MECHANISMS OF ERYTHROID GENE EXPRESSION

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The broad long-term objective of this proposal is to elucidate the mechanisms of transcriptional regulation of globins and other red blood cell-specific genes. A better understanding of these processes is likely to suggest means of manipulating globin regulation, which would be of use in the treatment of hemoglobinopathies such as sickle cell disease and beta-thalassemia. The specific aims are to analyze the three key DNA elements (here termed GATA, CACC and AP-1/NFE-2) responsible for globin gene regulation. In the case of the GATA element, this will involve study of its role in promoter function; AP-1/NFE-2 elements will be studied in order to better comprehend the multiple transcription factors that may act on them. We will construct transgenic mice carrying minimal promoters consisting of combinations of GATA, CACC, and AP-1/NFE-2 driving expression of beta- galactosidase to test effects of these elements on tissue and developmental stage specificity. These studies may have implications for the design of gene therapy vectors. In addition, we will study the regulation of GATA cis-elements in transiently and stably transfected erythroid cell lines in order to discover whether GATA-1 increases the transcriptional activity of the promoter or rather acts stochastically to increase the likelihood of being active without affecting its level of activity. Recent work from this and other laboratories suggests that GATA elements (and presumable GATA-1) function to make erythroid promoters and enhancers more accessible to other factors rather than activating them directly. Finally, the complex regulation of AP-1/NFE-2 elements will be explored. While NFE-2 has been shown to bind these elements and be necessary for their activation, this factor (unlike AP-1) does not bind all AP-1/NFE-2 elements. The role of AP-1 in the regulation of AP-1/NFE-2 will be studied by assessing the composition of AP-1 in erythroid cells and manipulating that composition. In addition, since AP-1/NFE-2 elements resemble nuclear hormone response elements, these elements will be studied for regulation by members of the steroid hormone receptor family.