We have continued our studies of chromatin structure, in order to learn how DNA is packaged within eukaryotic nuclei, and in particular to learn how that structure is altered in the neighborhood of genes that are being expressed. We have studied the way in which chromatin containing expressed genes folds to form compact fibers, and we have shown that at high salt concentrations such chromatin is not capable of the full compaction that occurs in inactive chromatin. We have also continued our studies of the structure of chromatin in the neighborhood of the chicken adult Beta globin gene, isolated from the nuclei of erythrocytes in which the gene is expressed. We had earlier identified sites within the nuclease hypersensitive domain in the 5' flanking region of the active gene that are binding sites for DNA sequence-specific proteins which we had partially purified. We have now shown that there are at least 3 different proteins involved, each of which binds to a distinct portion of the 5' flanking region, and we have studied their appearance in red cells as a function of developmental stage. A similar analysis has been undertaken for one of the Alpha globin genes. In order to determine the biological function of these and other globin DNA sequences, we have developed a method for transfecting DNA into primary chicken erythrocytes at various stages of development. The method makes use of controlled, specific red cell lysis to obtain high levels of expression of transfected DNA. The method has led to the detection of a new regulatory region with the properties of an enhancer in the 3' flanking region of the Beta globin gene. The region with enhancer activity is the site of another hypersensitive domain we had previously identified. We have identified specific protein factors that bind to the region, and we have used footprinting methods to determine the binding sites precisely.
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