Figure 1.

Properties of the chicken β-globin 5′HS4 compound insulator element (IE). (A) Diagram of the locus, showing the position of the insulator relative to the globin gene cluster, the condensed chromatin region upstream of the cluster, and further upstream the erythroid-specific folate receptor gene. (B) Enhancer-blocking assay using a locus control region (LCR) element as an enhancer to drive a neomycin-resistance gene (Neo) in the presence of an ~250-bp element from 5′HS4 (INS) or a small noninsulating DNA fragment derived from λ bacteriophage. Increased insulator activity results in fewer neomycin-resistant colonies (Chung et al. 1993). (C) Barrier function assay using fluorescence-activated cell sorter (FACS) analysis to detect the expression levels of a fragment of the IL2 receptor. The fragment was expressed from a reporter construct carrying a globin-specific promoter and enhancer, stably transformed in each case into the avian erythroid cell line 6C2. (Left panel) Uninsulated reporter, (right panel) reporter surrounded by two copies on each side of the ~250-bp IE from 5′HS4 (Pikaart et al. 1998). (D) The five distinct protein-binding domains within the IE, the proteins that bind to them, and their functions. At FIV, the USF1/USF2 heterodimer binds to DNA and recruits a protein complex containing SET1 and another containing PRMT1 (Huang et al. 2007). Histone acetyltransferases are also present in each complex, and the complexes are probably not bound simultaneously. The overlaps between factors shown are not intended to signify physical contacts between them.