Exploiting Nucleotide Composition to Engineer Promoters

Abstract

The choice of promoter is a critical step in optimizing the efficiency and stability of recombinant protein production in mammalian cell lines. Artificial promoters that provide stable expression across cell lines and can be designed to the desired strength constitute an alternative to the use of viral promoters. Here, we show how the nucleotide characteristics of highly active human promoters can be modelled via the genome-wide frequency distribution of short motifs: by overlapping motifs that occur infrequently in the genome, we constructed contiguous sequence that is rich in GC and CpGs, both features of known promoters, but lacking homology to real promoters. We show that snippets from this sequence, at 100 base pairs or longer, drive gene expression in vitro in a number of mammalian cells, and are thus candidates for use in protein production. We further show that expression is driven by the general transcription factors TFIIB and TFIID, both being ubiquitously present across cell types, which results in less tissue- and species-specific regulation compared to the viral promoter SV40. We lastly found that the strength of a promoter can be tuned up and down by modulating the counts of GC and CpGs in localized regions. These results constitute a “proof-of-concept” for custom-designing promoters that are suitable for biotechnological and medical applications.

Promoter and Terminator Discovery and Engineering

“…In addition, mammalian promoter regions have been modeled at the sequence level using an "alpha score," which describes the likelihood that a genomic region contains a promoter based on its nucleotide composition. Remodeling the X-linked gene cancer/testis antigen 1A promoter to have twice the alpha score improved expression in a non-quantitative manner [95]. Although predictive of high expression, these techniques are limited as they cannot design promoters de novo with prescribed expression.…”

Cell line development for biomanufacturing processes: recent advances and an outlook

“…These endogenous promoters offer the possibility of more predictable expression levels, and dynamic control of the transgene expression with various growth stages. In addition, synthetic promoters have been explored to modulate gene expression levels (Hartenbach and Fussenegger 2006;Grabherr et al 2011;Brown et al 2014). As the customized combinations of these regulatory motifs do not occur naturally, they tend to be less tissue-specific and species-restricted, and could possibly offer more stable and consistent gene expression than natural ones.…”

Artificially designed promoters

“…We previously reported that artificial DNA sequences do not have to share homology with existing promoters to drive strong expression of a reporter gene in vitro, 8 as long as these sequences are rich in the di-nucleotide CpG, even in the absence of canonical transcription factor binding sites (TFBS). Such sequences, we found, could be bound by the generic transcription factors TFIIB and TFIID, which would then recruit the polymerase II complex and initiate transcription.…”

“…Modified artificial promoter constructs. We edited promoter ArS232-d2 8 by replacing three (ArS232-d2_3inh) and four (ArS232-d2_4inh) CpGrich regions in local areas (25,20,20, and 25 nucleotides in size) with CpG-less sequences (A). This results in more than 3-fold increased in vitro activity for ArS232-d2_3inh, while ArS232-d2_4inh reduces activity to the original level.…”

“…8 The CpGs are fairly evenly spaced, with seven nucleotides being the largest distance between two consecutive instances. To determine the possible role of these motifs, we decided to alter this sequence's organization.…”