Cody T Mowery, Jacob W Freimer, Zeyu Chen, Salvador Casaní-Galdón, Jennifer M Umhoefer, Maya M Arce, Ketrin Gjoni, Bence Daniel, Katalin Sandor, Benjamin G Gowen, Vinh Nguyen, Dimitre R Simeonov, Christian M Garrido, Gemma L Curie, Ralf Schmidt, Zachary Steinhart, Ansuman T Satpathy, Katherine S Pollard, Jacob E Corn, Bradley E Bernstein, Chun Jimmie Ye, Alexander Marson
Nature Genetics, 29 May 2024

Cis-regulatory elements (CREs) interact with trans regulators to orchestrate gene expression, but how transcriptional regulation is coordinated in multi-gene loci has not been experimentally defined. We sought to characterize the CREs controlling dynamic expression of the adjacent costimulatory genes CD28, CTLA4 and ICOS, encoding regulators of T cell-mediated immunity. Tiling CRISPR interference (CRISPRi) screens in primary human T cells, both conventional and regulatory subsets, uncovered gene-, cell subset- and stimulation-specific CREs. Integration with CRISPR knockout screens and assay for transposase-accessible chromatin with sequencing (ATAC-seq) profiling identified trans regulators influencing chromatin states at specific CRISPRi-responsive elements to control costimulatory gene expression. We then discovered a critical CCCTC-binding factor (CTCF) boundary that reinforces CRE interaction with CTLA4 while also preventing promiscuous activation of CD28. By systematically mapping CREs and associated trans regulators directly in primary human T cell subsets, this work overcomes longstanding experimental limitations to decode context-dependent gene regulatory programs in a complex, multi-gene locus critical to immune homeostasis.