Point mutations in the WD40 domain of Eed block its interaction with Ezh2.
The Polycomb group proteins are involved in maintenance of the silenced state of several developmentally regulated genes. These proteins form large aggregates with different subunit compositions. To explore the nature of these complexes and their function, we used the full-length Eed (embryonic ectoderm development) protein, a mammalian homolog of the Drosophila Polycomb group protein Esc, as a bait in the yeast two-hybrid screen. Several strongly interacting cDNA clones were isolated. The cloned cDNAs all encoded the 150- to 200-amino-acid N-terminal fragment of the mammalian homolog of the Drosophila Enhancer of zeste [E(z)] protein, Ezh2. The full-length Ezh2 bound strongly to Eed in vitro, and Eed coimmunoprecipitated with Ezh2 from murine 70Z/3 cell extracts, confirming the interaction between these proteins observed in yeast. Mutations T1031A and T1040C in one of the WD40 repeats of Eed, which account for the hypomorphic and lethal phenotype of eed in mouse development, blocked binding of Ezh2 to Eed in a two-hybrid interaction in yeast and in mammalian cells. These mutations also blocked the interaction between these proteins in vitro. In mammalian cells, the Gal4-Eed fusion protein represses the activity of a promoter bearing Gal4 DNA elements. The N-terminal fragment of the Ezh2 protein abolished the transcriptional repressor activity of Gal4-Eed protein when they were coexpressed in mammalian cells. Eed and Ezh2 were also found to bind RNA in vitro, and RNA altered the interaction between these proteins. These findings suggest that Polycomb group proteins Eed and Ezh2 functionally interact in mammalian cells, an interaction that is mediated by the WD40-containing domain of Eed protein.
Denisenko
O
O
Department of Medicine, University of Washington, Seattle, Washington 98195, USA.
Shnyreva
M
M
Suzuki
H
H
Bomsztyk
K
K
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002460:Cell Line; D029721:Drosophila Proteins; D000071221:Enhancer of Zeste Homolog 2 Protein; D015870:Gene Expression; D006801:Humans; D019169:Jurkat Cells; D051379:Mice; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D009693:Nucleic Acid Hybridization; D017354:Point Mutation; D063151:Polycomb Repressive Complex 2 / Q000235:genetics* / Q000378:metabolism*; D012313:RNA; D051381:Rats; D012097:Repressor Proteins / Q000235:genetics* / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D015003:Yeasts
D029721:Drosophila Proteins; C491488:EED protein, human; C107275:Eed protein, mouse; D009687:Nuclear Proteins; D012097:Repressor Proteins; D012313:RNA; C083329:E(z) protein, Drosophila; C578253:EZH2 protein, rat; D000071221:Enhancer of Zeste Homolog 2 Protein; D063151:Polycomb Repressive Complex 2