Targeted deletion of the chicken beta-globin regulatory elements reveals a cooperative gene silencing activity

The chicken beta-globin locus represents a well characterized system to study the role of both proximal and distal regulatory elements in a eukaryotic multigene domain. The function of the chicken beta(A)/epsilon-intergenic enhancer and upstream regulatory elements 5'-HS1 and 5'-HS2 were studied using a gene targeting approach in chicken DT40 cells followed by microcell-mediated chromosome transfer into human erythroleukemia cells (K562). These regulatory elements all repressed expression of the rho- and beta(H)-chicken globin genes in the chromosome transfer assay. No rho- or beta(H)-globin gene expression was detected in K562 cells containing the chicken chromosome without deletions, whereas rho- and beta(H)-mRNA was activated in K562 cells containing chicken chromosomes with deletions of the intergenic enhancers, 5'-HS1 and 5'-HS2. Transcriptional activation of the rho- and beta(H)-globin genes correlated with hyperacetylation of histones H3 and H4, loss of histone H3 lysine 9 methylation, and binding of RNA polymerase II to the gene promoters. Surprisingly, the status of CpG dinucleotide methylation at the promoters did not correlate with the transcriptional status of the genes. Our results using a chromosomal transfer assay demonstrate an identical silencing function for these regulatory elements, which suggests they function as part of a common silencing pathway or complex.