Transcriptional regulation of the beta-globin gene cluster is dependent upon distal sequences that may activate the nucleosomal structure of very large chromosomal domains. These sequences, termed distal control regions (DCR), function in a highly tissue-specific manner and can confer erythroid-specificity upon genes not normally expressed in this cell type. Once the chromosomal domain is activated, individual globin genes are differentially transcribed during the course of erythroid development. The regulation of individual genes within the activated domain depends upon multiple proteins that act locally to affect expression from gene-specific promoters and enhancers. The applicants have previously analyzed the developmental regulation of the beta-globin promoter by determining how proteins that bind to this control region alter nucleosomal structure and activate transcription. In this proposal, the applicants wish to continue the studies on how the beta-globin gene is developmentally regulated by analyzing the mechanism of action of two distal control elements, the DCR and the 3' enhancer. First, experiments are designed to examine enhancer function by studying an erythroid-specific factor that binds to both the promoter and enhancer and which may mediate interaction between these two elements. The effect of this factor on enhancer activity will be analyzed by in vitro transcription using nucleosome-reconstituted beta-globin gene templates since the enhancer is not required on free DNA. Second, the mechanism of DCR action will be assessed by monitoring its effects on beta-globin expression and chromatin structure using both free DNA and nucleosome reconstitutes in in vitro assays. Third, the function of the enhancer and the DCR will be examined in the context of the entire beta-globin gene cluster using a cosmid DNA clone. The use of large chromosomal regions could reveal aspects of long-range transcriptional regulation that may not be observable with isolated genes in vitro. Finally, cosmids containing the beta-globin cluster will be reconstituted into synthetic nuclei with Xenopus egg extracts. Reconstitution in the presence of erythroid proteins from different stages of embryogenesis may enable one to approximate the nuclear structure of the beta-globin cluster as it exists during specific times in erythroid development. The transcriptional capacity of different globin genes within these synthetic organelles will then be examined in vitro to analyze the mechanisms involved in hemoglobin gene switching. In this way, the applicants hope to begin to reproduce the erythroid-specific regulation of a family of genes within a large chromosomal domain and analyze the function of distal control regions and other parameters of nuclear organization in this process. If successful, this experimental approach will have wide-spread application for other genes and may be applied to the study of any disease that results from abnormal gene expression at the level of transcription.
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