Systematic decoding of cis gene regulation defines context-dependent control of the multi-gene costimulatory receptor locus in human T cells

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.

CRISPR Knockout Screens CRISPR knockout screens were performed as previously described47 so as to accompany the published CTLA4 data.Cells were isolated and activated as above.One day after stimulation, cells were transduced with concentrated sgRNA library lentivirus produced as described above.Lentivirus was washed from cells after 24 hours in culture.Subsequently, Cas9 RNPs were prepared with lyophilized Edit-R crRNA nontargeting Control 3 (Dharmacon, #U-007503-01-05).crRNAs and Edit-R CRISPR-Cas9 Synthetic tracrRNA (Dharmacon #U-002005-20) were resuspended to 160mM in nuclease-free duplex buffer (IDT #11-05-01-03), mixed at a 1:1 ratio for a 80 mM solution, and incubated at 37°C 30 minutes.Single-stranded donor oligonucleotides enhancer (ssODN, CM_oligo_7) was added at a 1:1 molar ratio of the final Cas9-Guide complex, mixed well by pipetting, and incubated for an additional 5 minutes at 37°C.Cas9 protein (UCB MacroLab, 40uM) was added at a 1:1 ratio, mixed thoroughly by pipetting, and incubated at 37°C for 15 minutes.Prepared Cas9 ribonucleoproteins (RNPs) were distributed into a 96-well plate.On day 3, stimulated cells were pelleted at 90g for 10 minutes in a 25°C centrifuge, the supernatant removed, and resuspended at 1e6 cells per 20uL Buffer P3 (Lonza #V4SP-3096).Prepared cells were distributed into the plate with RNPs, mixed gently, and transferred to the 96-well Nucleocuvette Plate (Lonza) for nucleofection (DS-137, Amaxa Nucleofector 96-well Shuttle System).Cells were nucleofected using the pulse code EH-115.Immediately after electroporation, 90uL cRPMI prewarmed to 37°C was added to each well and incubated at 37°C 15 minutes.Cells were pooled, transferred to incubation flasks, and diluted with additional medium to a final concentration of 1e6 cells/mL.On day 6 after electroporation, cells were fixed, stained, and sorted for CD28 (unstimulated) and ICOS (24 hours restimulation) staining as described above.sgRNA libraries were generated and sequenced as for the CRISPRi screens.

Perturb-ATAC-Seq
Treg cells from two human donors were isolated and subjected to FOXP3 and AAVS1 knockout with CRISPR as described above.Nine days after the initial isolation and stimulation, 15000 resting Treg cells per sample were resuspended in ATAC Lysis Buffer (10mM Tris-HCl pH7.4,10mM NaCl, 3mM MgCl2, 0.1% IGEPAL) and nuclei subjected to tagmentation using Nextera DNA Library Preparation Kit (Illumina).Tagmentation DNA was purified with the MinElute PCR Purification Kit (Qiagen #28004) and amplified with Phusion High-Fidelity PCR Master Mix (NEB #F531L) using 16 PCR cycles.Amplified libraries were re-purified.Fragment distribution of libraries was assessed with Agilent Bioanalyzer and libraries sequenced low-depth on Illumina NextSeq 500 followed by deep sequencing on Illumina NovaSeq X using paired-end 150bp read configuration.Sequencing was performed at the UCSF CAT.
Randomization Primary human T cells were isolated from peripheral blood leukopaks provided by Stemcell Technologies isolated from human donors >18 years old without regard for demographics.All comparison conditions (e.g.control vs KO) were performed in donor-matched cells and thus internally controlled.

Blinding
All samples were handled equally and unblinded given that data from multiple biological and/or technical replicates were pooled for analysis.
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Novel plant genotypes
Describe the methods by which all novel plant genotypes were produced.This includes those generated by transgenic approaches, gene editing, chemical/radiation-based mutagenesis and hybridization.For transgenic lines, describe the transformation method, the number of independent lines analyzed and the generation upon which experiments were performed.For gene-edited lines, describe the editor used, the endogenous sequence targeted for editing, the targeting guide RNA sequence (if applicable) and how the editor was applied.

Seed stocks
Report on the source of all seed stocks or other plant material used.If applicable, state the seed stock centre and catalogue number.If plant specimens were collected from the field, describe the collection location, date and sampling procedures.
were selected.For Treg sorts, Foxp3-positive Helios-positive gates (CRISPRi screens) and CD25-High CD127-Low gates (arrayed validations) were set according to donor-matched Tconv samples.Treg gates were included, where applicable, and then all samples were gated on BFP positivity for sgRNA transduction based on untransduced control cells.
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