Engineering Epigenetic Regulation Using Synthetic Read-Write Modules

Abstract

Graphical Abstract HIGHLIGHTS d A synthetic epigenetic regulatory system in human cells using m6A DNA modification d Engineered writers and readers of m6A enable construction of regulatory circuits d Read-write circuits drive spatial propagation and hallmarks of chromatin spreading d Read-write circuits enable epigenetic memory of transcriptional states A synthetic, modular, and programmable read-write system allows isolated and orthogonal epigenetic control in mammalian cells. SUMMARYChemical modifications to DNA and histone proteins are involved in epigenetic programs underlying cellular differentiation and development. Regulatory networks involving molecular writers and readers of chromatin marks are thought to control these programs. Guided by this common principle, we established an orthogonal epigenetic regulatory system in mammalian cells using N6-methyladenine (m6A), a DNA modification not commonly found in metazoan epigenomes. Our system utilizes synthetic factors that write and read m6A and consequently recruit transcriptional regulators to control reporter loci. Inspired by models of chromatin spreading and epigenetic inheritance, we used our system and mathematical models to construct regulatory circuits that induce m6A-dependent transcriptional states, promote their spatial propagation, and maintain epigenetic memory of the states. These minimal circuits were able to program epigenetic functions de novo, conceptually validating ''read-write'' architectures. This work provides a toolkit for investigating models of epigenetic regulation and encoding additional layers of epigenetic information in cells.

μDamID: a microfluidic approach for imaging and sequencing protein-DNA interactions in single cells

“…We also performed RNA sequencing 213 in bulk cells that were untreated or transfected with Dam-only, Dam-LMNB1, or m6 A-Tracer, and 214 we found only two differentially expressed genes (Supplementary Figure 3). This corroborates 215 similar published findings by others showing that Dam expression and adenine methylation have 216 little or no effect on gene expression in HEK293T cells (Park et al 2018). 217…”

Transcriptional kinetic synergy: a complex landscape revealed by integrating modelling and synthetic biology

“…In order to probe the extent of kinetic synergy experimentally achievable under this setup, we built a reporter platform based upon synthetic TF fusions (Khalil et al, 2012;Park et al, 2019b). We selected 5 activation domains of mammalian TFs with a described diversity of functions in the literature, depicted in Fig.…”

“…1A, Fig. 3A) (Park et al, 2019b). This allows us to specifically recruit the activation domains to a reporter to assess their effects on transcription, while minimizing confounding effects from native TFs acting on the reporter.…”

Protein assembly systems in natural and synthetic biology

“…These can be broadly divided between epigenetic (transcriptional feedback loops, heritable chromatin changes, etc.) [227][228][229] and inducible DNA mutations/alterations [230][231][232][233][234]. Taken together, this work has yielded foundational synthetic elements for building more sophisticated biological systems that enable cell state changes, memory of gene expression states, and cellular devices that record lineage and environmental information.…”