5-Hydroxymethylcytosine Remodeling Precedes Lineage Specification during Differentiation of Human CD4+ T Cells

• 5hmC remodeling is widespread during human CD4+ T cell differentiation
• Early 5hmC gains predict loss of DNA methylation in differentiated cells
• Early 5hmC remodeling in vitro predicts loss of DNA methylation in vivo
• 5hmC loci are enriched for functional T cell disease-associated genetic variants

Summary5-methylcytosine (5mC) is converted to 5-hydroxymethylcytosine (5hmC) by the TET family of enzymes as part of a recently discovered active DNA de-methylation pathway. 5hmC plays important roles in regulation of gene expression and differentiation and has been implicated in T cell malignancies and autoimmunity. Here, we report early and widespread 5mC/5hmC remodeling during human CD4+ T cell differentiation ex vivo at genes and cell-specific enhancers with known T cell function. We observe similar DNA de-methylation in CD4+ memory T cells in vivo, indicating that early remodeling events persist long term in differentiated cells. Underscoring their important function, 5hmC loci were highly enriched for genetic variants associated with T cell diseases and T-cell-specific chromosomal interactions. Extensive functional validation of 22 risk variants revealed potentially pathogenic mechanisms in diabetes and multiple sclerosis. Our results support 5hmC-mediated DNA de-methylation as a key component of CD4+ T cell biology in humans, with important implications for gene regulation and lineage commitment.

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