Cooperative Silencing of Chicken ρ- and β^H- Globin Gene Expression by Chicken β-Globin Regulatory Elements: Involvement of GATA-1, NF-E2, and RNA Polymerase II.

The chicken β-globin locus represents a well characterized, model system where the relationship between chromatin structure, transcription and DNA replication can be studied. The locus contains several regulatory elements including an intergenic enhancer as well as upstream regulatory elements that may function either alone or in combination with the intergenic enhancer as an LCR. The availability of the recombination proficient chicken B cell line DT40 has allowed the introduction of mutations into the endogenous chicken β-globin locus and phenotypic analysis after microcell mediated chromosome transfer into human erythroleukemia (K562) cells. Using this system, we have introduced deletions in the chicken β-globin intergenic enhancer as well as 5′ HS 1,2, and 3. A chicken chromosome marked by insertion of a selectable marker gene into the upstream folate receptor gene locus or 5′ HS1 followed by Cre recombinase mediated removal of the selectable marker gene served as control “wild type” chromosomes. Expression of the embryonic ρ and fetal β^H chicken globin genes were repressed by the intergenic enhancer, 5′ HS1, or 5′HS2. No ρ or β^H globin gene expression was detected in K562 cells containing control chicken chromosomes, while ρ and β^H mRNA were activated when the intergenic enhancer, 5′ HS1, or 5′HS2 were deleted. Transcriptional activation of the ρ and β^H genes correlated with hyperacetylation of histones H3 and H4, loss of methylated K9 histone H3, and binding of RNA polymerase II to the gene promoters. The status of methylated CpG dinucleotides at the promoters did not correlate with the transcriptional status of the genes. The phenotype of the 5′ HS1, and HS2 deletions were surprisingly similar to that of the intergenic enhancer, which suggested these elements may function either in the same pathway or complex. Chromatin immunoprecipitation (ChIP) experiments that assayed RNA polmerase II (pol II), GATA-1 and NF-E2 p45/ p18 binding at regulatory elements and gene promoters in targeted cell lines supported this hypothesis and suggested a potential role for 5′HS3 in gene activation. However, targeted deletion of 5′ HS3, unlike the other chicken β-globin regulatory elements, showed no transcriptional phenotype. Our results demonstrate the intergenic enhancer, 5′HS1, and 5′ HS2 function through a common silencing mechanism involving pol II, GATA-1, and NF-E2/P18.