Faculty of Chemistry and Chemical Biology, TU Dortmund University, Otto-Hahn Str. 6, 44227, Dortmund, Germany.
International Max Planck Research School, Max Planck Institute of Molecular Physiology, Otto-Hahn Str. 10, 44227, Dortmund, Germany.
Angew Chem Int Ed Engl. 2020 Jun 2;59(23):8927-8931. doi: 10.1002/anie.202001935. Epub 2020 Apr 7.
We report programmable receptors for the imaging-based analysis of 5-methylcytosine (5mC) in user-defined DNA sequences of single cells. Using fluorescent transcription-activator-like effectors (TALEs) that can recognize sequences of canonical and epigenetic nucleobases through selective repeats, we imaged cellular SATIII DNA, the origin of nuclear stress bodies (nSB). We achieve high nucleobase selectivity of natural repeats in imaging and demonstrate universal nucleobase binding by an engineered repeat. We use TALE pairs differing in only one such repeat in co-stains to detect 5mC in SATIII sequences with nucleotide resolution independently of differences in target accessibility. Further, we directly correlate the presence of heat shock factor 1 with 5mC at its recognition sequence, revealing a potential function of 5mC in its recruitment as initial step of nSB formation. This opens a new avenue for studying 5mC functions in chromatin regulation in situ with nucleotide, locus, and cell resolution.
我们报告了可编程受体，可用于基于成像的单个细胞中用户定义的 DNA 序列 5-甲基胞嘧啶 (5mC) 的分析。使用可以通过选择性重复识别经典和表观遗传核碱基序列的荧光转录激活效应物 (TALE)，我们对核应激体 (nSB) 的起源细胞 SATIII DNA 进行了成像。我们在成像中实现了天然重复序列的高核碱基选择性，并通过工程重复序列证明了通用核碱基结合。我们使用仅在一个这样的重复中不同的 TALE 对在共染色中检测 SATIII 序列中的 5mC，具有核苷酸分辨率，而与靶标可及性的差异无关。此外，我们直接将热休克因子 1 的存在与它在其识别序列上的 5mC 相关联，揭示了 5mC 在其募集作为 nSB 形成初始步骤中的潜在功能。这为研究染色质调节中的 5mC 功能开辟了新途径，具有核苷酸、基因座和细胞分辨率。
