Enhancers are developmentally controlled transcriptional regulatory regions 
whose activities are modulated through histone modifications or histone variant 
deposition. In this study, we show by genome-wide mapping that the newly 
discovered deoxyribonucleic acid (DNA) modification 5-hydroxymethylcytosine 
(5hmC) is dynamically associated with transcription factor binding to distal 
regulatory sites during neural differentiation of mouse P19 cells and during 
adipocyte differentiation of mouse 3T3-L1 cells. Functional annotation reveals 
that regions gaining 5hmC are associated with genes expressed either in neural 
tissues when P19 cells undergo neural differentiation or in adipose tissue when 
3T3-L1 cells undergo adipocyte differentiation. Furthermore, distal regions 
gaining 5hmC together with H3K4me2 and H3K27ac in P19 cells behave as 
differentiation-dependent transcriptional enhancers. Identified regions are 
enriched in motifs for transcription factors regulating specific cell fates such 
as Meis1 in P19 cells and PPARγ in 3T3-L1 cells. Accordingly, a fraction of 
hydroxymethylated Meis1 sites were associated with a dynamic engagement of the 
5-methylcytosine hydroxylase Tet1. In addition, kinetic studies of cytosine 
hydroxymethylation of selected enhancers indicated that DNA hydroxymethylation 
is an early event of enhancer activation. Hence, acquisition of 5hmC in 
cell-specific distal regulatory regions may represent a major event of enhancer 
progression toward an active state and participate in selective activation of 
tissue-specific genes.