Open AccessDesign of genetically encoded sensors to detect nucleosome ubiquitination in live cells
Author(s) -
Carolina dos Santos Passos,
YunSeok Choi,
Christopher D. Snow,
Tingting Yao,
Robert E. Cohen
Publication year - 2021
Publication title -
the journal of cell biology/the journal of cell biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.414
H-Index - 380
eISSN - 1540-8140
pISSN - 0021-9525
DOI - 10.1083/jcb.201911130
Subject(s) - biology , ubiquitin , histone , histone h2b , microbiology and biotechnology , chromatin , nucleosome , histone h2a , dna repair , f box protein , dna , genetics , gene , ubiquitin ligase
Histone posttranslational modifications (PTMs) are dynamic, context-dependent signals that modulate chromatin structure and function. Ubiquitin (Ub) conjugation to different lysines of histones H2A and H2B is used to regulate diverse processes such as gene silencing, transcriptional elongation, and DNA repair. Despite considerable progress made to elucidate the players and mechanisms involved in histone ubiquitination, there remains a lack of tools to monitor these PTMs, especially in live cells. To address this, we combined an avidity-based strategy with in silico approaches to design sensors for specifically ubiquitinated nucleosomes. By linking Ub-binding domains to nucleosome-binding peptides, we engineered proteins that target H2AK13/15Ub and H2BK120Ub with Kd values from 10−8 to 10−6 M; when fused to fluorescent proteins, they work as PTM sensors in cells. The H2AK13/15Ub-specific sensor, employed to monitor signaling from endogenous DNA damage through the cell cycle, identified and differentiated roles for 53BP1 and BARD1 as mediators of this histone PTM.