Understanding how gene expression is controlled in a temporal and tissue specific manner is a basic problem in developmental biology. The individual members of the human globin family are temporally regulated so as to bring about the sequential production of embryonic, fetal, and adult hemoglobins during ontogeny. We are interested in the changes in chromatin structure that these genes undergo when they are activated during development. We have used the electrophoretic mobility shift assay to detect interactions between putative trans-acting regulatory factors present in the nuclei of cells actively transcribing globin genes and cis-acting sequences flanking the epsilon and gamma globin genes. We detected the formation of several complexes between K562 nuclear protein and a fragment of the human epsilon globin promoter. DNAse I footprinting and exonuclease protection assays suggested some of these interactions occurred at elements common to many regulated eukaryotic genes, i.e., at the CCAAT and ATA sequences and over the major transcription initiation site. One strong binding site for the general eukaryotic transcription factor Spl was observed in the epsilon-globin promoter. The proteins participating in these interactions could be partially resolved on DNA-agarose columns. however, none was erythroid specific. Strong binding sites for an erythroid specific protein present in K562 nuclear extracts were located in the 3' flanking regions of the epsilon-, gamma-, and beta-globin genes. The gamma- and beta-globin sites corresponded to regions which have been shown to possess enhancer activity for their respective genes. These sites are situated in regions of DNA which display tissue and developmental stage specific DNAse 1 hypersensitive sites when the genes are expressed. However, the factor is present in erythroid cells of different developmental stages. We will pursue studies aimed at understanding the structural and functional significance of the binding of this factor to DNA.
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