Abstract
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.
| Original language | English (US) |
|---|---|
| Article number | e36654 |
| Journal | eLife |
| Volume | 7 |
| DOIs | |
| State | Published - Aug 9 2018 |
Bibliographical note
Funding Information:This work was inspired by insightful discussions with Rolf Sternglanz, Caryn Outten, and Steven Glynn. We thank Nancy Hollingsworth and Erwin London for critical comments of the manuscript, members of the Luk laboratory for helpful suggestions, Fred Winston for sharing the HTA1-HTB1 plasmid (JH55) and the yeast strain (FY406) and Carl Wu for sharing the anti-H3 antibody. This work is supported by research grants from the National Institutes of Health (RO1 GM104111 to EL and RO1 CA200652 to CTL).
Funding Information:
This work was inspired by insightful discussions with Rolf Sternglanz, Caryn Outten, and Steven Glynn. We thank Nancy Hollingsworth and Erwin London for critical comments of the manuscript, members of the Luk laboratory for helpful suggestions, Fred Winston for sharing the HTA1-HTB1 plasmid (JH55) and the yeast strain (FY406) and Carl Wu for sharing the anti-H3 antibody. This work is supported by research grants from the National Institutes of Health (RO1.GM104111 to EL and RO1.CA200652 to CTL). National Institute of General Medical Sciences RO1 GM104111 Ed Luk National Cancer Institute RO1 CA200652 Cheuk T Leung
Publisher Copyright:
© Mohan et al.