CPG ISLANDS FROM THE ALPHA-GLOBIN GENE-CLUSTER INCREASE GENE-EXPRESSION IN AN INTEGRATION-DEPENDENT MANNER

In contrast to other globin genes, the human and rabbit a globin genes are expressed in transfected erythroid and nonerythroid cells in the absence of an enhancer, This enhancer-independent expression of the al pha-globin gene requires extensive sequences not only from the 5' flan king sequence but also from the intragenic region, However, the featur es of these internal sequences that are responsible for their positive effect are unclear, We tested several possible determinants of this a ctivity, One possibility is that a previously identified array of disc rete binding sites for known and potential regulatory proteins within the alpha-globin gene comprise an intragenic enhancer specific for the alpha-globin promoter, but directed rearrangements of the sequences s how that this is not the case, Alternatively, the promoter may extend into the gene, with the function of the discrete binding sites being d ependent on maintenance of their proper positions and orientations rel ative to the 5' flanking sequence, However, the positive effects obser ved in gene fusions do not localize to a discrete region of the alpha- globin gene and the results of internal deletions and point mutations argue against a required role of the targeted discrete binding sites. A third possibility is that the CpG island, which includes both the 5' flanking and intragenic regions associated with the positive activity , may itself have a more general effect on expression in transfected c ells, Indeed, we show that the size of the CpG island in constructs co rrelates with the level of gene expression, Furthermore, the alpha-glo bin promoter is more active in the contest of a previously inactive Cp G island than in an A+T-rich context, showing that the CpG island prov ides an environment more permissive for expression, These effects are seen only after integration, suggesting a possible mechanism at the le vel of chromatin structure.