The genes POU5F1 and SOX2 are critical for pluripotency and reprogramming, yet 
the chromosomal organization around these genes remains poorly understood. We 
assayed long-range chromosomal interactions on putative enhancers of POU5F1 and 
SOX2 genes in human embryonic stem cells (hESCs) using 4C-Seq technique. We 
discovered that their frequent interacting regions mainly overlap with early DNA 
replication domains. The interactomes are associated with active histone marks 
and enriched with 5-hydroxymethylcytosine sites. In hESCs, genes within the 
interactomes have elevated expression. Additionally, some genes associated with 
the POU5F1 enhancer contribute to pluripotency. Binding sites for multiple DNA 
binding proteins, including ATF3, CTCF, GABPA, JUND, NANOG, RAD21 and YY1, are 
enriched in both interactomes. The RARG locus, frequently interacting with the 
POU5F1 locus, has abundant RAD21 binding sites co-localized with other protein 
binding sites. Thus the interactomes of these two pluripotency genes could be an 
important part of the regulatory network in hESCs.