In order to gain further insights of the regulatory mechanisms of human β-like globin gene switch during erythroid development, we have studied protein-DNA interaction in vivo at the human adult β and fetal γ globin promoters and their upstream enhancer, 5'HS-2, in purified human adult erythroblasts, in which the β, but not γ or ε, globin gene is actively transcribing. This genomic footprinting analysis of adult erythroblasts was carried out in conjunction with those of different non-erythroid human tissues, an embryonic/fetal erythroid cell line K562, and several non- erythroid human cell lines. Protein-DNA binding in the β globin promoter, in particular at the two CACC promoter boxes and the CCAAT box, is detectable only in the adult erythroblasts. As expected, the γ globin promoters were bound with specific nuclear factors in the expressing K562 cells, but not in non-erythroid tissues or cell lines. Relatively weak protein binding could also be detected in the vicinities of the two CCAAT boxes of the inactive γ 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 undetected factor-binding motif of 5'HS-2 that is protected only in the adult erythroblasts. This motif is identical in sequence to the 3'-CACC box of the human β globin promoter, and it is well conserved at the same location among all mammalian 5'HS-2 enhancers, suggesting an important regulatory role of this element in human β globin gene transcription in adult erythroblasts. All of the above four motifs of 5'HS-2 are free of nuclear factor binding in non-erythroid tissues, but two of them, NF-E2/AP1 and GT-I, are bound with factors in some non-erythroid cell lines but not in others. The functional implications of these genomic footprinting data and the tissue-specific CpG methylation patterns of the β- like globin promoters we obtained by genomic sequencing are discussed in terms of positive and negative regulation of the human β-like globin switch during erythroid development.
|Number of pages||9|
|Journal||Journal of Biological Chemistry|
|Publication status||Published - 1994|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology