Design of genetically encoded sensors to detect nucleosome ubiquitination in live cells
| Autor: | Carolina Dos Santos Passos, Tingting Yao, Robert E. Cohen, Yun-Seok Choi, Christopher D. Snow |
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| Rok vydání: | 2021 |
| Předmět: |
Chromatin or Epigenetics
DNA damage DNA repair Ubiquitin-Protein Ligases In silico Biology Biochemistry Cell Line Tools Histones 03 medical and health sciences 0302 clinical medicine Ubiquitin Humans Nucleosome 030304 developmental biology 0303 health sciences Histone ubiquitination Tumor Suppressor Proteins Ubiquitination Cell Biology Nucleosomes Cell biology Chromatin Histone biology.protein Tumor Suppressor p53-Binding Protein 1 030217 neurology & neurosurgery |
| Zdroj: | The Journal of Cell Biology |
| ISSN: | 1540-8140 0021-9525 |
| Popis: | Ubiquitin conjugation to histone lysines regulates diverse processes such as gene silencing, transcriptional elongation, and DNA repair. Combining an avidity-based design strategy with molecular dynamics simulations, dos Santos Passos et al. develop genetically encoded sensors for H2AK13/15 or H2BK120 ubiquitinated nucleosomes and use them in live cells to monitor ubiquitin-dependent signaling. 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. |
| Databáze: | OpenAIRE |
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