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A synthetic circuit for buffering gene dosage variation between individual mammalian cells.

ABSTRACT: Precise control of gene expression is critical for biological research and biotechnology. However, transient plasmid transfections in mammalian cells produce a wide distribution of copy numbers per cell, and consequently, high expression heterogeneity. Here, we report plasmid-based synthetic circuits - Equalizers - that buffer copy-number variation at the single-cell level. Equalizers couple a transcriptional negative feedback loop with post-transcriptional incoherent feedforward control. Computational modeling suggests that the combination of these two topologies enables Equalizers to operate over a wide range of plasmid copy numbers. We demonstrate experimentally that Equalizers outperform other gene dosage compensation topologies and produce as low cell-to-cell variation as chromosomally integrated genes. We also show that episome-encoded Equalizers enable the rapid generation of extrachromosomal cell lines with stable and uniform expression. Overall, Equalizers are simple and versatile devices for homogeneous gene expression and can facilitate the engineering of synthetic circuits that function reliably in every cell.

SUBMITTER: Yang J

PROVIDER: S-EPMC8257781 | biostudies-literature | 2021 Jul

REPOSITORIES: biostudies-literature

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A synthetic circuit for buffering gene dosage variation between individual mammalian cells.

Yang Jin J Lee Jihwan J Land Michelle A MA Lai Shujuan S Igoshin Oleg A OA St-Pierre François F

Nature communications 20210705 1

Precise control of gene expression is critical for biological research and biotechnology. However, transient plasmid transfections in mammalian cells produce a wide distribution of copy numbers per cell, and consequently, high expression heterogeneity. Here, we report plasmid-based synthetic circuits - Equalizers - that buffer copy-number variation at the single-cell level. Equalizers couple a transcriptional negative feedback loop with post-transcriptional incoherent feedforward control. Comput ...[more]

PMID: 34226556

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Project description:Driven essentially by random genetic drift, subfunctionalization has been identified as a possible non-adaptive mechanism for the retention of duplicate genes in small-population species, where widespread deleterious mutations are likely to cause complementary loss of subfunctions across gene copies. Through subfunctionalization, duplicates become indispensable to maintain the functional requirements of the ancestral locus. Yet, gene duplication produces a dosage imbalance in the encoded proteins and thus, as investigated in this paper, subfunctionalization must be subject to the selective forces arising from the fitness bottleneck introduced by the duplication event.We show that, while arising from random drift, subfunctionalization must be inescapably subject to selective forces, since the diversification of expression patterns across paralogs mitigates duplication-related dosage imbalances in the concentrations of encoded proteins. Dosage imbalance effects become paramount when proteins rely on obligatory associations to maintain their structural integrity, and are expected to be weaker when protein complexation is ephemeral or adventitious. To establish the buffering effect of subfunctionalization on selection pressure, we determine the packing quality of encoded proteins, an established indicator of dosage sensitivity, and correlate this parameter with the extent of paralog segregation in humans, using species with larger population -and more efficient selection- as controls.Recognizing the role of subfunctionalization as a dosage-imbalance buffer in gene duplication events enabled us to reconcile its mechanistic nonadaptive origin with its adaptive role as an enabler of the evolution of genetic redundancy. This constructive role was established in this paper by proving the following assertion: If subfunctionalization is indeed adaptive, its effect on paralog segregation should scale with the dosage sensitivity of the duplicated genes. Thus, subfunctionalization becomes adaptive in response to the selection forces arising from the fitness bottleneck imposed by gene duplication.

Dataset Information

A synthetic circuit for buffering gene dosage variation between individual mammalian cells.

Publications

A synthetic circuit for buffering gene dosage variation between individual mammalian cells.

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