Terminal differentiation of erythroid cells is associated with generalized heterochromatinization and gene repression, reflected in decreased nuclear size and visibly more compact chromatin structure as red cells mature prior to enucleation. In the midst of this repressive environment, however, high-level expression of a select group of genes - especially the ?- and ?-globins - must be established and maintained. The mechanisms by which this is accomplished represent a basic question with implications both for studies of tissue-specific gene regulation and for clinical gene therapies, in which transgene expression must similarly be maintained in differentiating cells. Abundant evidence has been presented to establish the role of covalent histone modifications, such as acetylation, in the process of gene activation. For the most part, histone hyperacetylation is confined to promoter-proximal regions, where it presumably contributes to chromatin decondensation, relaxation of histone-DNA interactions, and stabilization of transcription factor interactions. At certain genes, however, histone hyperacetylation extends across much broader regions. In erythroid cells, the ?- and ?-globin loci exhibit such """"""""domains"""""""" of hyperacetylation, and we have characterized several additional erythroid-specific gene loci that have them as well. Our results, along with studies of other gene loci, suggest that such domains are a crucial feature of their activation, and that they reflect the activity of distal enhancer elements required for high-level expression. As yet, however, the mechanisms by which such domains are formed, and the precise role they play in gene expression during terminal differentiation, are unknown. We propose to characterize cis-acting DNA sequences required for domain formation at the p-globin and other loci in erythroid cells, and eventually to identify the trans-acting factors that bind to these sequences and mediate domain formation. In this way, we will establish systems that can be used to elucidate the formation and function of hyperacetylated domains. Public Health Statement: This research, while highly basic in nature, is designed to elucidate mechanisms that underlie gene expression during terminal cellular differentiation. As such, it has relevance to disorders in which tissue-specific gene expression is disrupted - which include a number of cancers - as well as gene therapy approaches involving stable transgene expression in specific cell types. ? ? ?
