed of transcription elongation and the size of yeast genes. We note that RNAPII takes about 5 s to transcribe through a nucleosome, a time that resembles the residence time of chromatin remodelers (Kim et al, 2021) but much shorter than the observed residence time of FACT. Second, despite FACT being very abundant, the number of FACT molecules stably associated with chromatin at a given time is estimated at 450-1,500 (3% of the total number of FACT molecules), similar to the number of elongating RNAPII molecules (690-1,380) (Pelechano et al, 2010).…”

“…We tagged Spt16 with the HaloTag (Los et al, 2008) and labeled mid-log growing cells with Janelia Fluor 552 (JF 552 ) (Zheng et al, 2019). We imaged single molecules at 10 ms resolution and tracked their positions to establish two-dimensional (2D) projections of trajectories through nuclear space (Kim et al, 2021;Nguyen et al, 2021). From these ''fasttracking'' movies, we calculated the mean squared displacement (MSD) for each trajectory to extract the molecule's apparent diffusion coefficient (D).…”