INTRONIC AND FLANKING SEQUENCES ARE REQUIRED TO SILENCE ENHANCEMENT OF AN EMBRYONIC BETA-TYPE GLOBIN GENE

In the course of studying regulatory elements that affect avian embryo nic rho-globin gene expression, the multipotential hematopoietic cell line K562 was transiently transfected with various rho-globin gene con structs containing or lacking an avian erythroid enhancer element. Enh anced levels of rho gene expression were seen from those constructs co ntaining an enhancer element and minimal 5' or 3' flanking rho sequenc es but were not seen from enhancer-containing constructs that included extensive 5' and 3' flanking sequences. Deletion analysis localized 5 ' and 3' ''enhancer-silencing elements'' to -2140 to -2000 and +1865 t o +2180 relative to the mRNA cap site. A third element required for en hancer silencing was identified within the second intron of the rho ge ne. The treatment of K562 cells with hemin, which induces erythroid di fferentiation, partially alleviated the enhancer-silencing effect. The silencer elements were able to block enhancement from a murine erythr oid enhancer, but not from a nonerythroid enhancer. Electrophoretic mo bility shift assays demonstrated that the transcription factor YY1 is able to bind both the 5' and 3' enhancer silencer elements; a point mu tation of the single overlapping YY1/NF-Y binding site in the 3' eleme nt completely abolished the enhancer-silencing effect. These results d emonstrate a complex enhancer silencer that requires 5' flanking, intr onic, and 3' flanking sequences for a single regulatory effect on a eu karyotic gene.