Comparative genetic screens in human cells reveal new regulatory mechanisms in WNT signaling

  1. Andres M Lebensohn
  2. Ramin Dubey
  3. Leif R Neitzel
  4. Ofelia Tacchelly-Benites
  5. Eungi Yang
  6. Caleb D Marceau
  7. Eric M Davis
  8. Bhaven B Patel
  9. Zahra Bahrami-Nejad
  10. Kyle J Travaglini
  11. Yashi Ahmed
  12. Ethan Lee
  13. Jan E Carette  Is a corresponding author
  14. Rajat Rohatgi  Is a corresponding author
  1. Stanford University School of Medicine, United States
  2. Vanderbilt University Medical Center, United States
  3. Geisel School of Medicine at Dartmouth College, United States
  4. University of Colorado, Boulder, United States

Abstract

The comprehensive understanding of cellular signaling pathways remains a challenge due to multiple layers of regulation that may become evident only when the pathway is probed at different levels or critical nodes are eliminated. To discover regulatory mechanisms in canonical WNT signaling, we conducted a systematic forward genetic analysis through reporter-based screens in haploid human cells. Comparison of screens for negative, sensitizing and positive regulators of WNT signaling, mediators of R-spondin-dependent signaling and suppressors of constitutive signaling induced by loss of the tumor suppressor APC or casein kinase 1α uncovered new regulatory features at many levels of the pathway. These include a requirement for the transcription factor TFAP4, a role for the DAX domain of AXIN2 in controlling β-catenin activity, a contribution of GPI anchor biosynthetic enzymes and glypicans to R-spondin-potentiated signaling, and two different mechanisms that regulate signaling when distinct components of the β-catenin destruction complex are lost.

Data availability

The following data sets were generated

Article and author information

Author details

  1. Andres M Lebensohn

    Department of Biochemistry, Stanford University School of Medicine, Stanford, United States
    Competing interests
    The authors declare that no competing interests exist.
  2. Ramin Dubey

    Department of Biochemistry, Stanford University School of Medicine, Stanford, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Leif R Neitzel

    Department of Cell and Developmental Biology, Vanderbilt University Medical Center, Nashville, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Ofelia Tacchelly-Benites

    Department of Molecular and Systems Biology, Geisel School of Medicine at Dartmouth College, Hanover, United States
    Competing interests
    The authors declare that no competing interests exist.
  5. Eungi Yang

    Department of Molecular and Systems Biology, Geisel School of Medicine at Dartmouth College, Hanover, United States
    Competing interests
    The authors declare that no competing interests exist.
  6. Caleb D Marceau

    Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, United States
    Competing interests
    The authors declare that no competing interests exist.
  7. Eric M Davis

    Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, Boulder, United States
    Competing interests
    The authors declare that no competing interests exist.
  8. Bhaven B Patel

    Department of Biochemistry, Stanford University School of Medicine, Stanford, United States
    Competing interests
    The authors declare that no competing interests exist.
  9. Zahra Bahrami-Nejad

    Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, United States
    Competing interests
    The authors declare that no competing interests exist.
  10. Kyle J Travaglini

    Department of Biochemistry, Stanford University School of Medicine, Stanford, United States
    Competing interests
    The authors declare that no competing interests exist.
  11. Yashi Ahmed

    Department of Molecular and Systems Biology, Geisel School of Medicine at Dartmouth College, Hanover, United States
    Competing interests
    The authors declare that no competing interests exist.
  12. Ethan Lee

    Department of Cell and Developmental Biology, Vanderbilt University Medical Center, Nashville, United States
    Competing interests
    The authors declare that no competing interests exist.
  13. Jan E Carette

    Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, United States
    For correspondence
    carette@stanford.edu
    Competing interests
    The authors declare that no competing interests exist.
  14. Rajat Rohatgi

    Department of Biochemistry, Stanford University School of Medicine, Stanford, United States
    For correspondence
    rrohatgi@stanford.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7609-8858

Funding

National Institutes of Health (DP2 AI104557,DP2 GM105448,R01 GM081635,R01 GM103926,RO1 CA105038)

  • Yashi Ahmed
  • Ethan Lee
  • Jan E Carette
  • Rajat Rohatgi

National Science Foundation (DBI-1039423)

  • Yashi Ahmed

David and Lucile Packard Foundation (Fellow Award)

  • Jan E Carette

Helen Hay Whitney Foundation (Novartis Fellowship)

  • Andres M Lebensohn

Stanford University School of Medicine (Josephine Q. Berry Fellowship)

