FUNCTION OF BETA GLOBIN DNA BINDING PROTEINS

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DESCRIPTION (Adapted from Applicant's Abstract): Transcriptional regulation of the chick beta-globin gene cluster requires long-range interaction between distal DNA sequences to tissue-specifically activate the chromosomal locus and to differentially regulate individual genes using erythroid development. One control element that functions in both of these capacities is the beta-epsilon enhancer. This region residues between the adult beta(A)-and embryonic epsilon globin genes and separately activates each promoter at distinct times in red cell maturation. In addition, the beta-epsilon enhancer also has Locus Control Region (LCR) activity since it is required to establish an active chromosomal domain when linked to the beta(A)-globin gene or gene cluster. In view of the widespread importance of tissue-specific chromosomal domain activation and enhancer-dependent transcriptional regulation in many different gene system, the beta-epsilon enhancer represents an excellent model in which to analyze long-range DNA interactions. The investigator previously demonstrated that betaA-globin transcription is completely dependent upon the 3' beta-epsilon enhancer, acting at a distance of 2 kb, using genes reconstituted into chromatin or synthetic nuclei in the presence of erythroid proteins. In this proposal, the investigator wishes to continue the studies of tissue- specific enhancer function by conducting a detailed examination of the mechanism by which it occurs. The approach is to use in vitro transcription and chromatin reconstitution systems to reproduce critical aspects of enhancer-regulated betaA-globin gene expression. First, experiments are designed to define the DNA structure required to confer beta- epsilon enhancer activity in vitro. Specifically, whether nucleosome assembly or DNA architectural proteins are necessary to generate this topology. Second, the minimum transcription proteins required for enhancer-dependent betaA-globin expression will be identified by using purified or recombinant erythroid DNA binding proteins and general initiation factors as well as unknown proteins obtained by chromatography of erythroid extracts. Third, the ability of enhancers to either activate or depress promoters will be examined by a combination of biochemical and structural studies. These include order- of-addition transcription experiments using purified components and plasmid footprinting of enhancer-dependent and -independent betaA- globin genes. Fourth, the investigator will examine whether enhancer regulation involves DNA looping or protein tracking using electron microscopy and "protein blockage" in vitro transcription experiments. Finally, promoter switching by the beta- epsilon enhancer will be examined in DNA templates containing linked betaA and epsilon globin genes. The analysis of betaA-globin enhancer function in this in vitro system should provide useful information that is applicable not only to other tissue-specific genes but to long-range genetic effects that are particularly relevant to health related issues. These include the demonstrated, but poorly understood, role of enhancers in controlling immunoglobulin and T cells receptor gene rearrangement and the observation that in a variety of lymphomas and leukemias, oncogene deregulation is associated with an aberrant translocation into the vicinity of active enhancers.