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

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.