Brown, A.J., Gibson, S.J., Hatton, D. et al. (1 more author) (2017) In silico design of context-responsive mammalian promoters with user-defined functionality. Nucleic Acids Research, 45 (18). pp. 10906-10919. ISSN 0305-1048
Abstract
Comprehensive de novo-design of complex mammalian promoters is restricted by unpredictable combinatorial interactions between constituent transcription factor regulatory elements (TFREs). In this study, we show that modular binding sites that do not function cooperatively can be identified by analyzing host cell transcription factor expression profiles, and subsequently testing cognate TFRE activities in varying homotypic and heterotypic promoter architectures. TFREs that displayed position-insensitive, additive function within a specific expression context could be rationally combined together in silico to create promoters with highly predictable activities. As TFRE order and spacing did not affect the performance of these TFRE-combinations, compositions could be specifically arranged to preclude the formation of undesirable sequence features. This facilitated simple in silico-design of promoters with context-required, user-defined functionalities. To demonstrate this, we de novo-created promoters for biopharmaceutical production in CHO cells that exhibited precisely designed activity dynamics and long-term expression-stability, without causing observable retroactive effects on cellular performance. The design process described can be utilized for applications requiring context-responsive, customizable promoter function, particularly where co-expression of synthetic TFs is not suitable. Although the synthetic promoter structure utilized does not closely resemble native mammalian architectures, our findings also provide additional support for a flexible billboard model of promoter regulation.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com |
Keywords: | Architecture; binding sites; chinese hamster ovary cell; mammals; oncogenes; host (organism) |
Dates: |
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Institution: | The University of Sheffield |
Academic Units: | The University of Sheffield > Faculty of Engineering (Sheffield) > Department of Chemical and Biological Engineering (Sheffield) |
Depositing User: | Symplectic Sheffield |
Date Deposited: | 06 Oct 2017 11:13 |
Last Modified: | 07 Nov 2023 08:22 |
Status: | Published |
Publisher: | Oxford University Press |
Refereed: | Yes |
Identification Number: | 10.1093/nar/gkx768 |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:122147 |