We reported previously that well-characterized enhancers but not promoters for 
typical tissue-specific genes, including the classic Alb1 gene, contain 
unmethylated CpG dinucleotides and evidence of pioneer factor interactions in 
embryonic stem (ES) cells. These properties, which are distinct from the 
bivalent histone modification domains that characterize the promoters of genes 
involved in developmental decisions, raise the possibility that genes expressed 
only in differentiated cells may need to be marked at the pluripotent stage. 
Here, we demonstrate that the forkhead family member FoxD3 is essential for the 
unmethylated mark observed at the Alb1 enhancer in ES cells, with FoxA1 
replacing FoxD3 following differentiation into endoderm. Up-regulation of FoxD3 
and loss of CpG methylation at the Alb1 enhancer accompanied the reprogramming 
of mouse embryonic fibroblasts (MEFs) into induced pluripotent stem (iPS) cells. 
Studies of two genes expressed in specific hematopoietic lineages revealed that 
the establishment of enhancer marks in ES cells and iPS cells can be regulated 
both positively and negatively. Furthermore, the absence of a pre-established 
mark consistently resulted in resistance to transcriptional activation in the 
repressive chromatin environment that characterizes differentiated cells. These 
results support the hypothesis that pluripotency and successful reprogramming 
may be critically dependent on the marking of enhancers for many or all 
tissue-specific genes.