IN-VIVO ASSAY FOR PROTEIN-PROTEIN INTERACTIONS USING DROSOPHILA CHROMOSOMES

The ability of a chimeric HP1-Polycomb (Pc) protein to bind both to he terochromatin and to euchromatic sites of Pc protein binding was explo ited to detect stable protein-protein interactions in vivo. Previously , we showed that endogenous Pc protein was recruited to ectopic hetero chromatic binding sites by the chimeric protein. Here, we examine the association of other Pc group (Pc-G) proteins. We show that Posterior sex combs (Pac) protein also is recruited to heterochromatin by the ch imeric protein, demonstrating that Psc protein participates in direct protein-protein interaction with Pc protein or Pc-associated protein. In flies carrying temperature-sensitive alleles of Enhancer of zeste[E (z)] the general decondensation of polytene chromosomes that occurs at the restrictive temperature is associated with loss of binding of end ogenous Pc and chimeric HP1-Polycomb protein to euchromatin, but bindi ng of HP1 and chimeric HP1-Polycomb protein to the heterochromatin is maintained. The E(z) mutation also results in the loss of chimera-depe ndent binding to heterochromatin by endogenous Pc and Psc proteins at the restrictive temperature, suggesting that interaction of these prot eins is mediated by E(z) protein. A myc-tagged full-length Suppressor 2 of zeste [Su(z)2] protein interacts poorly or not at all with ectopi c Pc-C complexes, but a truncated Su(z)2 protein is strongly recruited to all sites of chimeric protein binding. Trithorax protein is not re cruited to the heterochromatin by the chimeric HP1-Polycomb protein, s uggesting either that this protein does not interact directly with Pc- G complexes or that such interactions are regulated. Ectopic binding o f chimeric chromosomal proteins provides a useful tool for distinguish ing specific protein-protein interactions from specific protein-DNA in teractions important for complex assembly in vivo.