Deciphering the Interactome of Histone Marks in Living Cells via Genetic Code Expansion Combined with Proximity Labeling.
Anal Chem
; 94(30): 10705-10714, 2022 08 02.
Article
em En
| MEDLINE
| ID: mdl-35862615
ABSTRACT
Deciphering the endogenous interactors of histone post-translational modifications (hPTMs, also called histone marks) is essential to understand the mechanisms of epigenetic regulation. However, most of the analytical methods to determine hPTM interactomes are in vitro settings, lacking interrogating native chromatin. Although lysine crotonylation (Kcr) has recently been considered an important hPTM for the regulation of gene transcription, the interactors of Kcr still remain to be explored. Herein, we present a general approach relying upon a genetic code expansion system, APEX2 (engineered peroxidase)-mediated proximity labeling, and quantitative proteomics to profile interactomes of the selected hPTMs in living cells. We genetically fused APEX2 to the recombinant histone H3 with a crotonyl lysine inserted site specifically to generate APEX2-H3K9cr that incorporated into native chromatin. Upon activation, APEX2 triggered in vivo biotin labeling of H3K9cr interactors that can then be enriched with streptavidin beads and identified by mass spectrometry. Proteomic analysis further revealed the endogenous interactomes of H3K9cr and confirmed the reliability of the method. Moreover, DPF2 was identified as a candidate interactor, and the binding interaction of DPF2 to H3K9c was further characterized and verified. This study provides a novel strategy for the identification of hPTM interactomes in living cells, and we envision that this is key to elucidating epigenetic regulatory pathways.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Código das Histonas
/
Lisina
Tipo de estudo:
Prognostic_studies
Idioma:
En
Revista:
Anal Chem
Ano de publicação:
2022
Tipo de documento:
Article