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. 1998 Oct;18(10):5634-42.
doi: 10.1128/MCB.18.10.5634.

Point mutations in the WD40 domain of Eed block its interaction with Ezh2

O Denisenko  1 , M Shnyreva, H Suzuki, K Bomsztyk
Affiliations

Point mutations in the WD40 domain of Eed block its interaction with Ezh2

O Denisenko et al. Mol Cell Biol. 1998 Oct.

Abstract

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.

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Figures

FIG. 1
FIG. 1
Screening of cDNA libraries using Eed as a bait in yeast two-hybrid system. (A) cDNA libraries from mouse embryos 9.5 to 10.5 days postcoitum or Jurkat cells were screened in yeast against the LexA-Eed bait construct as described in Materials and Methods. The cDNA plasmids from primary positive clones were purified and transformed into yeast strains carrying either LexA-Eed wild-type or LexA-T1040C mutant Eed bait. Three colonies from each transformation were grown on plate with selective media (Leu−, Trp−) and then transferred onto nitrocellulose filter and assayed for β-galactosidase activity. The blue color was developed for 3 h at 30°C. (B) The transformants were also checked for expression of the bait constructs. Cells transformed with pBT116 (Vector), LexA-Eed, and LexA-T1040C plasmids were grown overnight in selective medium, lysed in SDS sample buffer, electrophoresed, transferred onto a polyvinylidene difluoride membrane, and stained with anti-LexA rabbit serum followed by anti-rabbit immunoglobulin G-alkaline phosphatase conjugate. A membrane stained with 5-bromo-4-chloro-3-indolylphosphate/nitroblue tetrazolium phosphatase substrate is shown.
FIG. 2
FIG. 2
The Eed-interacting clones encode the N-terminal part of Ezh2 protein, a mammalian homolog of the Drosophila E(z) protein. The cDNA inserts from the positive clones were sequenced, and a search for sequence similarity in a database was carried out. All of the clones matched the N-terminal part of the Ezh2 protein. The alignment of the shortest clone with different members of the E(z) protein family is shown. DmE(z), D. melanogaster E(z) protein (20); mEnx1, mouse Enx1 protein (16); HsEzh2, human Ezh2 protein (6); HsEzh1, human Ezh1 protein (1). Only N-terminal parts of the proteins are shown. Identical positions are shaded.
FIG. 3
FIG. 3
Northern blot analysis reveals that the ezh2 and eed genes are coexpressed in cell lines and mouse tissues. Portions (10 μg) of total RNA from different sources, as indicated, were run in a 1.2% agarose gel containing 2.2 M formaldehyde, transferred onto a nylon membrane, and probed with either ezh2 (A and B) or eed (B) 32P-labeled probes. After hybridization, the membranes were washed and exposed to X-ray films. Before the hybridization procedure, the membranes were stained for RNA with methylene blue (A, lower panel) as a control for the amounts of RNA. 28S indicates 28S rRNA. The membrane in panel B was boiled in water–0.1% SDS for 10 min after use of the first probe and then rehybridized with the second probe. The positions of RNA size markers are shown on the left. The positions of bands corresponding to ezh2 and eed transcripts are indicated by arrows.
FIG. 4
FIG. 4
Ezh2 binds Eed in vitro. Ezh2 or Eed mRNAs were translated in a rabbit reticulocyte cell-free system in the presence of [35S]methionine. The translational products were incubated (1 h at 4°C) with glutathione-agarose beads bearing either the wild-type GST-Eed or GST-Eed mutants I287N (T1031A) and L290P (T1040C) (A), GST-Ezh2 (aa 1 to 192) (C and D) or GST-K (D). After binding, the beads were washed and boiled in SDS buffer, and eluted proteins were analyzed by SDS-PAGE and autoradiography. The Coomassie blue-stained gel of the experiment in panel A, lanes 1 to 3, is shown in panel B. (C) Eed and T1040C translational products were bound to GST-Ezh2 beads. Lanes 5 and 6 display the Coomassie blue-stained gel corresponding to lanes 3 and 4. (D) Eed, EedΔN, and EedΔC translational products were mixed and bound to either GST-Ezh2 or GST-K beads. (E) Eed constructs used in the experiments. Open box, GST (A) or His/T7 tag (pET28 vector) (C and D); shaded box, N-terminal part of Eed; solid box, C-terminal propeller domain of Eed.
