POINT MUTATIONS IN THE WD40 DOMAIN OF EED BLOCK ITS INTERACTION WITH EZH2

The Polycomb group proteins are involved in maintenance of the silence d state of several developmentally regulated genes. These proteins for m 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 fr agment of the mammalian homolog of the Drosphila Enhancer of zeste [E( z)] protein, Ezh2. The full-length Exh2 bound strongly to Eed in vitro , and Eed coimmunoprecipitated with Ezh2 from murine 70Z/3 cell extrac ts, confirming the interaction between these proteins observed in yeas t, Mutations T1031A and T1040C in one of the WD40 repeats of Eed, whic h account for the hypomorphic and lethal phenotype of eed in mouse dev elopment, blocked binding of Ezh2 to Eed in a two-hybrid interaction i n yeast and in mammalian cells. These mutations also blocked the inter action between these proteins in vitro. In mammalian cells, the Gal4-E ed fusion protein represses the activity of a promoter bearing Gal4 DN A 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. fed and Ezh2 were also found to bind R NA in vitro, and RNA altered the interaction between these proteins. T hese findings suggest that Polycomb group proteins Eed and Ezh2 functi onally interact in mammalian cells, an interaction that is mediated by the WD40-containing domain of Eed protein.