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VivosX, a disulfide crosslinking method to capture site-specific, protein-protein interactions in yeast and human cells

VivosX is an in vivo disulfide crosslinking approach that utilizes a pair of strategically positioned cysteines on two proteins to probe physical interactions within cells. Histone H2A.Z, which often replaces one or both copies of H2A in nucleosomes downstream of promoters, was used to validate Vivo...

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Autores principales: Mohan, Chitra, Kim, Lisa M, Hollar, Nicole, Li, Tailai, Paulissen, Eric, Leung, Cheuk T, Luk, Ed
Formato: Online Artículo Texto
Lenguaje:English
Publicado: eLife Sciences Publications, Ltd 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6107336/
https://www.ncbi.nlm.nih.gov/pubmed/30091702
http://dx.doi.org/10.7554/eLife.36654
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author Mohan, Chitra
Kim, Lisa M
Hollar, Nicole
Li, Tailai
Paulissen, Eric
Leung, Cheuk T
Luk, Ed
author_facet Mohan, Chitra
Kim, Lisa M
Hollar, Nicole
Li, Tailai
Paulissen, Eric
Leung, Cheuk T
Luk, Ed
author_sort Mohan, Chitra
collection PubMed
description VivosX is an in vivo disulfide crosslinking approach that utilizes a pair of strategically positioned cysteines on two proteins to probe physical interactions within cells. Histone H2A.Z, which often replaces one or both copies of H2A in nucleosomes downstream of promoters, was used to validate VivosX. Disulfide crosslinks between cysteine-modified H2A.Z and/or H2A histones within nucleosomes were induced using a membrane-permeable oxidant. VivosX detected different combinations of H2A.Z and H2A within nucleosomes in yeast cells. This assay correctly reported the change in global H2A.Z occupancy previously observed when the deposition and eviction pathways of H2A.Z were perturbed. Homotypic H2A.Z/H2A.Z (ZZ) nucleosomes accumulated when assembly of the transcription preinitiation complex was blocked, revealing that the transcription machinery preferentially disassembles ZZ nucleosomes. VivosX works in human cells and distinguishes ZZ nucleosomes with one or two ubiquitin moieties, demonstrating that it can be used to detect protein-protein interactions inside cells from different species.
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spelling pubmed-61073362018-08-27 VivosX, a disulfide crosslinking method to capture site-specific, protein-protein interactions in yeast and human cells Mohan, Chitra Kim, Lisa M Hollar, Nicole Li, Tailai Paulissen, Eric Leung, Cheuk T Luk, Ed eLife Biochemistry and Chemical Biology VivosX is an in vivo disulfide crosslinking approach that utilizes a pair of strategically positioned cysteines on two proteins to probe physical interactions within cells. Histone H2A.Z, which often replaces one or both copies of H2A in nucleosomes downstream of promoters, was used to validate VivosX. Disulfide crosslinks between cysteine-modified H2A.Z and/or H2A histones within nucleosomes were induced using a membrane-permeable oxidant. VivosX detected different combinations of H2A.Z and H2A within nucleosomes in yeast cells. This assay correctly reported the change in global H2A.Z occupancy previously observed when the deposition and eviction pathways of H2A.Z were perturbed. Homotypic H2A.Z/H2A.Z (ZZ) nucleosomes accumulated when assembly of the transcription preinitiation complex was blocked, revealing that the transcription machinery preferentially disassembles ZZ nucleosomes. VivosX works in human cells and distinguishes ZZ nucleosomes with one or two ubiquitin moieties, demonstrating that it can be used to detect protein-protein interactions inside cells from different species. eLife Sciences Publications, Ltd 2018-08-09 /pmc/articles/PMC6107336/ /pubmed/30091702 http://dx.doi.org/10.7554/eLife.36654 Text en © 2018, Mohan et al http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/This article is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use and redistribution provided that the original author and source are credited.
spellingShingle Biochemistry and Chemical Biology
Mohan, Chitra
Kim, Lisa M
Hollar, Nicole
Li, Tailai
Paulissen, Eric
Leung, Cheuk T
Luk, Ed
VivosX, a disulfide crosslinking method to capture site-specific, protein-protein interactions in yeast and human cells
title VivosX, a disulfide crosslinking method to capture site-specific, protein-protein interactions in yeast and human cells
title_full VivosX, a disulfide crosslinking method to capture site-specific, protein-protein interactions in yeast and human cells
title_fullStr VivosX, a disulfide crosslinking method to capture site-specific, protein-protein interactions in yeast and human cells
title_full_unstemmed VivosX, a disulfide crosslinking method to capture site-specific, protein-protein interactions in yeast and human cells
title_short VivosX, a disulfide crosslinking method to capture site-specific, protein-protein interactions in yeast and human cells
title_sort vivosx, a disulfide crosslinking method to capture site-specific, protein-protein interactions in yeast and human cells
topic Biochemistry and Chemical Biology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6107336/
https://www.ncbi.nlm.nih.gov/pubmed/30091702
http://dx.doi.org/10.7554/eLife.36654
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