FIG. 5
FIG. 5
Binding of Eed to Ezh2 in vitro is modulated by RNA. The in vitro binding assay was performed as described in the legend to Fig. 4. (A) The 35S-Eed translational product was bound to either GST-Ezh2 or GST-K beads in the presence of 20 μg of poly(G) (lane 3), poly(C) (lane 4), poly(U) (lane 5), or poly(A) (lane 6) per ml. −, no addition (lane 2). The Coomassie blue-stained gels are shown in the panels below the autoradiograms. Eed, position of 35S-Eed in the gel; GST-Ezh2 and GST-K, positions of the proteins in the Coomassie blue-stained gel. (B) Binding of 35S-Eed, 35S-K, and 35S-Ezh2 to different homopolyribonucleotides. The Eed, hnRNP-K, and Ezh2 translational products were incubated (1 h at 4°C) with agarose beads with covalently attached poly(G) (lane 2), poly(C) (lane 3), poly(U) (lane 4), or poly(A) (lane 5). After binding, the beads were washed and boiled in SDS buffer, and eluted proteins were analyzed by SDS-PAGE and autoradiography.
FIG. 6
FIG. 6
Eed and Ezh2 coprecipitate from 70Z/3 cell extracts. A 5-μl volume of 35S-labeled Ezh2 (A) or 100 μl of 70Z/3 cell extract (B) was incubated with 3 μl of anti-Ezh2 rabbit serum in a final volume of 1 ml of ELB buffer (13). The immunoglobulins were pulled down with protein A-Sepharose, and the beads were washed extensively with the ELB buffer and eluted with SDS loading buffer. The eluted proteins were separated by SDS-PAGE, transferred to a polyvinylidene difluoride membrane, and either exposed to X-ray film (A) or probed with anti-Eed polyclonal serum (B). The position of bands corresponding to Ezh2 and Eed are shown by arrows. IP, immunoprecipitate.
FIG. 7
FIG. 7
Western blot analysis of Gal4-Eed expressed in COS cells. Mammalian expression plasmid containing either wild-type or mutated Eed fused to Gal4 were transfected into COS cells. After 2 days, total cellular extracts were separated by SDS-PAGE and proteins were transferred from the gel onto a polyvinylidene difluoride membrane and probed with anti-Gal4 monoclonal antibodies (Santa Cruz). The membrane was developed with secondary antibodies conjugated with alkaline phosphatase and 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium substrate. The positions of molecular mass markers are shown on the left. Vector, cells were transfected with pM1 plasmid containing no inserts (see Materials and Methods).
FIG. 8
FIG. 8
Effect of Ezh2 on transcriptional activity of Gal4-Eed. Jurkat cells were transfected with a mixture of reporter and expression plasmids. (A) Plasmids used in the transfection experiments. The reporter plasmid was a luciferase gene pGL3-promoter vector containing an SV40 promoter with five Gal4-binding elements. The expression plasmids were as follows. The mammalian vector, pM1, was used for expression of either the Gal4 DNA-binding domain alone (Gal4) or a fusion of Gal4 DNA-binding domain with the wild-type Eed (Gal4-Eed). Plasmid pM1 expressing the N-terminal fragment of the human Ezh2 protein (N-Ezh2, aa 1 to 203) instead of Gal4 was also used. (B) Result of the transfection experiments. Expression plasmids (1 μg of Gal4 or Gal4-Eed and 3 μg of N-Ezh2) with 0.3 μg of luciferase reporter plasmid were used for cotransfections. The total amount of DNA, 4.3 μg, per transfection was adjusted with pM1. Two days after transfection, cells were analyzed for luciferase activity. The data shown represent the means ± standard errors calculated from three independent experiments.
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