Specific chromatin structural changes are associated with the transcriptional activation of the adult Beta-globin gene in developing chick erythrocytes. These changes include the appearance of nuclease hypersensitive sites in the 5' promoter and 3' enhancer regions. Each region interacts with a complex of specific proteins whose composition appears to be tissue-specific and developmentally regulated. Erythrocyte extracts containing these proteins can confer nuclease hyersensitivity on histone- assembled Beta-globin genes in vitro. In this proposal, experiments are designed to study the function of these sequence-specific DNA binding proteins to further our understanding of Beta-globin gene activation. First, the proteins will be analyzed in an in vitro transcription system to test their effect on Beta-globin gene expression. Using partially purified factors and genes deleted in portions of the 5' and 3' control regions, it should be possible to identify those proteins required for transcription. Secondly, a similar analysis will be conducted on reconstituted Beta-globin genes to identify and factors that allow the gene to be transcribed when associated with histones. In this way, factors acting at the level of transcription can be distinguished from those that modify chromatin structure to facilitate transcription from histone-repressed genes. How the two types of factors function together to produce an active chromatin template is the focus of this proposal. The role of nuclease hypersensitivity in transcription will also be examined using histone-reconstituted templates. These studies should provide insight into how the binding of specific proteins can alter chromatin structure to activate a developmentally regulated gene. Such information is relevant to the processes involved in abnormal gene expression which is characteristic of many human diseases.