Design of synthetic epigenetic circuits featuring memory effects and reversible switching based on DNA methylation

Johannes A H Maier,Albert Jeltsch,Raphael Möhrle

doi:10.1038/ncomms15336

Abstract

Epigenetic systems store information in DNA methylation patterns in a durable but reversible manner, but have not been regularly used in synthetic biology. Here, we designed synthetic epigenetic memory systems using DNA methylation sensitive engineered zinc finger proteins to repress a memory operon comprising the CcrM methyltransferase and a reporter. Triggering by heat, nutrients, ultraviolet irradiation or DNA damaging compounds induces CcrM expression and DNA methylation. In the induced on-state, methylation in the operator of the memory operon prevents zinc finger protein binding leading to positive feedback and permanent activation. Using an mf-Lon protease degradable CcrM variant enables reversible switching. Epigenetic memory systems have numerous potential applications in synthetic biology, including life biosensors, death switches or induction systems for industrial protein production. The large variety of bacterial DNA methyltransferases potentially allows for massive multiplexing of signal storage and logical operations depending on more than one input signal.

Highlights

Epigenetic systems store information in DNA methylation patterns in a durable but reversible manner, but have not been regularly used in synthetic biology
Using a multi-step design process, we set up an epigenetic memory system in E. coli aiming to use orthologous components that do not interfere with E. coli physiology
Key components of our system are a DNA methyltransferase (MTase), a zinc finger (ZnF) protein that binds to DNA in a methylation sensitive manner and serves as a repressor, coiled-coil elements for controlled protein oligomerization and a targeted protein degradation system

Summary

Introduction

Epigenetic systems store information in DNA methylation patterns in a durable but reversible manner, but have not been regularly used in synthetic biology. We designed synthetic epigenetic memory systems using DNA methylation sensitive engineered zinc finger proteins to repress a memory operon comprising the CcrM methyltransferase and a reporter. Inspired by reports describing engineered zinc finger (ZnF) proteins and TALE repressors that are sensitive to cytosine-C5 methylation[18,19], we engineered an artificial adenine-N6 methylation dependent DNA binding protein using the ZnF chassis. Employing this ZnF protein and the CcrM methyltransferase from Caulobacter crescentus, which introduces DNA-(adenine N6)-methylation at GANTC sites[20,21], we constructed a bistable basic module for epigenetic memory systems, which can exist in two states, an onstate and an off-state. Coupling different trigger systems to this memory device, we were able to detect and memorize physical properties like temperature or ultraviolet radiation and the presence of soluble metabolites or toxic agents

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