Globin genes are expressed exclusively in the erythroid lineage, and at distinct stages of development. The mechanisms by which the globin genes are regulated are of interest because a more detailed knowledge of the process is likely to contribute to therapies for severe hemoglobinopathies such as thalassemia and sickle cell disease, and because the globin genes are classic models of complex multigene regulation. Our long-term goal is to understand how the globin genes are expressed only in red blood cells, and how the individual genes are controlled through development. During erythroid differentiation, there is a progressive condensation of chromatin in the nucleus culminating in complete cessation of transcription; the globin genes continue to be transcribed until late in this process. We study a nuclear factor, GATA- 1, that appears to have a central role in the expression of globins and other typically erythroid genes in the terminal phase of erythroid differentiation at all stages of development. We have developed a model in which GATA-1 serves to keep globins and other erythroid genes in an active state while genes not essential for the erythroid program are repressed. To test this hypothesis we have developed an assay that uses the beta-geo reporter to distinguish effects on the level of gene expression from effects on epigenetic stability; previous studies of gene regulation have not separated these two parameters. We propose to investigate the interaction of erythroid transcriptional control elements with chromatin, using the beta-geo assay in conjunction with site- specific recombination to control for position effects. We will dissect the gamma-globin promoter, asking which elements within it regulate the level of gene expression and which counteract repression. We will continue our study of the 5'HS2 globin enhancer, in which we have shown that it acts primarily to relieve repression, and focus on the contribution of its component elements to its function. We will also analyze the role of the chromatin factor HMG-l in developmental regulation of the gamma-globin gene, and the expression of repressive chromatin proteins in the terminal phase. Ultimately we hope that these studies will lead to better understanding of developmental regulation of the globin genes and improved therapies for the hemoglobinopathies..