  • Rajat Rohatgi

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

Copyright

© 2016, Lebensohn 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

  • 7,094
    views
  • 1,406
    downloads
  • 52
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

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. Andres M Lebensohn
  2. Ramin Dubey
  3. Leif R Neitzel
  4. Ofelia Tacchelly-Benites
  5. Eungi Yang
  6. Caleb D Marceau
  7. Eric M Davis
  8. Bhaven B Patel
  9. Zahra Bahrami-Nejad
  10. Kyle J Travaglini
  11. Yashi Ahmed
  12. Ethan Lee
  13. Jan E Carette
  14. Rajat Rohatgi
(2016)
Comparative genetic screens in human cells reveal new regulatory mechanisms in WNT signaling
eLife 5:e21459.
https://doi.org/10.7554/eLife.21459

Share this article

https://doi.org/10.7554/eLife.21459

Further reading

    1. Computational and Systems Biology
    2. Genetics and Genomics
    Fangluo Chen, Dylan C Sarver ... G William Wong
    Research Article

    Obesity is a major risk factor for type 2 diabetes, dyslipidemia, cardiovascular disease, and hypertension. Intriguingly, there is a subset of metabolically healthy obese (MHO) individuals who are seemingly able to maintain a healthy metabolic profile free of metabolic syndrome. The molecular underpinnings of MHO, however, are not well understood. Here, we report that CTRP10/C1QL2-deficient mice represent a unique female model of MHO. CTRP10 modulates weight gain in a striking and sexually dimorphic manner. Female, but not male, mice lacking CTRP10 develop obesity with age on a low-fat diet while maintaining an otherwise healthy metabolic profile. When fed an obesogenic diet, female Ctrp10 knockout (KO) mice show rapid weight gain. Despite pronounced obesity, Ctrp10 KO female mice do not develop steatosis, dyslipidemia, glucose intolerance, insulin resistance, oxidative stress, or low-grade inflammation. Obesity is largely uncoupled from metabolic dysregulation in female KO mice. Multi-tissue transcriptomic analyses highlighted gene expression changes and pathways associated with insulin-sensitive obesity. Transcriptional correlation of the differentially expressed gene (DEG) orthologs in humans also shows sex differences in gene connectivity within and across metabolic tissues, underscoring the conserved sex-dependent function of CTRP10. Collectively, our findings suggest that CTRP10 negatively regulates body weight in females, and that loss of CTRP10 results in benign obesity with largely preserved insulin sensitivity and metabolic health. This female MHO mouse model is valuable for understanding sex-biased mechanisms that uncouple obesity from metabolic dysfunction.

    1. Genetics and Genomics
    2. Neuroscience
    Akanksha Bafna, Gareth Banks ... Patrick M Nolan
    Research Article

    The mammalian suprachiasmatic nucleus (SCN), situated in the ventral hypothalamus, directs daily cellular and physiological rhythms across the body. The SCN clockwork is a self-sustaining transcriptional-translational feedback loop (TTFL) that in turn coordinates the expression of clock-controlled genes (CCGs) directing circadian programmes of SCN cellular activity. In the mouse, the transcription factor, ZFHX3 (zinc finger homeobox-3), is necessary for the development of the SCN and influences circadian behaviour in the adult. The molecular mechanisms by which ZFHX3 affects the SCN at transcriptomic and genomic levels are, however, poorly defined. Here, we used chromatin immunoprecipitation sequencing to map the genomic localization of ZFHX3-binding sites in SCN chromatin. To test for function, we then conducted comprehensive RNA sequencing at six distinct times-of-day to compare the SCN transcriptional profiles of control and ZFHX3-conditional null mutants. We show that the genome-wide occupancy of ZFHX3 occurs predominantly around gene transcription start sites, co-localizing with known histone modifications, and preferentially partnering with clock transcription factors (CLOCK, BMAL1) to regulate clock gene(s) transcription. Correspondingly, we show that the conditional loss of ZFHX3 in the adult has a dramatic effect on the SCN transcriptome, including changes in the levels of transcripts encoding elements of numerous neuropeptide neurotransmitter systems while attenuating the daily oscillation of the clock TF Bmal1. Furthermore, various TTFL genes and CCGs exhibited altered circadian expression profiles, consistent with an advanced in daily behavioural rhythms under 12 h light–12 h dark conditions. Together, these findings reveal the extensive genome-wide regulation mediated by ZFHX3 in the central clock that orchestrates daily timekeeping in mammals.