The Polycomb Group gene esc encodes an evolutionarily conserved protein required for transcriptional silencing of the homeotic genes. Unlike other Polycomb Group genes, esc is expressed and apparently required only during early embryogenesis, suggesting it is required for the initial establishment of silencing but not for its subsequent maintenance. We present evidence that the ESC protein interacts directly with E(Z), another Polycomb Group protein required for silencing of the homeotic genes. We show that the most highly conserved region of ESC, containing seven WD motifs that are predicted to fold into a beta-propeller structure, mediate its binding to a conserved N-terminal region of E(Z). Mutations in the WD region that perturb ESC silencing function in vivo also perturb binding to E(Z) in vitro. The entire WD region forms a trypsin-resistant structure, like known beta -propeller domains, and mutations that would affect the predicted ESC beta-propeller perturb its trypsin-resistance, while a putative structure-conserving mutation does not. We show by co-immunoprecipitation that ESC and E(Z) are directly associated in vivo and that they also co-localize at many chromosomal binding sites. Since E(Z) is required for binding of other Polycomb Group proteins to chromosomes, these results suggest that formation of an E(Z):ESC complex at Polycomb Response Elements may be an essential prerequisite for the establishment of silencing.

REFERENCES

REFERENCES
Alkema
M. J.
,
Bronk
M.
,
Verhoeven
E.
,
Otte
A.
,
van't Veer
L. J.
,
Berns
A.
,
van Lohuizen
M.
(
1997
)
Identification of Bmi1-interacting proteins as constituents of a multimeric mammalian Polycomb complex.
Genes Dev
11
,
226
240
Bornemann
D.
,
Miller
E.
,
Simon
J.
(
1996
)
The Drosophila Polycomb group gene Sex comb on midleg (Scm) encodes a zinc finger protein with similarity to polyhomeotic protein.
Development
122
,
1621
1630
Breen
T. R.
,
Duncan
I. M.
(
1986
)
Maternal expression of genes that regulate the bithorax complex of Drosophila melanogaster.
Dev. Biol
118
,
442
456
Brunk
B. P.
,
Martin
E. C.
,
Adler
P. N.
(
1991
)
Drosophila genes Posterior Sex Combs and Suppressor two of zeste encode proteins with homology to the murine bmi-1 oncogene.
Nature
353
,
351
353
Buchenau
P.
,
Hodgson
J.
,
Strutt
H.
,
Arndt-Jovin
D. J.
(
1998
)
The distribution of Polycomb-Group proteins during cell division and development in Drosophila embryos: impact on models for silencing.
J. Cell Biol
141
,
469
481
Bunker
C. A.
,
Kingston
R. E.
(
1994
)
Transcriptional repression by Drosophila and mammalian Polycomb group proteins in transfected mammalian cells.
Mol. Cell. Biol
14
,
1721
1732
Busturia
A.
,
Wightman
C. D.
,
Sakonju
S.
(
1997
)
A silencer is required for maintenance of transcriptional repression throughout Drosophila development.
Development
124
,
4343
4350
Campbell
R. B.
,
Sinclair
D. A.
,
Couling
M.
,
Brock
H. W.
(
1995
)
Genetic interactions and dosage effects of Polycomb group genes of Drosophila.
Mol. Gen. Genet
246
,
291
300
Carrington
E. A.
,
Jones
R. S.
(
1996
)
The Drosophila Enhancer of zeste gene encodes a chromosomal protein: examination of wild-type and mutant protein distribution.
Development
122
,
4073
4083
Chan
C. S.
,
Rastelli
L.
,
Pirrotta
V.
(
1994
)
A Polycomb response element in the Ubx gene that determines an epigenetically inherited state of repression.
EMBO J
13
,
2553
2564
Chen
H.
,
Rossier
C.
,
Antonarakis
S. E.
(
1996
).
Cloning of a human homolog of the DrosophilaEnhancer of zeste gene (EZH2) that maps to chromosome 21q22.2.
Genomics
38
,
30
37
Chinwalla
V.
,
Jane
E. P.
,
Harte
P. J.
(
1994
)
The Drosophila trithorax protein binds to specific chromosomal sites and is co-localized with Polycomb at many sites.
EMBO J
14
,
2056
2065
DeCamillis
M.
,
Cheng
N. S.
,
Pierre
D.
,
Brock
H. W.
(
1992
)
The polyhomeotic gene of Drosophila encodes a chromatin protein that shares polytene chromosome-binding sites with Polycomb.
Genes Dev
6
,
223
232
Denisenko
O. N.
,
Bomstyk
K.
(
1997
)
The product of the murine homolog of the Drosophila extra sex combs gene displays transcriptional repressor activity.
Mol. Cell. Biol
17
,
4707
4717
Eissenberg
J. C.
,
James
T. C.
,
Foster-Hartnett
D. M.
,
Hartnett
T.
,
Ngan
V.
