CRISPRi and KO screens reveal integrated cis- and trans-regulation of the CD28, CTLA4, and ICOS locus in primary human T cells
Authors: Mowery Cody , Freimer Jacob , Umhoefer Jennifer , Garrido Christian , Schmidt Ralf , Steinhart Zachary , Gowen Benjamin , Curie Gemma , Corn Jacob , Ye Jimmie Chun , Marson Alexander 
Published: January 2023

Abstract
Costimulation is essential for complete T cell activation, requiring the coordinated expression of various surface proteins including those encoded by CD28 family members CD28, CTLA4, and ICOS. Interestingly, these three genes lie adjacently in a topologically-associating domain (TAD) and are believed to have arisen through duplications of CD28, though they have evolved opposing functions and exhibit expression differences across cell states and T cell subsets. We hypothesized that distinct cis- and trans-regulatory circuits emerged to uniquely govern the expression of each gene. Therefore, we tiled CRISPR interference (CRISPRi) across the 1.44Mb TAD in primary human conventional (Tconv) and regulatory T (Treg) cells to functionally profile cis-regulatory elements (CREs) controlling expression of CD28, CTLA4, and ICOS. We find that inhibiting the transcriptional start site of one gene (but not gene KO) enhances the expression of an adjacent gene, potentially due to redirection of shared CREs to the un-inhibited promoter. Moreover, we find distinct CREs responsible for constitutive expression of CTLA4 in Treg cells but stimulation-responsiveness in Tconv cells. We also find evidence of a single CRE that negatively regulates CD28 at baseline but positively regulates CTLA4 upon restimulation. Lastly, we characterize mechanisms of CRE control by identifying upstream regulators of target genes via CRISPR knockout screening. Our work nominates ZNF217, a zinc finger protein with no known function in human T cells, as a novel negative regulator altering chromatin accessibility in the locus. Here, we present a framework for functional characterization of regulatory modules controlling loci of interest in primary human CD4+ T cells.
CM is supported by NIAID F30 AI157167-01 and is a UCSF ImmunoX Computational Immunology Fellow. ZS is supported by the Parker Institute for Cancer Immunology scholarship. RS was supported by fellowships of the Austrian Exchange Service and the Austrian Society of Laboratory Medicine. C.J.Y. and AM are Chan Zuckerberg Biohub Investigators and members of the PICI. C.J.Y. is further supported by the National Institutes of Health (NIH) grant nos. R01AR071522, R01AI136972 and R01HG011239. AM is supported by National Institute of Diabetes and Digestive and Kidney Diseases DP3DK111914-01 (AM) Simons Foundation (AM) Burroughs Wellcome Fund, Career Award for Medical Scientists (AM) The Cancer Research Institute (CRI) Lloyd J. Old STAR grant (AM) Parker Institute for Cancer Immunotherapy, PICI (AM) Innovative Genomics Institute (IGI) National Institutes of Health grant P30 DK063720 (Parnassus Flow Cytometry Core, AM) National Institutes of Health grant S10 1S10OD021822-01 (Parnassus Flow Cytometry Core, AM) Investigator at the Chan Zuckerberg Biohub (AM) Gifts from Brook Byers, Barbara Bakar, Karen Jordan, Elena Radutzky