FOXP3 expression depends on cell-type-specific cis-regulatory elements and transcription factor circuitry

Jennifer M Umhoefer; Maya M Arce; Sivakanthan Kasinathan; Sean Whalen; Rama Dajani; Sanjana Subramanya; Laine Goudy; Julia A Belk; Royce Zhou; Minh T N Pham; Wenxi Zhang; Rosmely Hernandez; Carinna Tran; Nikhita Kirthivasan; Jacob W Freimer; Cody T Mowery; Vinh Nguyen; Mineto Ota; Benjamin G Gowen; Dimitre R Simeonov; Gemma L Curie; Zhongmei Li; Andy Y Chen; Jacob E Corn; Howard Y Chang; Qizhi Tang; Luke A Gilbert; Ansuman T Satpathy; Katherine S Pollard; Alexander Marson

doi:10.1016/j.immuni.2025.10.020

FOXP3 expression depends on cell-type-specific cis-regulatory elements and transcription factor circuitry

Immunity. 2026 Jan 13;59(1):129-144.e13. doi: 10.1016/j.immuni.2025.10.020. Epub 2025 Nov 13.

Authors

Jennifer M Umhoefer¹, Maya M Arce², Sivakanthan Kasinathan³, Sean Whalen⁴, Rama Dajani⁵, Sanjana Subramanya⁵, Laine Goudy², Julia A Belk⁶, Royce Zhou⁷, Minh T N Pham⁸, Wenxi Zhang⁹, Rosmely Hernandez⁵, Carinna Tran⁵, Nikhita Kirthivasan¹⁰, Jacob W Freimer¹¹, Cody T Mowery², Vinh Nguyen¹², Mineto Ota¹¹, Benjamin G Gowen¹³, Dimitre R Simeonov¹⁴, Gemma L Curie¹³, Zhongmei Li¹⁵, Andy Y Chen⁸, Jacob E Corn¹⁶, Howard Y Chang¹⁷, Qizhi Tang¹⁸, Luke A Gilbert¹⁹, Ansuman T Satpathy²⁰, Katherine S Pollard²¹, Alexander Marson²²

