In vivo assay for protein-protein interactions using Drosophila chromosomes.
The ability of a chimeric HP1-Polycomb (Pc) protein to bind both to heterochromatin and to euchromatic sites of Pc protein binding was exploited to detect stable protein-protein interactions in vivo. Previously, we showed that endogenous Pc protein was recruited to ectopic heterochromatic binding sites by the chimeric protein. Here, we examine the association of other Pc group (Pc-G) proteins. We show that Posterior sex combs (Psc) protein also is recruited to heterochromatin by the chimeric 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 endogenous Pc and chimeric HP1-Polycomb protein to euchromatin, but binding of HP1 and chimeric HP1-Polycomb protein to the heterochromatin is maintained. The E(z) mutation also results in the loss of chimera-dependent binding to heterochromatin by endogenous Pc and Psc proteins at the restrictive temperature, suggesting that interaction of these proteins 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 ectopic Pc-G complexes, but a truncated Su(z)2 protein is strongly recruited to all sites of chimeric protein binding. Trithorax protein is not recruited to the heterochromatin by the chimeric HP1-Polycomb protein, suggesting either that this protein does not interact directly with Pc-G complexes or that such interactions are regulated. Ectopic binding of chimeric chromosomal proteins provides a useful tool for distinguishing specific protein-protein interactions from specific protein-DNA interactions important for complex assembly in vivo.
Platero
J S
JS
Cell and Molecular Biology Program, Saint Louis University Health Science Center, 1402 S. Grand Blvd., St. Louis, MO 63104, USA.
Sharp
E J
EJ
Adler
P N
PN
Eissenberg
J C
JC
D000818:Animals; D000090266:Chromobox Protein Homolog 5; D002868:Chromosomal Proteins, Non-Histone / Q000737:chemistry*; D002875:Chromosomes / Q000235:genetics; D004330:Drosophila / Q000235:genetics*; D004347:Drug Interactions; D017344:Genes, Insect; D006570:Heterochromatin / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000502:physiology
D002868:Chromosomal Proteins, Non-Histone; D006570:Heterochromatin; D011993:Recombinant Fusion Proteins; D000090266:Chromobox Protein Homolog 5