Abstract
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.
Highlights
The chicken -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 A/⑀-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)
The availability of the recombination-proficient chicken B cell line DT40 has allowed the development of a system where mutations in the regulatory elements can be introduced into the endogenous chicken -globin locus, and the erythroid phenotype can be analyzed after microcell-mediated chromosome transfer into human erythroleukemia cells
Summary
The chicken -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 A/⑀-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). The insulator element is located at the 5Ј-boundaries of DNase I sensitivity and histone acetylation (7) The functions of these regulatory elements have been studied in transient and stable transfection assays (14 –16) and in transgenic mice (3, 5, 17) but not in their endogenous chromosomal location like the human and mouse -globin elements (18). Chicken B cell lines such as DT40 cells have been described to be able to undergo homologous recombination at high frequency They have been used to study both the functions of genes and regulatory elements (19, 20). Our results demonstrate that these regulatory elements possess identical gene silencing activity
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