Function and identification of a novel silencer in 5′ flanking distal region of human β globin gene

In order to inquire into the possible function of the sequence in the 5' flanking distal region and/or delta-beta intergene of human beta-globin gene, several recombinant retrovirus vectors containing beta-globin gene with different length of 5' flanking sequences were constructed and transfected into Hela cells. The levels of beta-globin gene expression determined quantitatively by Northern blotting and RT-cPCR revealed that there is a strong silencer activity in a DNA sequence(723 bp) between - 2282 bp and - 1559 bp upstream from the cap site of beta-globin gene. The silencer activity was further identified to be possibly located mainly on a 310 bp DNA restriction fragment by using the competitive gel retardation assay(cGRA), and many known regulatory elements were found present in it as well. Luciferase reporter gene expression system was adopted to study the function of this 310 bp fragment, and data showed that the fragment exhibited significant inhibitory effect on each of the recombinant vectors with alpha, beta or delta gene promoters. The above results suggest that this silencer may be involved in a potential regulatory network between delta and beta globin genes, and may play an important role in the regulation of globin gene expression.