Platero J S, Sharp E J, Adler P N, Eissenberg J C
Cell and Molecular Biology Program, Saint Louis University Health Science Center, 1402 S. Grand Blvd., St. Louis, MO 63104, USA.
Chromosoma. 1996 Mar;104(6):393-404. doi: 10.1007/BF00352263.
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.
嵌合的HP1-多梳（Pc）蛋白既能结合异染色质，又能结合Pc蛋白结合的常染色质位点，利用这一特性来检测体内稳定的蛋白质-蛋白质相互作用。此前，我们发现内源性Pc蛋白可被嵌合蛋白招募至异位异染色质结合位点。在此，我们研究其他多梳组（Pc-G）蛋白的关联情况。我们发现后胸梳（Psc）蛋白也能被嵌合蛋白招募至异染色质，这表明Psc蛋白参与了与Pc蛋白或Pc相关蛋白的直接蛋白质-蛋白质相互作用。在携带zeste增强子[E(z)]温度敏感等位基因的果蝇中，在限制温度下发生的多线染色体普遍解聚与内源性Pc和嵌合的HP1-多梳蛋白与常染色质的结合丧失有关，但HP1和嵌合的HP1-多梳蛋白与异染色质的结合得以维持。E(z)突变还导致在限制温度下内源性Pc和Psc蛋白通过嵌合体依赖的方式与异染色质的结合丧失，这表明这些蛋白的相互作用是由E(z)蛋白介导的。带有myc标签的全长zeste抑制因子2 [Su(z)2]蛋白与异位Pc-G复合物的相互作用较弱或根本不相互作用，但截短的Su(z)2蛋白能被强烈招募至嵌合蛋白结合的所有位点。三体胸蛋白不会被嵌合的HP1-多梳蛋白招募至异染色质，这表明该蛋白要么不直接与Pc-G复合物相互作用，要么这种相互作用受到调控。嵌合染色体蛋白的异位结合为区分对体内复合物组装重要的特定蛋白质-蛋白质相互作用与特定蛋白质-DNA相互作用提供了一个有用的工具。
