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Live-cell single-molecule tracking highlights requirements for stable Smc5/6 chromatin association in vivo.

ABSTRACT: The essential Smc5/6 complex is required in response to replication stress and is best known for ensuring the fidelity of homologous recombination. Using single-molecule tracking in live fission yeast to investigate Smc5/6 chromatin association, we show that Smc5/6 is chromatin associated in unchallenged cells and this depends on the non-SMC protein Nse6. We define a minimum of two Nse6-dependent sub-pathways, one of which requires the BRCT-domain protein Brc1. Using defined mutants in genes encoding the core Smc5/6 complex subunits, we show that the Nse3 double-stranded DNA binding activity and the arginine fingers of the two Smc5/6 ATPase binding sites are critical for chromatin association. Interestingly, disrupting the single-stranded DNA (ssDNA) binding activity at the hinge region does not prevent chromatin association but leads to elevated levels of gross chromosomal rearrangements during replication restart. This is consistent with a downstream function for ssDNA binding in regulating homologous recombination.

SUBMITTER: Etheridge TJ

PROVIDER: S-EPMC8075580 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Live-cell single-molecule tracking reveals co-recognition of H3K27me3 and DNA targets polycomb Cbx7-PRC1 to chromatin.

Project description:The Polycomb PRC1 plays essential roles in development and disease pathogenesis. Targeting of PRC1 to chromatin is thought to be mediated by the Cbx family proteins (Cbx2/4/6/7/8) binding to histone H3 with a K27me3 modification (H3K27me3). Despite this prevailing view, the molecular mechanisms of targeting remain poorly understood. Here, by combining live-cell single-molecule tracking (SMT) and genetic engineering, we reveal that H3K27me3 contributes significantly to the targeting of Cbx7 and Cbx8 to chromatin, but less to Cbx2, Cbx4, and Cbx6. Genetic disruption of the complex formation of PRC1 facilitates the targeting of Cbx7 to chromatin. Biochemical analyses uncover that the CD and AT-hook-like (ATL) motif of Cbx7 constitute a functional DNA-binding unit. Live-cell SMT of Cbx7 mutants demonstrates that Cbx7 is targeted to chromatin by co-recognizing of H3K27me3 and DNA. Our data suggest a novel hierarchical cooperation mechanism by which histone modifications and DNA coordinate to target chromatin regulatory complexes.

| S-EPMC5056789 | biostudies-literature

Live-cell 3D single-molecule tracking reveals how NuRD modulates enhancer dynamics

Project description:Enhancer-promoter dynamics are crucial for the spatiotemporal control of gene expression, but it remains unclear whether these dynamics are controlled by chromatin regulators, such as the nucleosome remodelling and deacetylase (NuRD) complex. The NuRD complex binds to all active enhancers to modulate transcription and here we use Hi-C experiments to show that it blurs TAD boundaries and increases the proximity of intermediate-range (~1 Mb) genomic sequences and enhancer-promoter interactions. To understand whether NuRD alters the dynamics of 3D genome structure, we developed an approach to segment and extract key biophysical parameters from 3D single-molecule trajectories of the NuRD complex determined using live-cell imaging. Unexpectedly, this revealed that the intact NuRD complex decompacts chromatin structure and makes NuRD-bound enhancers move faster, increasing the overall volume of the nucleus that these key regulatory regions explore. Interestingly, we also uncovered a rare fast-diffusing state of NuRD bound enhancers that exhibits directed motion. The NuRD complex reduces the amount of time that enhancers remain in this fast-diffusing state, which we propose might otherwise re-organise enhancer-promoter proximity.

2023-05-31 | GSE179007 | GEO

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Tracking live-cell single-molecule dynamics enables measurements of heterochromatin-associated protein-protein interactions

2024-05-01 | GSE263472 | GEO

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Live-cell 3D single-molecule tracking reveals how NuRD modulates enhancer/promoter dynamics [ChIP-seq]

Project description:Enhancer-promoter dynamics are critical for the spatiotemporal control of gene expression, but it remains unclear how these dynamics are controlled by the nucleosome remodelling and deacetylase (NuRD) complex that is found at nearly all active enhancers and promoters. We show that MBD3-dependent assembly of the intact NuRD complex increases CTCF/Cohesin binding (using ChIP-seq) and the probability of the interaction of intermediate-range (~1Mb) genomic sequences (using population Hi-C). Using single-molecule imaging, we then revealed that these changes are related to how the chromatin moves, and that the intact NuRD complex increases how fast the nucleus is explored by NuRD-bound enhancers and promoters.

2021-04-01 | GSE147789 | GEO

Single-Molecule Tracking of Chromatin-Associated Proteins in the <i>C. elegans</i> Gonad.

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