Eukaryotes chromosomes are subdivided into functionally and structurally autonomous domains. While this was first suggested by cytological studies on the lampbrush chromosomes of amphibian oocytes and the polytene chromosomes of insects more than a half century ago, only little progress was made on this problem until 10-15 years ago. Since then a combination of genetic, molecular and biochemical experiments has provided convincing evidence for the domain organization of eukaryotic chromosomes. These studies have shown that the domain organization of the chromatin fiber is of critical importance not only for the packaging of chromosomes inside the nucleus, but also for the proper regulation of gene activity. The subdivision of eukaryotic chromosome into discrete domains requires a mechanism to separate one domain from another. Special elements called boundaries or insulators have been shown to serve this purpose. Elements that function as boundaries of chromatin domains were first identified in Drosophila, and they have now been found in a diverse array of organisms including from yeast to mammals. These elements delimit high order chromatin domains and function to establish independent units of gene activity, insulating genes or regulatory elements within a domain from the action of regulatory elements located outside in adjacent domains. The major aims of this proposal are to learn more about the biological functions of boundaries, to characterize the factors that confer insulator activity of several BX-C boundaries (including apparent stage and perhaps tissue specific factors) and to elucidate the mechanisms that enable boundaries to establish independent units of genetic activity.