The overall goal of this program is to understand the domain structure of heterochromatin and the mechanism underlying the function of heterochromatin boundary elements in the model organism Saccharomyces cerevisiae. In S. cerevisiae, transcriptionally silent chromatin at the HML and HMR loci is the yeast equivalent of metazoan heterochromatin, and is established by cis-acting silencers. To address how heterochromatin is restricted to limited regions, the domain structure of the silent HML locus will be defined by examining the profile of repression of a reporter gene inserted in positions within and around HML. Moreover, the mechanism underlying why certain silencers establish silent chromatin only in one direction but others function in both directions will be examined by testing if this is determined by the sequence features of the silencer or its genomic context. To understand the molecular mechanism of chromatin boundary elements that delimit chromosome domains, the function of yeast heterochromatin boundary elements that can block the spread of silent chromatin will be extensively studied. This will be achieved by examining the functions of factors involved in the boundary activity and testing models for boundary activity. The possible functional conservation of boundary elements between yeast and higher eukaryotes will also be investigated by examining if yeast boundary elements function in higher cells and vice versa. These studies should shed light on the mechanism of heterochromatin related gene regulation in higher eukaryotes. One such regulation is chromosomal imprinting in mammals that plays an important role in embryonic development and underlies the variable expression of a variety of human disorders and sporadic cancers. Chromatin boundary elements can be very useful in a variety of practical applications including shielding reporter genes against the repressive effect of heterochromatin in cells and transgenic animals, as well as the construction of viral vectors for gene therapy.