We are interested in how the temporal and tissue specific expression of a gene is brought about during development. The orderly activation and repression of individual members of the human globin gene family is a prominent example of this phenomenon. We are studying the changes in chromatin structure that these genes undergo when they are activated. We have used DNAse I footprinting and the electrophoretic mobility shift assay to detect, in vitro, interactions between nuclear proteins from erythroid and non-erythroid cells and DNA sequences from regulatory regions both 5' and 3' to the human epsilon-globin gene. At least one of the interactions we observed was erythroid specific and corresponded to binding of EryF1/NF-E1/GF-1 to two sites in the promoter region. This protein also has strong binding sites in the region 3' to the gene. Other interactions are in the process of being analyzed. To investigate the functional significance of these protein-DNA interactions transient assays in K562 cells and HeLa cells are being employed in which transcription from the epsilon-globin promoter is monitored via a chloramphenicol acetyltransferase reporter gene. An episomal vector system is being developed in order to study in vivo the effect of these protein DNA interactions on higher order chromatin structure and transcription of the epsilon-globin gene. Preliminary studies suggest that an EB virus-based vector can be maintained as an amplified episome in K562 cells. A marked epsilon-globin gene is being inserted into the vector and its regulation will be studied.
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