Modular fluorescence complementation sensors for live cell detection of epigenetic signals at endogenous genomic sites

Cristiana Lungu,Julian Broche,Sabine Pinter,Albert Jeltsch,Philipp Rathert

doi:10.1038/s41467-017-00457-z

Abstract

Investigation of the fundamental role of epigenetic processes requires methods for the locus-specific detection of epigenetic modifications in living cells. Here, we address this urgent demand by developing four modular fluorescence complementation-based epigenetic biosensors for live-cell microscopy applications. These tools combine engineered DNA-binding proteins with domains recognizing defined epigenetic marks, both fused to non-fluorescent fragments of a fluorescent protein. The presence of the epigenetic mark at the target DNA sequence leads to the reconstitution of a functional fluorophore. With this approach, we could for the first time directly detect DNA methylation and histone 3 lysine 9 trimethylation at endogenous genomic sites in live cells and follow dynamic changes in these marks upon drug treatment, induction of epigenetic enzymes and during the cell cycle. We anticipate that this versatile technology will improve our understanding of how specific epigenetic signatures are set, erased and maintained during embryonic development or disease onset.

Highlights

Investigation of the fundamental role of epigenetic processes requires methods for the locus-specific detection of epigenetic modifications in living cells
Since the epigenetic landscape is highly dynamic during cellular differentiation and pathological development, a meaningful interpretation of epigenetic signaling cascades can only be obtained by combining the static information on the locus-specific status of epigenetic marks with a real-time readout of their changes
To develop a BiAD system to monitor histone tail marks at specific genomic loci, we focused on the detection of heterochromatic H3K9me[3], which is introduced by the SUV39H1 and SUV39H2 protein lysine methyltransferases[45] and abundantly decorates facultative and constitutive heterochromatin[46]

Summary

Introduction

Investigation of the fundamental role of epigenetic processes requires methods for the locus-specific detection of epigenetic modifications in living cells. To overcome these methodological limitations, we engineered an epigenetic detection method for dynamic and direct readout of locus-specific epigenetic signals in live mammalian cells using modular fluorescence complementation-based BiAD (Bimolecular Anchor Detector) sensors consisting of anchor modules for programmable sequence-specific DNA binding and detector domains for chromatin mark recognition. The punctuate and virtually background-free BiFC signal observed in HEK293 cells upon cotransfection of the anchor/detector modules highlights the advantage of the BiAD approach over co-localization methods such as immunofluorescence-FISH (Supplementary Fig. 13a vs b).

Results

Conclusion