The globin gene family has been serving for a long time as a model for studying tissue-specific and developmental stage-specific regulation of expression. The K562 human erythroleukemia continuous cell line is a useful tool for examining globin gene expression as these cells express embryonic epsilon - and fetal gamma -, but not adult beta-globin genes. It has been shown that regulation of expression of globin gene family involves cis-acting DNA sequences in the vicinity of the transcription initiation site, and those play an important role in constitutive expression of the genes, as well as more upstream and downstream sequences which are thought to be necessary for tissue and developmental specific regulation. Although the role and the structure of different DNA sequences and DNA-binding proteins have been investigated very intensively, the molecular mechanisms governing developmental switching and tissue specific regulation of globin genes are still unknown. Our goals are to define functionally active DNA sequences in 5' flanking region of human epsilon- globin gene, to show the specific interaction of regulatory protein(s) with these regions, and to isolate and to characterize these specific DNAbinding protein(s). Functional analysis of regulatory role of epsilon-globin gene promoter region will be performed by deletion mutations using luciferase as a reporter gene. The significance of 5' flanking DNA sequences in transcriptional regulation will be further proven by site-directed mutagenesis. Experiments leading to the detection of protein interaction site(s) will include DNase footprinting and gel retardation assays. Finally, attempts will be undertaken to isolate and characterize regulatory protein(s) which specifically interact with DNA sequences of the 5' flanking epsilon-globin region.
