DNA methylation profiles at the human Th2 locus in BAC transgenic mice point to novel putative regulatory elements (IRM6P.720)

Most cis-regulatory elements in the mouse Th2 cytokine locus were initially identified based on their evolutionary conservation. In contrast, the regulatory landscape of the human Th2 locus, which is ~40 kB larger than the mouse syntenic region, remains poorly defined because recently arisen putative regulatory elements are less readily identifiable by bioinformatics and cannot be functionally interrogated in humans. To address these limitations we created BAC transgenic (TG) mice carrying 160 kB of the human Th2 locus including RAD50, IL13, and IL4. These mice appeared normal and demonstrated faithful transcriptional regulation of human Th2 cytokine genes. Because dynamic changes in chromatin architecture typically mark functional non-coding sequences, we used next generation bisulfite sequencing to compare CpG methylation of DNA isolated from TG naive CD4+ T cells and in vitro polarized Th2 or Th1 cells. All major regulatory elements previously characterized in mice were clearly identifiable by their CpG methylation profile in the human TG Th2 locus. Moreover, we identified at least three other non-coding regions (conserved highly in primates and moderately in species of the clade Laurasiatheria) that were differentially methylated in Th2, Th1 and naive T cells, and are located in the RAD50/IL13 and IL13/IL4 intergenic areas. Our data suggest these regions contain novel cis-regulatory elements involved in the transcriptional regulation of human Th2 cytokines expression.