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.
Highlights
To uncover the mechanism of a molecular pathway, one approach is to understand how individual components interact within the physiological context of a cell
We show that H2A-to-H2A, H2A.Z-to-H2A, and H2A.Z-to-H2A.Z crosslinking can be used to infer the levels of AA, AZ and ZZ nucleosomal species in yeast cells, demonstrating that VivosX can be used as a general strategy for probing interactions of other protein pairs in cells when structural information exists to allow strategic placement of the cysteine probes
To identify a cysteine substitution in the L1 region of H2A.Z that can crosslink to the same site on the opposite H2A.Z molecule within a ZZ nucleosome (Figure 1A), the six codons in the L1 region of
Summary
To uncover the mechanism of a molecular pathway, one approach is to understand how individual components interact within the physiological context of a cell. Existing cell-based, protein-protein interaction assays, such as yeast two-hybrid or protein-fragment complementation, do not provide information about the site of interaction or the proportion of free versus bound species (Fields and Sternglanz, 1994; Michnick et al, 2007). We introduce a methodology called VivosX (in vivo disulfide crosslinking) which is a simple, quantifiable assay for reporting site-specific interactions that occur inside the nucleus or the cytosol. VivosX can be applied to detect the oligomerization status of nuclear and cytosolic factors based on the proportion of crosslinked and uncrosslinked species, providing a simple strategy to study, for example, how transcription factors or signaling molecules dimerize in response to cellular cues. As a proof of concept, VivosX was used to detect interactions between specific histone proteins within the nucleosomes of yeast and human cells
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