,
Elgin
S. C.
(
1990
)
Mutation in a heterochromatin-specific chromosomal protein is associated with suppression of position-effect variegation in Drosophila melanogaster.
Proc. Natl. Acad. Sci. USA
87
,
9923
9927
Eissenberg
J. C.
,
Morris
G. D.
,
Reuter
G.
,
Hartnett
T.
(
1992
)
The heterochromatin-associated protein HP-1 is an essential protein in Drosophila with dosage-dependent effects on position-effect variegation.
Genetics
131
,
345
352
Farkas
G.
,
Galloni
M.
,
Gausz
J.
,
Reuter
G.
,
Gyurkovics
H.
,
Karch
F.
(
1994
)
Trithorax-like, the gene encoding the Drosophila GAGA factor, regulates homeotic loci and suppresses heterochromatin-induced position effect variegation.
Nature
371
,
806
808
Fong
H. K. W.
,
Hurley
J. B.
,
Hopkins
R. S.
,
Miake-Lye
R.
,
Johnson
M. S.
,
Doolittle
R. F.
,
Simon
M. I.
(
1986
)
Repetitive segmental structure of the transducinsubunit: homology with the CDC4 gene and identification of related mRNAs.
Proc. Natl. Acad. Sci. USA
83
,
2162
2166
Franke
A.
,
DeCamillis
M.
,
Zink
D.
,
Cheng
N.
,
Brock
H. W.
,
Paro
R.
(
1992
)
Polycomb and polyhomeotic are constituents of a multimeric protein complex in chromatin of Drosophila melanogaster.
EMBO J
11
,
2941
2950
Frei
E.
,
Baumgartner
S.
,
Edström
J. E.
,
Noll
M.
(
1985
)
Cloning of the extra sex combs gene of Drosophila and its identification by P element-mediated gene transfer.
EMBO J
4
,
979
987
Garcia-Higuera
I.
,
Fenoglio
J.
,
Li
Y.
,
Lewis
C.
,
Panchenko
M. P.
,
Reiner
O.
,
Smith
T. F.
,
Neer
E. J.
(
1996
)
Folding of proteins with WD repeats: comparison of six members of the WD-repeat superfamily to the G proteinsubunit.
Biochemistry
35
,
13985
13994
Gatti
M.
,
Baker
B. S.
(
1989
)
Genes controlling essential cell-cycle functions in Drosophila melanogaster.
Genes Dev
3
,
438
453
Goodrich
J.
,
Puangsomlee
P.
,
Martin
M.
,
Long
D.
,
Meyerowitz
E. M.
,
Coupland
G.
(
1997
)
A Polycomb-group gene regulates homeotic gene expression in Arabidopsis.
Nature
386
,
44
51
Grossniklaus
U.
,
Vielle-Calzada
J.-P.
,
Hoeppner
M. A.
,
Gagliano
W. B.
(
1998
)
Maternal control of embryogenesis by MEDEA, a Polycomb Group gene in Arabadopsis.
Science
280
,
446
450
Gunster
M. J.
,
Satijn
D. P.
,
Hamer
K. M.
,
den Blaauwen
J. L.
,
de Bruijn
D.
,
Alkema
M. J.
,
van Lohuizen
M.
,
van Driel
R.
,
Otte
A. P.
(
1997
)
Identification and characterization of interactions between the vertebrate Polycomb-Group protein BMI1 and human homologs of polyhomeotic.
Mol. Cell. Biol
17
,
2326
2335
Gutjahr
T.
,
Frei
E.
,
Spicer
C.
,
Baumgartner
S.
,
White
R. A. H.
,
Noll
M.
(
1995
)
The Polycomb-group gene, extra sex combs, encodes a nuclear member of the WD-40 repeat family.
EMBO J
14
,
4296
4306
Hagstrom
K.
,
Muller
M.
,
Schedl
P.
(
1997
)
A Polycomb and GAGA dependent silencer adjoins the Fab-7 boundary in the Drosophila bithorax complex.
Genetics
146
,
1365
1380
Hobert
O.
,
Jallal
B.
,
Ullrich
A.
(
1996
)
Interaction of Vav with ENX-1, a putative transcriptional regulator of homeobox gene expression.
Mol. Cell. Biol
16
,
3066
3073
Hobert
O.
,
Sures
I.
,
Ciossek
T.
,
Fuchs
M.
,
Ullrich
A.
(
1996
)
Isolation and developmental expression analysis of Enx-1, a novel mouse Polycomb group gene.
Mech. Dev
55
,
171
184
Jimenez
G.
,
Paroush
Z.
,
Ish-Horowicz
D.
(
1997
)
Groucho acts as a corepressor for a subset of negative regulators, including Hairy and Engrailed.
Genes and Development
11
,
3072
3417
Jones
R. S.
,
Gelbart
W. M.
(
1993
)
The Drosophila Polycomb-Group gene Enhancer of zeste shares a domain of sequence similarity with trithorax.
Mol. Cell. Biol
13
,
6357
6366
Jones
C. A.
,
Ng
J.
