Engineered TALE Repeats for Enhanced Imaging-Based Analysis of Cellular 5-Methylcytosine.
DNA methylation
epigenetics
imaging probes
membrane-less organelles
Journal
Chembiochem : a European journal of chemical biology
ISSN: 1439-7633
Titre abrégé: Chembiochem
Pays: Germany
ID NLM: 100937360
Informations de publication
Date de publication:
15 02 2021
15 02 2021
Historique:
received:
12
08
2020
revised:
29
09
2020
pubmed:
30
9
2020
medline:
28
9
2021
entrez:
29
9
2020
Statut:
ppublish
Résumé
Transcription-activator-like effectors (TALEs) are repeat-based, programmable DNA-binding proteins that can be engineered to recognize sequences of canonical and epigenetically modified nucleobases. Fluorescent TALEs can be used for the imaging-based analysis of cellular 5-methylcytosine (5 mC) in repetitive DNA sequences. This is based on recording fluorescence ratios from cell co-stains with two TALEs: an analytical TALE targeting the cytosine (C) position of interest through a C-selective repeat that is blocked by 5 mC, and a control TALE targeting the position with a universal repeat that binds both C and 5 mC. To enhance this approach, we report herein the development of novel 5 mC-selective repeats and their integration into TALEs that can replace universal TALEs in imaging-based 5 mC analysis, resulting in a methylation-dependent response of both TALEs. We screened a library of size-reduced repeats and identified several 5 mC binders. Compared to the 5 mC-binding repeat of natural TALEs and to the universal repeat, two repeats containing aromatic residues showed enhancement of 5 mC binding and selectivity in cellular transcription activation and electromobility shift assays, respectively. In co-stains of cellular SATIII DNA with a corresponding C-selective TALE, this selectivity results in a positive methylation response of the new TALE, offering perspectives for studying 5 mC functions in chromatin regulation by in situ imaging with increased dynamic range.
Identifiants
pubmed: 32991020
doi: 10.1002/cbic.202000563
pmc: PMC7894354
doi:
Substances chimiques
Molecular Probes
0
Transcription Activator-Like Effectors
0
5-Methylcytosine
6R795CQT4H
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
645-651Subventions
Organisme : European Research Council
ID : ERC-CoG EPICODE, grant no. 723863
Pays : International
Informations de copyright
© 2020 The Authors. ChemBioChem published by Wiley-VCH GmbH.
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