Manipulation of alternative splicing of IKZF1 elicits distinct gene regulatory responses in T cells

Genome-wide studies have identified significant allelic associations between genetic variants in or near the IKZF1 gene and multiple autoimmune disorders. IKZF1, encoding the transcription factor IKAROS, produces at least 10 distinct transcripts. To explore the impact of alternative splicing of IKZF1 on the function of mature T cells, we generated a panel of human T-cell clones with truncating mutations in IKZF1 exons 4, 6 or both. Differences in gene expression, chromatin accessibility, and protein abundance among clones were assessed by RNA-seq, ATAC-seq and immunoblotting. Clones with single targeting events clustered separately from double-targeted clones on multiple parameters, but overall, clone responses were highly heterogeneous. Perturbation of IKZF1 splicing resulted in significant differences in expression and chromatin accessibility of other autoimmunity-associated genes and elicited compensatory expression changes in other IKAROS family members. Our results suggest that even modest alterations of IKZF1 splicing can have significant effects on gene expression and function in mature T cells, potentially contributing to autoimmunity in susceptible individuals. Author SummaryRNA sequencing has revealed an unexpectedly large population of alternative transcripts produced by most human genes, but the functional significance of such transcripts has been debated, with some authors arguing that they represent non-functional "noise" and others arguing that they are largely functional and greatly expand the potential human proteome. Here, we explore these issues for the transcription factor IKZF1, which produces numerous alternative transcripts in human T cells and is significantly associated with risk for type 1 diabetes and other autoimmune disorders. We introduce stop codons at multiple sites in alternatively spliced exons and evaluate transcript levels and chromatin accessibility in individually targeted clones, allowing us to assay the broad impact of alternative splicing of IKZF1 in human T cells. Our studies reveal that perturbation of IKZF1 splicing results in significant differences in gene expression, chromatin accessibility and protein production of other autoimmunity-associated genes and elicits compensatory expression changes in other IKAROS family members. Even modest alterations in IKZF1 splicing had significant effects on gene expression and function in mature T cells, potentially contributing to autoimmunity in susceptible individuals and suggesting that transcript isoforms produced by alternative splicing can and do have functional impacts.