Expression regulation by a methyl-CpG binding domain in anE. colibased, cell-free TX-TL system

Abstract

Cytosine methylation plays an important role in the epigenetic regulation of eukaryotic gene expression. The methyl-CpG binding domain (MBD) is common to a family of eukaryotic transcriptional regulators. How MBD, a stretch of about 80 amino acids, recognizes CpGs in a methylation dependent manner, and as a function of sequence, is only partly understood. Here we show, using an Escherichia coli cell-free expression system, that MBD from the human transcriptional regulator MeCP2 performs as a specific, methylation-dependent repressor in conjunction with the BDNF (brain-derived neurotrophic factor) promoter sequence. Mutation of either base flanking the central CpG pair changes the expression level of the target gene. However, the relative degree of repression as a function of MBD concentration remains unaltered. Molecular dynamics simulations that address the DNA B fiber ratio and the handedness reveal cooperative transitions in the promoter DNA upon MBD binding that correlate well with our experimental observations. We suggest that not only steric hindrance, but also conformational changes of the BDNF promoter as a result of MBD binding are required for MBD to act as a specific inhibitory element. Our work demonstrates that the prokaryotic transcription machinery can reproduce features of epigenetic mammalian transcriptional regulatory elements.

A bead-based method for the removal of the amino acid lysine from cell-free transcription-translation systems

“…This plasmid is optimized for our cell-free system. It can be derived from the commercially available plasmid pBest-Or2-Or1-Pr-UTR1-deGFP-T500 (Addgene Cat# 40019) (Finkler and Ott, 2019;Schenkelberger et al, 2017;Shin and Noireaux, 2010). The gene for the high-lysine dummy protein (HLDP), DNA-sequence in Supplementary Fig.…”

“…In vitro transcription translation systems have numerous applications, among them the study of gene expression and regulation, biochemical reactions and metabolic pathways (Dudley et al, 2015;Hold and Panke, 2009;Rolf et al, 2019;Schenkelberger et al, 2017). While in vivo the concentrations of different biochemical compounds can often only be estimated, in vitro systems allow for adjustable concentrations by addition or removal (Hold and Panke, 2009;Rolf et al, 2019;Sullivan et al, 2016).…”

“…Our cell-free expression systems are derived from E. coli cell lysates. They include the entire endogenous transcription-translation machinery (Garenne et al, 2019;Schenkelberger et al, 2017;Worst et al, 2015). In principle lysine-free expression systems could be derived from lysine auxotrophic E. coli strains (Gao et al, 2019;Singh-Blom et al, 2014).…”

Bead-Based Assay for Spatiotemporal Gene Expression Control in Cell-Free Transcription–translation Systems

“…Stopping transcription requires complete removal of the inducer, which is difficult without affecting the expression system. A different method to control transcription uses repressing transcription factors recognizing specific DNA sequences or specific DNA modifications such as methylation [9]. However, due to basal transcription, complete suppression cannot be achieved [9,10].…”

“…A different method to control transcription uses repressing transcription factors recognizing specific DNA sequences or specific DNA modifications such as methylation [9]. However, due to basal transcription, complete suppression cannot be achieved [9,10]. A light-dependent gene switch enables transcription as a response to visible light [7,11,12], but the problem with basal transcription remains [12].…”