Single molecule DNA methylation reveals unique epigenetic identity profiles of T helper cells

Identity and plasticity of CD4 T helper (Th) cells are regulated in part by epigenetic mechanisms. Cytosine methylation in CpG context (5mCpG) and cytosine hydroxymethylation (5hmCpG) are DNA modifications that identify stable cell phenotypes. To assess transition states in Th cells, we developed a method based on Cas9-targeted single molecule nanopore sequencing and found that 5mCpG can be used as markers of cellular identity. Targeting as few as 10 mouse selected genomic loci, we were able to distinguish major differentiated T cell subtypes as well as intermediate phenotypes by their native DNA 5mCpG patterns. Moreover, by using off-target sequences we were able to infer transcription factor activities relevant to each cell subtype. Our data highlight the potential to exploit native DNA methylation profiling to study physiological and pathological Th transition states. Overall design: We polarized mouse naïve Th0 cells towards various Th fates in vitro. Th0 cells were isolated by FACs-sorting and activated under polarizing conditions towards the Th0, Th1, Th2, Th17 and Treg phenotypes using cytokine cocktails. To test the sensitivity of this approach to detect potential pathogenic Th cell phenotypes, naïve T cells were also polarized under Th17 conditions in the absence of TGFb, referred to as 'Th17-noTGFb' cells.