A genome-wide CRISPR screen identifies the TNRC18 gene locus as a novel regulator of inflammatory signaling [CUT&Tag]

Interleukin-1β (IL-1β) is dysregulated in chronic inflammatory diseases, yet the genetic factors influencing IL-1β production remain largely unknown. Myeloid-derived cells are the primary producers of IL-1β, which prompted a genome-wide CRISPR knockout screen in the human myeloid-derived U937 cell model treated with lipopolysaccharide (LPS) to mimic inflammatory conditions and sorted for high and low intracellular IL-1β levels. A total of 295 genes were identified as regulators of IL-1β production, including known mediators such as TLR4, JAK-STAT and IL-10 receptor. Notably, 57 out of the 295 genes overlapped with loci associated with human inflammatory diseases, including the TNRC18 gene locus associated with multiple diseases in the Finnish population. U937 cells engineered with the Finnish-enriched rs748670681 risk allele demonstrated decreased levels of mRNA for TNRC18 and an adjacent gene WIPI2, reduction in LPS-dependent gene activation and cytokine production, but elevation of interferon-responsive gene programs. Transcriptomic profiles for individual knockouts of TNRC18 and WIPI2 attributed the loss of LPS-dependent signaling primarily to TNRC18, which occurs through the modulation of H3K27 acetylation around inflammatory regulatory regions via TNRC18 and its protein interaction network. In contrast, the loss of WIPI2 is characterized by an exacerbation of interferon signaling. Collectively, these findings delineate the global regulatory mechanisms of IL-1β production and provide molecular insights to the role of the rs748670681 variant as a pleiotropic risk factor for inflammatory diseases. CUT&Tag was performed on two biological replicates per condition using the CUT&Tag-IT Assay Kit (Active Motif, 53172) following manufacturers recommendation. Briefly, 450,000 cells per replicate per condition were bound to the prepared Concanavalin A beads (+spike-in control, Active Motif, 53173) and incubated overnight at 4°C in a rotator with primary antibodies [1 µg of mouse anti-H3K27Ac (1:50, Active Motif, 91193), 1 ug of positive control mouse anti-H3K4me3 (1:50, Active Motif, 91263)]. The samples were then incubated with Rabbit Anti-Mouse secondary antibody (1:100, Active Motif, 53160) for 1 hour at room temperature on a rotator, followed by washes in Dig-Wash buffer. The beads were resuspended with CUT&Tag-IT Assembled pA-Tn5 Transposons and incubated at room temperature on a rotator, followed by washes with Dig-300 buffer. The beads were finally resuspended in tagmentation buffer, and the reactions incubated at 37°C for 60 minutes. The DNA was extracted as detailed in the manufacturer's instructions. Libraries were PCR-amplified using i5 and i7 indexed primers (14 cycles) and primer dimers were removed with AmpureXP bead (Beckman Coulter, A63881) purification. The final libraries were evaluated on Agilent 4200 Tapestation (quality) and Thermo Qubit (quantity) and sequenced on the Illumina NextSeq550 platform (75-bp paired-end reads). Data analysis was performed using HOMER and DeepTools. Briefly, raw FASTQ read pairs were pre-processed where adapter, low-quality sequences, duplicates were removed using cutadapt, rmdup. Preprocessed reads were aligned to human genome (hg38) using Bowtie2 and Drosophila genome (spike in control) to generate the normalization factor. Bedtools were used to sort and index, and HOMER to generate TagDirectory, peak calls and bedgraph for visualization.