Selective recognition of N 4-methylcytosine in DNA by engineered transcription-activator-like effectors

Abstract

The epigenetic DNA nucleobases 5-methylcytosine (5mC) and N4-methylcytosine (4mC) coexist in bacterial genomes and have important functions in host defence and transcription regulation. To better understand the individual biological roles of both methylated nucleobases, analytical strategies for distinguishing unmodified cytosine (C) from 4mC and 5mC are required. Transcription-activator-like effectors (TALEs) are programmable DNA-binding repeat proteins, which can be re-engineered for the direct detection of epigenetic nucleobases in user-defined DNA sequences. We here report the natural, cytosine-binding TALE repeat to not strongly differentiate between 5mC and 4mC. To engineer repeats with selectivity in the context of C, 5mC and 4mC, we developed a homogeneous fluorescence assay and screened a library of size-reduced TALE repeats for binding to all three nucleobases. This provided insights into the requirements of size-reduced TALE repeats for 4mC binding and revealed a single mutant repeat as a selective binder of 4mC. Employment of a TALE with this repeat in affinity enrichment enabled the isolation of a user-defined DNA sequence containing a single 4mC but not C or 5mC from the background of a bacterial genome. Comparative enrichments with TALEs bearing this or the natural C-binding repeat provides an approach for the complete, programmable decoding of all cytosine nucleobases found in bacterial genomes.This article is part of a discussion meeting issue 'Frontiers in epigenetic chemical biology'.

Keywords: DNA methylation; affinity enrichment; epigenetics; programmable DNA recognition; transcription-activator-like effectors.

Figure 1.

Bacterial cytosine 5-methylation and features of the TALE scaffold, including recognition of C, 5mC and 4mC in DNA. (a) Conversion of the nucleobase C into the epigenetic nucleobases 5mC and 4mC by DNA methyltransferases (Dnmt) using S-adenosylmethionine as a cofactor. (b) Cartoon showing the general features of employed TALEs. The amino acid sequence of one representative TALE repeat is shown at the top, with RVD amino acids 12 and 13 marked with a grey box. TALE repeats with selectivity for canonical nucleobases are shown on the right with RVDs specified. (c) Interaction of RVD HD with cytosine (C) in a crystal structure of a TALE–DNA complex (pdb entry 3V6T) [28]. Hydrogen bonds are shown as dotted green lines. (d) Crystal structure of a 4mC-G base pair as found in a Z-DNA duplex (pdb entry 133D) [33]. (e) Target sequences used in this study. (f) EMSA assay conducted with 100 nM TALE_Hey2(HD) and 500 nM pre-hybridized DNA duplexes with sequences shown in (e) at 4°C.

Figure 2.

Homogeneous FRET assay for rapidly testing single-nucleobase selectivities of TALE mutants. (a) Assay principle. TALE mutant and Dnase I compete for binding to a Cy3/Cy5-labelled DNA duplex. Binding of a TALE mutant protects the duplex from Dnase I cleavage and leads to high FRET, which is read out as high Cy5 fluorescence intensity. Low TALE binding leads to DNA cleavage and low FRET/Cy5 fluorescence. (b) Evaluation of FRET assay using 250 nM TALE_Hey2(HD), 50 nM labelled DNA duplex containing a single C or 5mC and 0.5 U Dnase I with an incubation time of 55 min at 37°C (arb. units, arbitrary units). (c) Time course of FRET assay using TALE_Hey2(HD) and DNA containing a single C or 5mC with conditions as in (b). Data were normalized by subtraction of a control with Dnase I and without TALE (minimal FRET) and dividing by a control without Dnase I (maximal FRET). Experiments were performed in duplicate and the error bars indicate standard error.

Figure 3.

TALE RVD mutant library design and screening. (a) Crystal structure of a TALE repeat with RVD N* (* = deletion) bound to a C nucleobase in DNA (pdb entry 3V6T) [29]. Hydrogen bonds are shown as dotted green lines; position 12 of the RVD is marked with X (random position). Deletion position is marked with a star. (b) FRET screen of X* library conducted as described in figure 2b,c using 50 nM target DNA sequences containing a single C, 5mC or 4mC position and 250 nM of the mentioned TALEs as shown in figure 1e. Experiments were performed in duplicate and the error bars indicate standard error. Cy5 emission was measured at 665 nm. Arrows mark the TALE_Hey2(HD) control and the identified TALE_Hey2(T*) exhibiting selective binding of 4mC. For clarity, only the 60 min time point of the FRET time course is shown (see the electronic supplementary material for complete time courses).

Figure 4.

Selective binding of TALE_Hey2(T*) to DNA containing a single 4mC. (a) Evaluation of binding selectivity of TALE_Hey2(HD) to C, 5mC and 4mC by FRET assay described in figure 2a using varying TALE concentrations and 50 nM of DNA sequence containing a single C, 5mC or 4mC position. Experiments were performed in duplicate and the error bars indicate standard error. Cy5 emission was measured at 665 nm. Line graphs correspond to Hill fits. (b) The same experiment as in (a), but with TALE_Hey2(T*). (c) Table of data (K_i and maximal Cy5 fluorescence F_max) obtained from (a,b). AU, arbitrary units. (d) EMSA assay with TALE_Hey2(T*) and DNA duplexes with sequence shown in figure 1e with 100 nM TALE and 500 nM of DNA duplex containing a single C, 5mC or 4mC at position 6. (e) Principle of TALE-controlled primer extension. Competitive, methylation-dependent binding of TALE to DNA inhibits primer extension by KF(exo⁻). TALE is shown as a cartoon, with RVD targeting the variable nucleobase in black. (f) Binding analysis of TALE_Hey2(T*) to DNA containing a single C, 5mC or 4mC by primer extension as shown in (e). Reactions contained 8.325 nM primer/template complex, five equivalents TALE, 12.5 mU KF(exo⁻) and 100 µM of each dNTP, and were incubated for 15 min at room temperature. Experiments were conducted in duplicate and the error bars indicate standard error. (g) Workflow of TALE-based affinity enrichment of user-defined genomic DNA sequences with selectivity for single 4mC or 5mC nucleobases. (h) Affinity enrichment experiments with TALE_Hey2(HD) as shown in (g), using PCR products containing a single C, 5mC or 4mC position spiked in E. coli genomic DNA at a concentration representing a single genomic locus. Target DNA copies obtained from enrichments were quantified by qPCR. Affinity enrichment experiments were conducted in triplicate and each quantified by duplicate qPCRs. Error bars indicate standard error. (i) Affinity enrichment experiments conducted as in (h), but with TALE_Hey2(T*).