This proposal addresses the mechanism for assembling the specialized chromatin found at the centromeres and telomeres of eukaryotic chromosomes. Because this chromatin differs from most in not becoming decondensed during interphase, the term, heterochromatin has been used to describe it. The roles of heterochromatin are multi-faceted and range from structural functions in chromosome mechanics and nuclear architecture to gene regulation. Abnormalities in chromosome segregation and gene regulation are often the root causes of human genetic disease. The array of functions for heterochromatin in the nucleus make it pertinent both to understanding and treating these diseases. This proposal describes experiments we are using to dissect the process of heterochromatin assembly in Drosophila. Through biochemistry and cytogenetics, we have uncovered a role for the origin recognition complex (ORC) in recruiting a highly conserved heterochromatin-associated protein (HP1) to heterochromatin. We have also identified a novel protein that is a component of a putative HP1 recruitment complex containing HP1 and ORC protein (HP1/ORC-associated protein, HOAP). Preliminary biochemical and genetic data indicate that it is also required for heterochromatin assembly. The function of HOAP in this process will be further examined. We are using chromatin immunoprecipitations to retrieve the binding sequences for ORC and HOAP in heterochromatin to which HP1 is being recruited. The authenticity of these sequences as binding sites for these proteins will be tested by in situ hybridizations to mitotic chromosomes coupled with immunostaining. Studies to characterize differentially phosphorylated populations of HP1 in interphase nuclei are also planned. One of these populations is specifically associated with ORC and HOAP and has been proposed to have a specific function in nucleating heterochromatin assembly, whereas the other populations may be extended from these hypothetical nucleation sites. We will be preparing immunospecific reagents and mutant HP1 transgenes to address the functions of these populations.
