title: A conserved insulator that recruits CTCF and cohesin exists between the closely related but divergently regulated interleukin-3 and granulocyte-macrophage colony-stimulating factor genes.
abstract: 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.
authors.affiliation: Experimental Haematology, Leeds Institute of Molecular Medicine, University of Leeds, St. James's University Hospital, Leeds LS9 7TF, United Kingdom.
authors.forename: Sarion R
authors.initials: SR
authors.lastname: Bowers
authors.forename: Fabio
authors.initials: F
authors.lastname: Mirabella
authors.forename: Fernando J
authors.initials: FJ
authors.lastname: Calero-Nieto
authors.forename: Stephanie
authors.initials: S
authors.lastname: Valeaux
authors.forename: Suzana
authors.initials: S
authors.lastname: Hadjur
authors.forename: Euan W
authors.initials: EW
authors.lastname: Baxter
authors.forename: Matthias
authors.initials: M
authors.lastname: Merkenschlager
authors.forename: Peter N
authors.initials: PN
authors.lastname: Cockerill
mesh_terms: D000818:Animals; D020029:Base Pairing; D001483:Base Sequence; D001665:Binding Sites; D000076246:CCCTC-Binding Factor; D018797:Cell Cycle Proteins / Q000378:metabolism; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism; D017124:Conserved Sequence; D018983:DNA Footprinting; D019175:DNA Methylation; D004268:DNA-Binding Proteins / Q000378:metabolism*; D003851:Deoxyribonucleases / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D004742:Enhancer Elements, Genetic; D005786:Gene Expression Regulation; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics*; D006801:Humans; D038101:Insulator Elements / Q000235:genetics*; D007377:Interleukin-3 / Q000235:genetics*; D019169:Jurkat Cells; D051379:Mice; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D011485:Protein Binding; D012097:Repressor Proteins / Q000378:metabolism*; D000097722:Cohesins
chemical_list: D000076246:CCCTC-Binding Factor; D018797:Cell Cycle Proteins; D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins; D003851:Deoxyribonucleases; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D007377:Interleukin-3; D009687:Nuclear Proteins; D010750:Phosphoproteins; D012097:Repressor Proteins; D000097722:Cohesins; C000619697:CTCF protein, human; C000619698:Ctcf protein, mouse; C429022:RAD21 protein, human