A precisely adjustable, variation-suppressed eukaryotic transcriptional controller to enable genetic discovery

  1. Asli Azizoglu  Is a corresponding author
  2. Roger Brent  Is a corresponding author
  3. Fabian Rudolf  Is a corresponding author
  1. ETH Zurich, Switzerland
  2. Fred Hutchinson Cancer Research Center, United States


Conditional expression of genes and observation of phenotype remain central to biological discovery. Current methods enable either on/off or imprecisely controlled graded gene expression. We developed a 'well-tempered' controller, WTC846, for precisely adjustable, graded, growth condition independent expression of genes in Saccharomyces cerevisiae. Controlled genes are expressed from a strong semisynthetic promoter repressed by the prokaryotic TetR, which also represses its own synthesis; with basal expression abolished by a second, 'zeroing' repressor. The autorepression loop lowers cell-to-cell variation while enabling precise adjustment of protein expression by a chemical inducer. WTC846 allelic strains in which the controller replaced the native promoters recapitulated known null phenotypes (CDC42, TPI1), exhibited novel overexpression phenotypes (IPL1), showed protein dosage-dependent growth rates and morphological phenotypes (CDC28, TOR2, PMA1 and the hitherto uncharacterized PBR1), and enabled cell cycle synchronization (CDC20). WTC846 defines an 'expression clamp' allowing protein dosage to be adjusted by the experimenter across the range of cellular protein abundances, with limited variation around the setpoint.

Data availability

All relevant sequences are included in the supporting files for reproducibility. All raw flow cytometry data is publicly available at doi.org/10.3929/ethz-b-000488967. All other source data is included in the manuscript and supporting files.

Article and author information

Author details

  1. Asli Azizoglu

    BSSE, ETH Zurich, Basel, Switzerland
    For correspondence
    Competing interests
    The authors declare that no competing interests exist.
  2. Roger Brent

    Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, United States
    For correspondence
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8398-3273
  3. Fabian Rudolf

    D-BSSE, ETH Zurich, Basel, Switzerland
    For correspondence
    Competing interests
    The authors declare that no competing interests exist.


Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (NCCR Molecular Systems Engineering)

  • Asli Azizoglu
  • Fabian Rudolf

National Cancer Institute (R21CA223901)

  • Roger Brent

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Naama Barkai, Weizmann Institute of Science, Israel

Publication history

  1. Received: April 19, 2021
  2. Accepted: August 2, 2021
  3. Accepted Manuscript published: August 3, 2021 (version 1)
  4. Version of Record published: September 6, 2021 (version 2)


© 2021, Azizoglu et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.


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  1. Asli Azizoglu
  2. Roger Brent
  3. Fabian Rudolf
A precisely adjustable, variation-suppressed eukaryotic transcriptional controller to enable genetic discovery
eLife 10:e69549.

Further reading

    1. Chromosomes and Gene Expression
    2. Neuroscience
    Bradley M Colquitt, Kelly Li ... Michael S Brainard
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    Sensory feedback is required for the stable execution of learned motor skills, and its loss can severely disrupt motor performance. The neural mechanisms that mediate sensorimotor stability have been extensively studied at systems and physiological levels, yet relatively little is known about how disruptions to sensory input alter the molecular properties of associated motor systems. Songbird courtship song, a model for skilled behavior, is a learned and highly structured vocalization that is destabilized following deafening. Here, we sought to determine how the loss of auditory feedback modifies gene expression and its coordination across the birdsong sensorimotor circuit. To facilitate this system-wide analysis of transcriptional responses, we developed a gene expression profiling approach that enables the construction of hundreds of spatially-defined RNA-sequencing libraries. Using this method, we found that deafening preferentially alters gene expression across birdsong neural circuitry relative to surrounding areas, particularly in premotor and striatal regions. Genes with altered expression are associated with synaptic transmission, neuronal spines, and neuromodulation and show a bias toward expression in glutamatergic neurons and Pvalb/Sst-class GABAergic interneurons. We also found that connected song regions exhibit correlations in gene expression that were reduced in deafened birds relative to hearing birds, suggesting that song destabilization alters the inter-region coordination of transcriptional states. Finally, lesioning LMAN, a forebrain afferent of RA required for deafening-induced song plasticity, had the largest effect on groups of genes that were also most affected by deafening. Combined, this integrated transcriptomics analysis demonstrates that the loss of peripheral sensory input drives a distributed gene expression response throughout associated sensorimotor neural circuitry and identifies specific candidate molecular and cellular mechanisms that support the stability and plasticity of learned motor skills.

    1. Chromosomes and Gene Expression
    Sarah E London

    In songbirds, deafening leads to changes in gene expression which have now been mapped at the single-cell level across the neural circuit involved in song production.