SUMMARY
FOXP3 is a lineage-defining transcription factor (TF) for immune-suppressive regulatory T cells (Tregs). While mice exclusively express FOXP3 in Tregs, humans also transiently express FOXP3 in stimulated conventional CD4+ T cells (Tconvs). Mechanisms governing these distinct expression patterns remain unknown. Here, we performed CRISPR screens tiling the FOXP3 locus and targeting TFs in human Tregs and Tconvs to discover cis-regulatory elements (CREs) and trans-regulators of FOXP3. Tconv FOXP3 expression depended on a subset of Treg CREs and Tconv-selective positive (TcNS+) and negative (TcNS-) CREs. The CREs are occupied and regulated by TFs we identified as critical regulators of FOXP3. Finally, mutagenesis of murine TcNS- revealed that it is critical for restriction of FOXP3 expression to Tregs. We discover CRE and TF circuitry controlling FOXP3 expression and reveal evolution of mechanisms regulating a gene indispensable to immune homeostasis.
Highlights
Comprehensive CRISPR maps of CREs and TFs controlling FOXP3 in human Tregs and Tconvs
Key TFs that control FOXP3 directly occupy and regulate CREs forming TF-CRE circuits
A previously unknown negative CRE stringently restricts FOXP3 to Tregs in mice
Competing Interest Statement
A.M. is a cofounder of Site Tx, Arsenal Biosciences, Spotlight Therapeutics and Survey Genomics, serves on the boards of directors at Site Tx, Spotlight Therapeutics and Survey Genomics, is a member of the scientific advisory boards of Site Tx, Arsenal Biosciences, Cellanome, Spotlight Therapeutics, Survey Genomics, NewLimit, Amgen, and Tenaya, owns stock in Arsenal Biosciences, Site Tx, Cellanome, Spotlight Therapeutics, NewLimit, Survey Genomics, Tenaya and Lightcast and has received fees from Site Tx, Arsenal Biosciences, Cellanome, Spotlight Therapeutics, NewLimit, Abbvie, Gilead, Pfizer, 23andMe, PACT Pharma, Juno Therapeutics, Tenaya, Lightcast, Trizell, Vertex, Merck, Amgen, Genentech, GLG, ClearView Healthcare, AlphaSights, Rupert Case Management, Bernstein and ALDA. A.M. is an investor in and informal advisor to Offline Ventures and a client of EPIQ. The Marson laboratory has received research support from the Parker Institute for Cancer Immunotherapy, the Emerson Collective, Arc Institute, Juno Therapeutics, Epinomics, Sanofi, GlaxoSmithKline, Gilead and Anthem and reagents from Genscript and Illumina. C.T.M. is a Bio+Health Venture Fellow at Andreessen Horowitz. J.E.C. is a co-founder and SAB member of Serac Biosciences and an SAB member of Mission Therapeutics, Relation Therapeutics, Hornet Bio, and Kano Therapeutics. The lab of J.E.C. has funded collaborations with Allogene, Cimeio, and Serac. H.Y.C. is a co-founder of Accent Therapeutics, Boundless Bio, Cartography Biosciences, and Orbital Therapeutics, and is an advisor of 10x Genomics and Exai Bio. H.Y.C. was an advisor of Arsenal Bio and Chroma Medicine up to Dec. 15, 2024. H.Y.C. is an employee and stockholder of Amgen as of Dec. 16, 2024. J.W.F. was a consultant for NewLimit, is an employee of Genentech, and has equity in Roche. A.T.S. is a founder of Immunai, Cartography Biosciences, Santa Ana Bio, and Prox Biosciences, an advisor to 10x Genomics and Wing Venture Capital, and receives research funding from Astellas. J.M.U., A.M., M.A., L.G, and L.A.G. have filed patents related to this work.
Footnotes
Additional characterization of chromatin at the FOXP3 locus; Addition of regulatory T cell transcription factor screens and ChIP-seq; New characterization of FOXP3 CREs in mice.