Genome-wide CRISPR screen identifies non-canonical NF-κB signaling as a regulator of density-dependent proliferation

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Abstract

Epithelial cells possess intrinsic mechanisms to maintain an appropriate cell density for normal tissue morphogenesis and homeostasis. Defects in such mechanisms likely contribute to hyperplasia and cancer initiation. To identify genes that regulate the density-dependent proliferation of murine mammary epithelial cells, we developed a fluorescence-activated cell sorting assay based on FUCCI, which marks different stages of the cell cycle with distinct fluorophores. Using this powerful assay, we performed a genome-wide CRISPR/Cas9 knockout screen, selecting for cells that proliferate normally at low density but continue to divide at high density. Unexpectedly, one top hit was Traf3, a negative regulator of NF-κB signaling that has never previously been linked to density-dependent proliferation. We demonstrate that loss of Traf3 specifically activates non-canonical NF-κB signaling. This in turn triggers an innate immune response and drives cell division independently of known density-dependent proliferation mechanisms, including YAP/TAZ signaling and cyclin kinase inhibitors, by blocking entry into quiescence.

Data availability

Sequencing data have been deposited in GEO under accession code GSE147767All other data generated or analysed during this study are included in the manuscript and supporting files.

The following data sets were generated

1. Fomicheva M
2. Macara IG
(2020) RNA sequencing of NT control cells, Traf3 KO, and Traf3/p100 double KO cells
NCBI Gene Expression Omnibus, GSE147767.

https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE147767

Article and author information

Author details

Maria Fomicheva

Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, United States

Competing interests
The authors declare that no competing interests exist.
Ian G Macara

Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, United States

For correspondence
ian.g.macara@vanderbilt.edu

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0001-8546-5357

Funding

National Cancer Institute (R35 CA132898)

Maria Fomicheva

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

Ethics

Animal experimentation: All mouse experimental procedures were approved by Vanderbilt Institutional Animal Care and Use Committee; IACUC protocol number M1800045, Exp: 04/26/2021.

Copyright

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.