P027 - DIFFERENTIAL BINDING OF RBPJ AND CUX1 TO IBD CAUSAL SNP rs1887428 MODIFIES EXPRESSION OF JAK2
(Room Poster Hall)
19 Jan 18
5:30 PM
-
7:00 PM
Tracks:
Clinical and Research Challenges
Background Recently, genetic variants conferring risk for IBD were fine-mapped to single variant resolution. Eight of these causal variants resulted in amino acid substitutions while ten were non-coding. The mechanism by which these non-coding causal variants confer disease risk is unknown, though presumably they influence gene expression by differential binding of transcription factors. SNP rs1887428 is located in the promoter region of Janus kinase 2 (JAK2), an enzyme that interacts with STAT family transcription factors and cytokine receptors to regulate hematopoiesis and the immune response. Methods We constructed oligodeoxynucleotide baits containing risk or protective alleles of rs1887428 and the surrounding sequence. A nuclear extract was made from the T cell acute lymphoblastic leukemia line Jurkat. The baits were used in affinity purification of DNA-binding transcription factors from the extract and the recovered proteins were identified by mass spectrometry. In vitro assays using affinity purification, electrophoresis mobility shift (EMSA), and dual luciferase were used to confirm the allelic preference of the identified proteins. Results We found that transcription factor RBPJ, the downstream effector of the Notch signaling pathway, binds preferentially to the risk allele. The homeodomain transcriptional repressor CUX1 binds exclusively to the protective allele, which was confirmed by EMSA. Co-expression of the intracellular domain of NOTCH1, partner of RBPJ, increased expression of the JAK luciferase reporter while co-expression of CUX1 decreased expression. Conversely, knock-down of CUX1 with siRNA increased expression. Conclusion We have shown that a causal SNP in the promoter of JAK2 is regulated by transcriptional activators and repressors. These results suggest that the mechanism of IBD causal SNPs may not be reducible to a canonical binding site but may involve cooperative and competitive interactions of multiple nuclear proteins.