Affiliations

¹ Gladstone-UCSF Institute of Genomic Immunology, San Francisco, CA 94158, USA; Department of Medicine, University of California, San Francisco, San Francisco, CA 94143, USA; Biomedical Sciences Graduate Program, University of California, San Francisco, San Francisco, CA 94143, USA. Electronic address: jenny@sitetx.com.
² Gladstone-UCSF Institute of Genomic Immunology, San Francisco, CA 94158, USA; Department of Medicine, University of California, San Francisco, San Francisco, CA 94143, USA; Biomedical Sciences Graduate Program, University of California, San Francisco, San Francisco, CA 94143, USA.
³ Gladstone-UCSF Institute of Genomic Immunology, San Francisco, CA 94158, USA; Division of Allergy, Immunology, and Rheumatology, Department of Pediatrics, Stanford University School of Medicine, Stanford, CA 94305, USA.
⁴ Gladstone Institute of Data Science and Biotechnology, San Francisco, CA 94158, USA.
⁵ Gladstone-UCSF Institute of Genomic Immunology, San Francisco, CA 94158, USA.
⁶ Center for Personal Dynamic Regulomes, Stanford University School of Medicine, Stanford, CA 94305, USA.
⁷ Gladstone-UCSF Institute of Genomic Immunology, San Francisco, CA 94158, USA; Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305, USA; Molecular Medicine Program, Division of Gastroenterology, Department of Medicine, University of California, San Francisco, San Francisco, CA 94143, USA.
⁸ Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305, USA.
⁹ Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305, USA; Tetrad Graduate Program, University of California, San Francisco, San Francisco, CA 94158, USA.
¹⁰ Gladstone-UCSF Institute of Genomic Immunology, San Francisco, CA 94158, USA; Biomedical Sciences Graduate Program, University of California, San Francisco, San Francisco, CA 94143, USA.
¹¹ Gladstone-UCSF Institute of Genomic Immunology, San Francisco, CA 94158, USA; Department of Medicine, University of California, San Francisco, San Francisco, CA 94143, USA; Department of Genetics, Stanford University, Stanford, CA 94305, USA.
¹² Gladstone-UCSF Institute of Genomic Immunology, San Francisco, CA 94158, USA; Department of Medicine, University of California, San Francisco, San Francisco, CA 94143, USA; UCSF CoLabs, University of California, San Francisco, San Francisco, CA 94143, USA; Department of Surgery, University of California, San Francisco, San Francisco, CA 94143, USA; Diabetes Center, University of California, San Francisco, San Francisco, CA 94143, USA.
¹³ Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA 94720, USA; Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA.
¹⁴ Department of Medicine, University of California, San Francisco, San Francisco, CA 94143, USA; Biomedical Sciences Graduate Program, University of California, San Francisco, San Francisco, CA 94143, USA.
¹⁵ Gladstone-UCSF Institute of Genomic Immunology, San Francisco, CA 94158, USA; Department of Medicine, University of California, San Francisco, San Francisco, CA 94143, USA.
¹⁶ Department of Biology, Institute of Molecular Health Sciences, ETH Zürich, 8093 Zürich, Switzerland.
¹⁷ Center for Personal Dynamic Regulomes, Stanford University School of Medicine, Stanford, CA 94305, USA; Howard Hughes Medical Institute, Stanford University, Stanford, CA 94305, USA.
¹⁸ Department of Surgery, University of California, San Francisco, San Francisco, CA 94143, USA; Diabetes Center, University of California, San Francisco, San Francisco, CA 94143, USA.
¹⁹ Arc Institute, Palo Alto, CA 94304, USA; Department of Urology, University of California, San Francisco, San Francisco, CA 94143, USA.
²⁰ Gladstone-UCSF Institute of Genomic Immunology, San Francisco, CA 94158, USA; Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305, USA; Parker Institute for Cancer Immunotherapy, San Francisco, CA 94129, USA.
²¹ Gladstone Institute of Data Science and Biotechnology, San Francisco, CA 94158, USA; Institute for Human Genetics, University of California, San Francisco, San Francisco, CA 94143, USA; Department of Epidemiology and Biostatistics, University of California, San Francisco, San Francisco, CA 94143, USA; Chan Zuckerberg Biohub SF, San Francisco, CA 94158, USA.
²² Gladstone-UCSF Institute of Genomic Immunology, San Francisco, CA 94158, USA; Department of Medicine, University of California, San Francisco, San Francisco, CA 94143, USA; Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA 94720, USA; Parker Institute for Cancer Immunotherapy, San Francisco, CA 94129, USA; Institute for Human Genetics, University of California, San Francisco, San Francisco, CA 94143, USA; UCSF Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, CA 94158, USA; Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA 94143, USA. Electronic address: alex.marson@gladstone.ucsf.edu.

Abstract

FOXP3 is a lineage-defining transcription factor (TF) for immune-suppressive regulatory T cells (Treg cells). Although mice exclusively express FOXP3 in Treg cells, stimulated conventional CD4⁺ T cells (Tconv cells) also transiently express FOXP3 in humans. Mechanisms governing these distinct expression patterns need elucidation. Here, we performed CRISPR screens tiling the FOXP3 locus and targeting TFs in human Treg and Tconv cells to identify cis-regulatory elements (CREs) and trans-regulators of FOXP3. Tconv cell FOXP3 expression depended on a subset of Treg cell CREs, as well as Tconv-cell-selective positive (NS+) and negative (NS-) CREs. Combinatorial silencing of Tconv cell CREs revealed their epistatic logic. These CREs are occupied and regulated by TFs that we identified as FOXP3 regulators. Finally, mutagenesis of murine NS- CRE revealed its essentiality for restricting FOXP3 expression to Treg cells. We map CRE and TF circuitry to reveal distinct cell- and species-specific regulation of FOXP3 expression.

Keywords: CRISPR; FOXP3; T cells; enhancers; epigenetics; gene regulation; regulatory T cells; transcription factors.

MeSH terms

Animals
Forkhead Transcription Factors* / genetics
Forkhead Transcription Factors* / metabolism
Gene Expression Regulation*
Humans
Mice
Mice, Inbred C57BL
Regulatory Elements, Transcriptional* / genetics
Regulatory Sequences, Nucleic Acid* / genetics
T-Lymphocytes, Regulatory* / immunology
T-Lymphocytes, Regulatory* / metabolism
Transcription Factors* / genetics
Transcription Factors* / metabolism

Substances

Forkhead Transcription Factors
FOXP3 protein, human
Transcription Factors
Foxp3 protein, mouse

Abstract

MeSH terms

Substances

Grants and funding