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. 2021 Feb 15;22(4):645-651.
doi: 10.1002/cbic.202000563. Epub 2020 Nov 6.

Engineered TALE Repeats for Enhanced Imaging-Based Analysis of Cellular 5-Methylcytosine

Álvaro Muñoz-López  1 , Anne Jung  1 , Benjamin Buchmuller  1 , Jan Wolffgramm  1 , Sara Maurer  1 , Anna Witte  1 , Daniel Summerer  1
Affiliations

Engineered TALE Repeats for Enhanced Imaging-Based Analysis of Cellular 5-Methylcytosine

Álvaro Muñoz-López et al. Chembiochem. .

Abstract

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.

Keywords: DNA methylation; epigenetics; imaging probes; membrane-less organelles.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
DNA recognition of TALEs. a) Chemical structures of cytosine and 5‐methylcytosine. b) Features of a TALE used in this study. An example repeat sequence is on top with the RVD in box. 5′‐T nucleotide is bound by the noncanonical repeat 0 and is thus not counted in target sequences. c) Crystal structures of DNA‐bound TALE RVDs HD and NG, [9] and model of RVD G* [10] bound to C or 5 mC, respectively.
Figure 2
Figure 2
Library design and screening assay for the development of 5 mC‐selective TALE repeats. a) Positions targeted for deletion or randomization in a model of repeat G*; [10] *: deletion; X: random position. b) Repeat sequences for RVDs HD, G*, and library. RVD positions in gray box. c) DNase I competition assay using Cy3/Cy5 doubly labeled DNA oligonucleotide duplexes with variable nucleobase (○) opposite a mutant repeat (X). d) Results of screening with DNase I assay conducted in duplicate with 0.5 μM of each TALE (RVDs indicated below), 0.1 μM DNA and 1 unit DNase I. The Cy5 fluorescence 25 min after DNase I addition is shown, background‐corrected by subtracting a control without TALE and normalized to a control w/o DNase I.
Figure 3
Figure 3
Characterization of engineered TALE repeats. a) Principle of 5 mC‐selective luciferase reporter assay based on a TALE_1‐VP64 fusion construct. b) Luminescence data from a transcriptional activation assay using Hey2‐targeting TALE_1 versions and luciferase reporter plasmid with methylated target sequence in HEK 293T cells. Error bars show the standard deviation of three independent biological replicates. c) 5 mC selectivity of SATIII‐targeting TALE_2 versions with standard and engineered repeats opposite two target CpGs in EMSA. Fraction of DNA‐bound TALE_2 versions in EMSA was quantified for different stoichiometries of TALE_2 s and target DNA duplex containing either 5 mC or C, and ratio between the two is depicted (bars show standard error of six independent experiments).
Figure 4
Figure 4
Evaluation of NY* and NH* repeats in 5 mC analysis at user‐defined CpG by cellular imaging. a) Imaging of cells expressing an mClover3‐TALE_2 version targeting CpGs with HD repeats and mCherry‐HSF1 with or without heat‐shock. b) Experimental setup and employed TALE_2 versions for co‐staining and imaging‐based 5 mC analysis. c) Histogram of HD TALE_2 FI of foci from DNMTact/DNMTinact cells co‐stained with HD TALE_2 and (from left to right) TALE_2 G*, NY*, and NH*. For each TALE, log FI of each focus is normalized to the mean of log FI of all foci of DNMTinact cells. d) Histograms of control TALE FI from co‐stains with HD TALE (corresponding to Figure 4d). Histograms in both Figure 4c and d are cropped for clarity and do not show outliers; these are fully shown in box plots of Figure 4e. e) Box plots of data from Figure 4c and d. Unpaired t‐test with * p <0.05; ns: not significant. N=5, 4 and 4 independent biological experiments with each ∼250 cells and >2000 foci.
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