Epigenetic modifications: building a synthetic propagation system in human cells

Minhee Park, Ahmad S. Khalil, and their collaborators, at the University of Boston, studied a synthetic method capable to regulate epigenetic modification in humans cells (HEK293FT)[1]. This method was based on the introduction of the modification m6A (N6-methyl-adenine) by the E.coli DNA adenine methyltransferase (Dam), a system that is widely diffused in bacterial species [2]. This could provide a simple model to study epigenetic interactions. The synthetic machinery involves three main modules: a synthetic initiator (made by ZF-Dam), a read-write module (made by DpnI-Dam ) and a synthetic reader module (made by DpnI-transcriptional factor (VP64)). The system was applied on two different gene reporter coding for GFP protein. The aim of this work was to create a synthetic system capable to introduce a methylation, maintain it through time and cell division, and finally affect gene expression. In order to build the simplest model possible, they chose to transplant a bacterial methylation system to avoid random cross-talk or interference with pre-existing network in the cells.  Moreover, they build to kind of specific gene reporter to assess spatial propagation (clustered reporter) and temporal maintenance (interspersed reporter). The first had ZF harboring sequences preceding the GATC methylation motifs, while the second one has alternating ZF binding and GATC.

The first step of this work was to design a suitable initiator (SynI). The initiator had to be inducible and able to bind only specific regions. To achieve this goal they used a Dam library to find one with very impaired DNA binding system. The best Dam variant was found to be N132A. The initiator was then tested on the clustered reporter gene that showed an enrichment of the m6A. In order to make the initiator inducible they separated the ZF and Dam, binding them to two different transducing proteins, that can bind together only in presence of abscissic acid (ABA) [3]. Then they build an effector capable  (formed by the fusion  of DpnI and transcription factors) to read  the methylated sequence and regulate reporter transcription. They did so by comparing two different signals one of a transcriptional promoter and one of a transcriptional repressor, both guided by the reader with the same transcription factors, directly guided by a ZF motifs.

Furthermore they tried to characterize the read-write module (SynRW). From the screening of the phenotype, as before they build a synRW library and tested it on the interspersed reporter. Comparing cells in active division not having the RW module against the one who had it, they managed to demonstrate that the presence of RW module is essential to maintain epigenetic memory through cells division, thanks to a positive feedback mechanism. And it can do so, even in absence of an external inductor signal (ABA).  Moreover, the test was effective both in presence of a strong promoter (pUBC) and in the presence of weak promoter (pMinCMV). The best Dam variant for RW module was found to have an intermediate phenotype (R195A), between WT Dam (too much active) and Dam double mutant (not enough active).