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

Publisher: Oxford University Press Country of Publication: England NLM ID: 0411011 Publication Model: Print Cited Medium: Internet ISSN: 1362-4962 (Electronic) Linking ISSN: 03051048 NLM ISO Abbreviation: Nucleic Acids Res Subsets: MEDLINE

Publication: 1992- : Oxford : Oxford University Press
Original Publication: London, Information Retrieval ltd.

Schizosaccharomyces*/metabolism ; Schizosaccharomyces*/genetics ; Heterochromatin*/metabolism ; Schizosaccharomyces pombe Proteins*/metabolism ; Schizosaccharomyces pombe Proteins*/genetics ; Histones*/metabolism ; Chromosomal Proteins, Non-Histone*/metabolism ; Single Molecule Imaging*/methods ; Chromobox Protein Homolog 5*/metabolism ; Protein Binding*; Methylation ; Chromatin/metabolism ; Cell Nucleus/metabolism

Visualizing and measuring molecular-scale interactions in living cells represents a major challenge, but recent advances in single-molecule super-resolution microscopy are bringing us closer to achieving this goal. Single-molecule super-resolution microscopy enables high-resolution and sensitive imaging of the positions and movement of molecules in living cells. HP1 proteins are important regulators of gene expression because they selectively bind and recognize H3K9 methylated (H3K9me) histones to form heterochromatin-associated protein complexes that silence gene expression, but several important mechanistic details of this process remain unexplored. Here, we extended live-cell single-molecule tracking studies in fission yeast to determine how HP1 proteins interact with their binding partners in the nucleus. We measured how genetic perturbations that affect H3K9me alter the diffusive properties of HP1 proteins and their binding partners, and we inferred their most likely interaction sites. Our results demonstrate that H3K9 methylation spatially restricts HP1 proteins and their interactors, thereby promoting ternary complex formation on chromatin while simultaneously suppressing off-chromatin binding. As opposed to being an inert platform to direct HP1 binding, our studies propose a novel function for H3K9me in promoting ternary complex formation by enhancing the specificity and stimulating the assembly of HP1-protein complexes in living cells.
(© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Update of: bioRxiv. 2023 Oct 19:2023.03.08.531771. doi: 10.1101/2023.03.08.531771. (PMID: 36945633)

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RSG2211701DMC American Cancer Society; 2316281 Division of Emerging Frontiers; R35 GM137832 United States GM NIGMS NIH HHS; 140220 Division of Molecular and Cellular Biosciences; NSF; R35GM137832 United States GM NIGMS NIH HHS

Local Abstract: [plain-language-summary] Visualizing molecular-scale interactions in living cells is challenging, but advances in single-molecule super-resolution microscopy enable high-resolution imaging of molecular positions of proteins and their motions within cells. HP1 proteins bind to H3K9 methylated histones to form complexes that silence gene expression. Here, we tracked single HP1 proteins and their binding partners to measure when and where they form complexes in live fission yeast cells. Genetic perturbations enabled us to connect their motions to specific changes in their cellular properties. Surprisingly, we noted that HP1 proteins preferentially form ternary complexes with their binding partners at sites of H3K9me. This work proposes a novel function for chromatin and shows how H3K9 methylation spatially restricts HP1-associated complex formation while suppressing off-chromatin binding.

0 (Heterochromatin)
0 (Schizosaccharomyces pombe Proteins)
0 (Histones)
0 (Chromosomal Proteins, Non-Histone)
107283-02-3 (Chromobox Protein Homolog 5)
0 (Chromatin)

Date Created: 20240814 Date Completed: 20241014 Latest Revision: 20241016

20250114

PMC11472046

10.1093/nar/gkae692

39142658