title: 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.
authors.affiliation: Broad Institute, Cambridge, Massachusetts, United States of America.
authors.forename: Manfred G
authors.initials: MG
authors.lastname: Grabherr
authors.forename: Jens
authors.initials: J
authors.lastname: Pontiller
authors.forename: Evan
authors.initials: E
authors.lastname: Mauceli
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Ernst
authors.forename: Martina
authors.initials: M
authors.lastname: Baumann
authors.forename: Tara
authors.initials: T
authors.lastname: Biagi
authors.forename: Ross
authors.initials: R
authors.lastname: Swofford
authors.forename: Pamela
authors.initials: P
authors.lastname: Russell
authors.forename: Michael C
authors.initials: MC
authors.lastname: Zody
authors.forename: Federica
authors.initials: F
authors.lastname: Di Palma
authors.forename: Kerstin
authors.initials: K
authors.lastname: Lindblad-Toh
authors.forename: Reingard M
authors.initials: RM
authors.lastname: Grabherr
mesh_terms: D005818:Genetic Engineering; D009711:Nucleotides / Q000737:chemistry*; D011401:Promoter Regions, Genetic
chemical_list: D009711:Nucleotides