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, there is still much to learn about the molecular mechanisms by which FOXP3 orchestrates the tolerance process in Treg. Our genome wide analysis for FOXP3 target genes in human regulatory cells using chromatin immunoprecipitation (ChIP) combined with gene and micro RNA expression profiling identified over 1300 genes and more than 60 micro RNAs that are potential human FOXP3 target genes. However many of the FOXP3 binding sites we identifed could not be directly linked to a differentially expressed gene. An emerging concept is that long range interactions are mediated by DNA looping bringing distal regulatory regions such as enhancers and silencers together with gene promoters , and this is a key driver in tissue-specific expression. In addition DNA looping can also bring loci together to co regulate genes in transcriptionally active hubs. ChIP assays do not capture this type of conformation dependant regulation.
Chromosomal Conformation Capture (3C) assays and sequencing are now the method of choice to investigate the physical interaction of separated regulatory regions. We are establishing genome wide conformation capture techniques (ChIC/ ChIA-Pet) to investigate the chromosome contact profile in human Treg cells. Circular chromosomal conformation capture sequencing (4C-seq) assays are also being used to produce high resolution mapping of interactions at selected regions. Our preliminary analysis of the conformation dependant interaction of the SATB1 proximal promoter by 4C-seq has identifed a cluster of enriched contacts over 250kb upstream of the SATB1 transcriptional start site. These regions contain putative FOXP3 binding sites, display marks of active enhancer regions in human T cells (epigenomics roadmap) and are in close proximity to several autoimmune disease SNPs. Together these approaches will identify distal regulatory elements involved in Treg-specific gene regulation and provide mechanistic insights into how intragenic SNPs (GWAS) may alter gene expression in human disease.