CHD4 slides nucleosomes by decoupling entry- and exit-side DNA translocation

Abstract

Chromatin remodellers hydrolyse ATP to move nucleosomal DNA against histone octamers. The mechanism, however, is only partially resolved, and unclear if it is conserved among the four remodeller families. Here we use single-molecule assays to examine the mechanism of action of CHD4, which is part of the least well understood family of remodellers. We demonstrate that the binding energy for CHD4-nucleosome complex formation -even in the absence of nucleotidetriggers significant conformational changes in DNA at the entry side, effectively priming the system for remodelling. During remodelling, flanking DNA enters the nucleosome in a continuous, gradual manner but exits in concerted 4-6 base-pair steps. This decoupling of entry-and exit-side translocation suggests that ATP-driven movement of entry-side DNA builds up strain inside the nucleosome that is subsequently released at the exit side by DNA expulsion. We propose a mechanism for nucleosome sliding based on these and published data.

Nucleosome-CHD4 chromatin remodeler structure maps human disease mutations

“…DNA unwrapping is observed for Chd1 in structures and in solution and is independent of which ATP or transition state analogue is bound to the motor domain, indicating it is achieved with the use of binding energy only. Our observations are consistent with a single-molecule FRET study ( Zhong et al, 2019 ). This major difference in Chd1 and CHD4 molecular function is likely related to a striking difference in cellular function.…”

Single-molecule fluorescence-based approach reveals novel mechanistic insights into human small heat shock protein chaperone function

“…Single-molecule measurements were performed at room temperature (approx. 20˚C) on a custom-built TIRF microscope with a Sapphire, green (532 nm) laser that has been previously described (57). Images were acquired every 200 msec and singlemolecule fluorescence intensity time trajectories from multiple fields of view were generated and analysed using a Matlab-based software (MASH-FRET) (58).…”

RNA inhibits dMi-2/CHD4 chromatin binding and nucleosome remodelling

“…The (CA)20 RNA, on the other hand, failed to inhibit remodelling even at high concentrations (2 µM). We also measured inhibition of CHD4-mediated remodelling by (GA)20 RNA using a Real Time Fluorescence Assay (RTFA) ( (Zhong et al, 2020); Supplementary Figure 6H and 6I). Here, we used nucleosomes carrying an AF488 fluorophore on histone H2A and a BHQ1 quencher at one end of the DNA fragment (Supplementary Figure 6H).…”