Non-erythroid genes inserted on either side of human HS-40 impair the activation of its natural alpha-globin gene targets without being themselves preferentially activated

The human cu-globin gene complex includes three functional globin genes (5' -zeta 2-alpha 2-alpha 1-3') regulated by a common positive regulatory eleme nt named HS-40 displaying strong erythroid-specific enhancer activity. How this enhancer activity can be shared between different promoters present at different positions in the same complex is poorly understood. To address t his question, we used homologous recombination to target the insertion of m arker genes driven by cytomegalovirus or long terminal repeat promoters in both possible orientations either upstream or downstream from the HS-40 reg ion into the single human alpha-globin gene locus present in hybrid mouse e rythroleukemia cells. We also used CRE recombinase-mediated cassette exchan ge to target the insertion of a tagged alpha-globin gene at the same positi on downstream from HS-40. All these insertions led to a similar decrease in the HS-40-dependent transcription of downstream human alpha-globin genes i n differentiated cells. Interestingly, this decrease is associated with the strong activation of the proximal newly inserted alpha-globin gene, wherea s in marked contrast, the transcription of the non-erythroid marker genes r emains insensitive to HS-40. Taken together, these results indicate that th e enhancer activity of HS-40 can be trapped by non-erythroid promoters in b oth upstream and downstream directions without necessarily leading to their own activation.