Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2011;6(5):e20136.
doi: 10.1371/journal.pone.0020136. Epub 2011 May 18.

Exploiting nucleotide composition to engineer promoters

Manfred G Grabherr  1 , Jens Pontiller, Evan Mauceli, Wolfgang Ernst, Martina Baumann, Tara Biagi, Ross Swofford, Pamela Russell, Michael C Zody, Federica Di Palma, Kerstin Lindblad-Toh, Reingard M Grabherr
Affiliations

Exploiting nucleotide composition to engineer promoters

Manfred G Grabherr et al. PLoS One. 2011.

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.

PubMed Disclaimer

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Receiver operator characteristics (ROC) of mono- and di-nucleotides (a), and histogram of α-score in promoter regions (b).
(a) Several di-nucleotides are positive or negative classifiers distinguishing 1,746 experimentally confirmed promoters of genes active in human cerebellum tissue from random sequences, with CpG, GpC and CpC/GpG the strongest positive predictors and ApT, TpA, and ApA/TpT the strongest negative ones. Shown are also G/C content, as well as the α-scores summed over the promoter regions. (b) Shown is the distribution of regions over the sums of α-scores: 1,746 experimentally confirmed promoters of genes active in human cerebellum tissue in red (i), promoters chosen from the UCSC gene set in blue (ii), randomly chosen sequences as negative control in green (iii), and the respective overlaps between the distributions in purple, dark chartreuse and black.
Figure 2
Figure 2. Human promoter CTAG1A and modified constructs.
(a) The 535 base pair long promoter region of human gene CTAG1A is rich in CpGs and exhibits α-scores higher than the genomic distribution with pronounced peaks. Shown are the composite αk-scores (top), the individual αk-scores for different sizes of k in the middle graph (colour coded, blue = negative, red/orange = positive), and CpGs in yellow (bottom). The three strongest regions are marked by red bars. (b) In-vitro activity of the original CTAG1A promoter (hCTAG1A Promoter), the three strongest α-score regions deleted (hCTAG1A delta), the three strongest α-score regions replaced with sequences from the genomic concatomer (hCTAG1A replace), and the three strongest α-score regions replaced with sequences from the promoter-like concatomer (hCTAG1A UP). Also shown are results without any promoter (Negative CO) and the SV40 core promoter (SV40 Promoter AVG).
Figure 3
Figure 3. In-vitro promoter activity driven by artificial constructs.
Artificial constructs ArS110, ArS300, ArS201 and ArS232 exhibit strong promoter activity driving a reporter gene (firefly luciferase, internally normalized by renilla luciferase) in mammalian cell lines: (a) CHO/hamster, (b) P19/mouse, (c) VERO/monkey, (d) HEK293/human, but not in (e) the insect cell line Sf9/army worm. Also shown are the negative control (−) and the SV40 core promoter activity (+). (f) TATA-boxes 1 (left) and 2 (right) were deleted from construct ArS232: deletion of TATA-box 1 only (dT1) results in lack of activity, deletion of TATA-box 2 (dT2) does not change expression levels, while deletion of both (dT1&2) results in slightly increased expression levels.
Figure 4
Figure 4. Binding affinity of artificial promoter constructs to the transcription factors TFIIB and TBP.
The binding expressed as Δnm on the y-axis was monitored in real time as sec (x-axis), using the ForteBio Octet QK instrument. Binding was conducted in four phases: (i) loading of biotinylated DNA fragments to the streptavidin biosensor tip, (ii) washing in Kinetics Buffer, (iii) association of the transcription factor and (iii) dissociation of the transcription factor. (a) The promoter constructs ArS110, ArS201, ArS232 and ArS300 show similar binding affinities to the TFIIB protein. (b) The promoter constructs ArS232, ArS232 dT1, ArS232 dT2 and ArS232 dT12 exhibit sequence-specific binding to the TBP protein. ArS232 dT12 lacking two TATA-Boxes shows the lowest binding affinity compared to the other constructs. (c) TFIIB binding vs. a negative control, for which we chose a 85 bp long sequence from inside the coding region of the luciferase gene (pGL3-Basic Promoter Promega: 1314 bp–1399 bp).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Wurm FM. Production of recombinant protein therapeutics in cultivated mammalian cells. Nature Biotechnology. 2004;22:1393–1398. - PubMed
    1. Omasa T, Onitsuka M, Kim W-D. Cell engineering and cultivation of Chinese hamster ovary (CHO) cells. Curr Pharm Biotechnol. 2010;11:233–240. - PubMed
    1. Hossler P, Khattak SF, Li ZJ. Optimal and consistent protein glycosylation in mammalian cell culture. Glycobiology. 2009;19:936–949. - PubMed
    1. Butler J, Kadonaga J. The RNA polymerase II core promoter: a key component in the regulation of gene expression. Genes Dev. 2002;16:2583–2592. - PubMed
    1. Juven-Gershon T, Hsu J, Theisen J, Kadonaga J. The RNA polymerase II core promoter - the gateway to transcription. Curr Opin Cell Biol. 2008;20:253–259. - PMC - PubMed

Publication types

MeSH terms