The human interleukin-3 (IL-3) and granulocyte-macrophage 
colony-stimulating-factor (GM-CSF, or CSF2) gene cluster arose by duplication of 
an ancestral gene. Although just 10 kb apart and responsive to the same signals, 
the IL-3 and GM-CSF genes are nevertheless regulated independently by separate, 
tissue-specific enhancers. To understand the differential regulation of the IL-3 
and GM-CSF genes we have investigated a cluster of three ubiquitous DNase 
I-hypersensitive sites (DHSs) located between the two genes. We found that each 
site contains a conserved CTCF consensus sequence, binds CTCF, and recruits the 
cohesin subunit Rad21 in vivo. The positioning of these sites relative to the 
IL-3 and GM-CSF genes and their respective enhancers is conserved between human 
and mouse, suggesting a functional role in the organization of the locus. We 
found that these sites effectively block functional interactions between the 
GM-CSF enhancer and either the IL-3 or the GM-CSF promoter in reporter gene 
assays. These data argue that the regulation of the IL-3 and the GM-CSF 
promoters depends on the positions of their enhancers relative to the conserved 
CTCF/cohesin-binding sites. We suggest that one important role of these sites is 
to enable the independent regulation of the IL-3 and GM-CSF genes.