FOXP3 is essential for the formation and function of regulatory T cells (Tregs) as demonstrated by autoimmune disease in the scurfy mouse and in IPEX patients. However, the molecular basis of human FOXP3 function or the relationship between direct and indirect target genes in human Treg has yet to be fully characterised. Our genome wide analysis of human FOXP3 target genes in natural regulatory cells using chromatin immunoprecipitation (ChIP) combined with gene expression profiling and micro RNA profiling identified a core gene set which defines nTreg, and which includes transcription factors, key functional molecules and microRNAS. Mapping the targets of FOXP3 has revealed a number of key genes that are regulated by FOXP3 to enforce and maintain the suppressor phenotype, including SATB1. These key genes are also regulated by feed forward loops involving miRs, and it is this mechanism which may account for functional plasticity in vivo, as miRs provide fine tuning of gene dose (Beyer et al Nat Immunol 2011). As treg respond to thier environment via the cell surface, and the cell surface plays a major role in the function of Treg, we also investigated the genes expressed on the cell surface. We identified a candidate biomarker for hu Tcells, PI16, and have characterised its expressin on both Treg and Th cells. PI16 identifes a subset of both Treg and T helper cells with memory or effector memory phenotype, which share paired functional chacarcteristics matched to all Thelper lineages.
In an attempt to fully identify the FOXP3 interactome, and to identify all targets of miRs expressed by Treg we have established new genome wide approaches. As over 50% of the experimentally defined miR:mRNA targets are not predicted by bioinformatics tools, and many targets of miRs show small changes at the mRNA transcript level, but significant changes at the protein level, we have expanded our target discovery to include genome wide unbiased identification of Treg miR processing using individual-nucleotide crosslinking and immunoprecipitation (iCLIP). This relies on the biochemical trapping and isolation of miRs and mRNAs within the RNA-induced silencing complex (RISC), followed by deep sequencing to identify the associated RNA species. Many of the FOXP3 targets identified in our ChIP dataset are annotated by nearest neighbour principles and do not map to a differentially expressed gene, and it is now clear that many interactions between transcription factors and promoters or enhancers occur over large distances via DNA looping. We have established chromatin conformation capture to indentify all interactions between FOXP3 and the genome at any distance. This approach will potentially map the causal interactions of SNPs in autoimmune GWAS datasets.