Population-Optimized SNP Array Reveals RAP1A as a Novel Candidate Susceptibility Gene for Crohn's Disease in Japanese Individuals

Background:We recently published a deregulated gene network driven by the transcription factor FOXP3 in a clinical cohort of Crohn Disease (CD) patients.We found the majority of EZH2/FOXP3 gene targets in CD4 + lymphocytes isolated from CD lesions were significantly upregulated, at odds with this complex being an established gene repressor.These findings of upregulated gene expression cannot be explained by simple de-repression, but rather gain of an activation complex.A putative FOXP3 co-activator complex associated with Crohn's disease is unknown.Methods: The expression profile generated from 21 CD-affected subjects and 12 age/gender matched controls was analyzed utilizing multiple bioinformatics tools (MAPR-Seq, EdgeR, and Enrichr).5,328 statistically significant differentially expressed genes (DEGs) were examined for potential coordinated function between FOXP3 and epigenetic complexes.FOXP3 complex composition was assessed by immuno-precipitation. Treg and Jurkat cellular phenotyping was performed by flow cytometry, as was FOXP3 gene target activation (IL-2).Results: We report pathway analyses utilizing a FOXP3 deregulated gene set (n=260) to identify putative co-activators.We hypothesized that HIF1α regulates a FOXP3/p300 co-activator complex based upon; enrichment of our gene list within p300 bound gene sets (p= 6.2e -19 , ChEA2016, Enrichr) and enrichment of our gene list in key HIF1α regulatory pathways (mTOR signaling pathway, KEGG database, p=0.001 and Rapamycin dataset, SILAC phosphoproteomics, p=0.02).Using IL-2 as the model gene for FOXP3 transcriptional regulation, we demonstrated that HIF1α inhibition leads to IL-2 gene activation, while increased HIF1α protein levels leads to IL-2 gene repression.Biochemically, we demonstrated the FOXP3/p300 protein complex to be dependent upon HIF1α presence by immuno-precipitation utilizing HIF1α inhibition.Finally, we utilized an epigenetic mutant mouse model in which the FOXP3 co-repressor EZH2 is deleted resulting in severe systemic inflammation and unregulated FOXP3 gene network activation.Inhibition of p300 in vitro rescues CTLA-4, a previously validated marker of FOXP3 de-repression in Tregs.Conclusions: We utilized multiple biochemical approaches to confirm bioinformatics data suggesting HIF1α regulates a FOXP3/p300 activation complex.We postulate HIF1α availability limits access of epigenetic activator complexes to FOXP3, allowing for the PRC2/EZH2 repressor complex to silence target gene networks, thereby inhibiting the production of disease inducing lymphocytes.