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

Abstract

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
    asli.azizoglu@bsse.ethz.ch
    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
    rbrent@fhcrc.org
    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
    fabian.rudolf@bsse.ethz.ch
    Competing interests
    The authors declare that no competing interests exist.

Funding

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)

Copyright

© 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.

Metrics

  • 989
    Page views
  • 122
    Downloads
  • 1
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Asli Azizoglu
  2. Roger Brent
  3. Fabian Rudolf
(2021)
A precisely adjustable, variation-suppressed eukaryotic transcriptional controller to enable genetic discovery
eLife 10:e69549.
https://doi.org/10.7554/eLife.69549

Further reading

    1. Chromosomes and Gene Expression
    2. Genetics and Genomics
    Bethany Sump et al.
    Research Article

    For some inducible genes, the rate and molecular mechanism of transcriptional activation depends on the prior experiences of the cell. This phenomenon, called epigenetic transcriptional memory, accelerates reactivation and requires both changes in chromatin structure and recruitment of poised RNA Polymerase II (RNAPII) to the promoter. Memory of inositol starvation in budding yeast involves a positive feedback loop between transcription factor-dependent interaction with the nuclear pore complex and histone H3 lysine 4 dimethylation (H3K4me2). While H3K4me2 is essential for recruitment of RNAPII and faster reactivation, RNAPII is not required for H3K4me2. Unlike RNAPII-dependent H3K4me2 associated with transcription, RNAPII-independent H3K4me2 requires Nup100, SET3C, the Leo1 subunit of the Paf1 complex and, upon degradation of an essential transcription factor, is inherited through multiple cell cycles. The writer of this mark (COMPASS) physically interacts with the potential reader (SET3C), suggesting a molecular mechanism for the spreading and re-incorporation of H3K4me2 following DNA replication.

    1. Chromosomes and Gene Expression
    Faith C Fowler et al.
    Research Article

    DNA double-strand break (DSB) repair by homologous recombination is confined to the S and G2 phases of the cell cycle partly due to 53BP1 antagonizing DNA end resection in G1 phase and non-cycling quiescent (G0) cells where DSBs are predominately repaired by non-homologous end joining (NHEJ). Unexpectedly, we uncovered extensive MRE11- and CtIP-dependent DNA end resection at DSBs in G0 murine and human cells. A whole genome CRISPR/Cas9 screen revealed the DNA-dependent kinase (DNA-PK) complex as a key factor in promoting DNA end resection in G0 cells. In agreement, depletion of FBXL12, which promotes ubiquitylation and removal of the KU70/KU80 subunits of DNA-PK from DSBs, promotes even more extensive resection in G0 cells. In contrast, a requirement for DNA-PK in promoting DNA end resection in proliferating cells at the G1 or G2 phase of the cell cycle was not observed. Our findings establish that DNA-PK uniquely promotes DNA end resection in G0, but not in G1 or G2 phase cells, which has important implications for DNA DSB repair in quiescent cells.