GENOMIC FOOTPRINTING AND SEQUENCING OF HUMAN BETA-GLOBIN LOCUS - TISSUE-SPECIFICITY AND CELL-LINE ARTIFACT

In order to gain further insights of the regulatory mechanisms of huma n beta-like globin gene switch during erythroid development, we have s tudied protein-DNA interaction in vivo at the human adult beta and fet al gamma globin promoters and their upstream enhancer, 5'HS-2, in puri fied human adult erythroblasts, in which the beta, but not gamma or ep silon, globin gene is actively transcribing. This genomic footprinting analysis of adult erythroblasts was carried out in conjunction with t hose of different non-erythroid human tissues, an embryonic/fetal eryt hroid cell line K562, and several non-erythroid human cell lines. Prot ein-DNA binding in the beta globin promoter, in particular at the two CACC promoter boxes and the CCAAT box, is detectable only in the adult erythroblasts. As expected, the gamma globin promoters were bound wit h specific nuclear factors in the expressing K562 cells, but not in no n-erythroid tissues or cell lines. Relatively weak protein binding cou ld also be detected in the vicinities of the two CCAAT boxes of the in active gamma globin promoters in the adult erythroblasts. Although the patterns of nuclear factor-DNA interaction in vivo at the NF-E2/AP1, GATA-1, and GT-I motifs of 5'HS-2 enhancer in adult erythroblasts are similar to those in K562 cells, we have identified a previously undete cted factor-binding motif of 5'HS-2 that is protected only in the adul t erythroblasts. This motif is identical in sequence to the 3'-CACC bo x of the human beta globin promoter, and it is well conserved at the s ame location among all mammalian 5'HS-2 enhancers, suggesting an impor tant regulatory role of this element in human beta globin gene transcr iption in adult erythroblasts. All of the above four motifs of 5'HS-2 are free of nuclear factor tor binding in non-erythroid tissues, but t wo of them, NF-ES/AP1 and GT-I, are bound with factors in some non-ery throid cell lines but not in others. The functional implications of th ese genomic footprinting data and the tissue-specific CpG methylation patterns of the beta-like globin promoters we obtained by genomic sequ encing are discussed in terms of positive and negative regulation of t he human beta-like globin switch during erythroid development.