Probing the Role of Alternative Splicing of IKZF1 in Risk for Type 1 Diabetes

Genome wide association studies have identified a region at chromosome 7p12.2, containing the IKZF1 gene, that is associated with type 1 diabetes (T1D). IKZF1 encodes Ikaros, a transcription factor and critical regulator of lymphocyte development and homeostasis. The IKZF1 gene includes 9 exons that produce at least 10 distinct transcripts. Exons 3-5 encode 4 zinc finger motifs that mediate DNA binding, while the last exon encodes 2 zinc fingers that allow homo- or heterodimerization. We examined the expression of IKZF1 using RNA-seq data from different lymphocyte populations. Individual coding exons of IKZF1, notably exons 3 and 5, displayed significant differences in usage between CD4+ T cells, CD8+ T cells and CD19+ B cells among T1D cases as well as between cases and controls in memory CD4+ T cells. This suggested that specific transcript isoforms of IKZF1 are differentially expressed in lymphocyte subtypes as well as in T1D. To explore the role of alternative splicing of IKZF1 in T1D, CRISPR/cas9 was used to generate Jurkat T cell lines with truncating mutations in either IKZF1 exon 3, exon 5 or both. Clones were isolated by serial dilution and characterized by PCR amplification and DNA sequencing. Twenty clones with different IKZF1 mutations were identified. Immunoblots revealed shifts in Ikaros protein isoform abundance consistent with the exon targeting in individual clones. Mutation of exons 3 or 5 resulted in the upregulation of 218 genes and the downregulation of 309 genes, as assayed by RNA-seq. Interrogation of ENCODE project data showed that most of these differentially expressed genes have an IKZF1 ChIP-seq peak within 1kb of the gene, consistent with their being targets of Ikaros regulation. These results suggest that the number of zinc finger domains in Ikaros, rather than their sequence or structure, modulates their impact. More broadly, the differential expression of IKZF1 transcripts distinguishes the transcriptomes of specific lymphocyte populations in both T1D cases and controls. Disclosure L. Pastor palomo: Employee; Spouse/Partner; Cox Business. J. R. B. Newman: None. M. A. Atkinson: None. P. J. Concannon: Stock/Shareholder; Self; Amgen Inc. Funding National Institutes of Health (R01DK116954, P01AI042288)