,
Peterson
A. J.
,
Morgan
K.
,
Simon
J.
,
Jones
R. S.
(
1998
)
The Drosophila esc and E(z) proteins are direct partners in Polycomb Group-mediated repression.
Mol. Cell. Biol
18
,
2825
2834
Jurgens
G.
(
1985
)
A group of genes controlling the spatial expression of the Bithorax Complex in Drosophila.
Nature
316
,
153
155
Kassis
J. A.
(
1994
).
Unusual properties of regulatory DNA from the Drosophilaengrailed gene: three ‘pairing-sensitive’ sites within a 1.6 kb region.
Genetics
136
,
1025
1038
Komachi
K.
,
Johnson
A. D.
(
1997
)
Residues in the WD repeats of Tup1 required for interaction with2.
Mol. Cell. Biol
17
,
6023
6028
Komachi
K.
,
Redd
M. J.
,
Johnson
A. D.
(
1994
)
The WD repeats of Tup1 interact with the homeo domain protein2.
Genes Dev
8
,
2857
2867
Kuzin
B.
,
Tillib
S.
,
Sedkov
Y.
,
Mizrokhi
L.
,
Mazo
A.
(
1994
)
The Drosophilatrithorax gene encodes a chromosomal protein and directly regulates the region-specific homeotic gene fork head.
Genes Dev
8
,
2478
2490
Kyba
M.
,
Brock
H. W.
(
1998
)
The Drosophila Polycomb Group protein PSC contacts PH and PC through specific conserved domains.
Mol. Cell Biol
18
,
2712
2720
Laible
G.
,
Wolf
A.
,
Dorn
R.
,
Reuter
G.
,
Nislow
C.
,
Lebersorger
A.
,
Popkin
D.
,
Pillus
L.
,
Jenuwein
T.
(
1997
)
Mammalian homologues of the Polycomb-group gene Enhancer of zeste mediate gene silencing in Drosophila heterochromatin and at S. cerevisiae telomeres.
EMBO J
16
,
3219
3232
LaJeunesse
D.
,
Shearn
A.
(
1996
)
E(z): a Polycomb group gene or a trithorax group gene?.
Development
122
,
2189
2197
Lambright
D. G.
,
Sondek
J.
,
Bohm
A.
,
Skiba
N. P.
,
Hamm
H. E.
,
Sigler
P. B.
(
1996
).
The 2.0 Å crystal structure of a heterotrimeric G protein.
Science
379
,
311
319
Landecker
H. L.
,
Sinclair
D. A.
,
Brock
H. W.
(
1994
)
Screen for enhancers of Polycomb and Polycomblike in Drosophilamelanogaster.
Dev. Genet
15
,
425
434
Lonie
A.
,
D'Andrea
R.
,
Paro
R.
,
Saint
R.
(
1994
)
Molecular characterization of the Polycomblike gene of Drosophila melanogaster, a transacting negative regulator of homeotic gene expression.
Development
120
,
2629
2636
Martin
E. C.
,
Adler
P. N.
(
1993
)
The Polycomb group gene Posterior Sex Combs encodes a chromosomal protein.
Development
117
,
641
655
Matthews
K. A.
,
Miller
D. F.
,
Kaufman
T. C.
(
1989
)
Developmental distribution of RNA and protein products of the Drosophila alpha-tubulin gene family.
Dev. Biol
132
,
45
61
McCall
K.
,
Bender
W.
(
1996
)
Probes for chromatin accessibility in the Drosophila Bithorax Complex respond differently to Polycomb-mediated repression.
EMBO J
15
,
569
580
Moazed
D.
,
O'Farrell
P. H.
(
1992
)
Maintenance of the engrailed expression pattern by Polycomb group genes in Drosophila.
Development
116
,
805
810
Neer
E. J.
,
Schmidt
C. J.
,
Nambudripad
R.
,
Smith
T. F.
(
1994
)
The ancient regulatory-protein family of WD-repeat proteins.
Nature
371
,
297
300
Neer
E. J.
,
Smith
T. F.
(
1996
)
G protein heterodimers: new structures propel new questions.
Cell
84
,
175
178
Ng
J.
,
Li
R.
,
Morgan
K.
,
Simon
J.
(
1997
)
Evolutionary conservation and predicted structure of the Drosophila extra sex combs repressor protein.
Mol. Cell. Biol
17
,
6663
6672
O'Donnell
K. H.
,
Chen
C. T.
,
Wensink
P. C.
(
1994
)
Insulating DNA directs ubiquitous transcription of the Drosophila melanogaster alpha 1-tubulin gene.
Mol. Cell Biol
14
,
6398
6408
Orlando
V.
,
Paro
R.
(
1993
)
Mapping Polycomb -repressed domains in the bithorax complex using in vivo formaldehyde cross-linked chromatin.
Cell
75
,
1187
1198
Paro
R.
,
Hogness
D. S.
(
1991
)
The Polycomb protein shares a homologous domain with a heterochromatin-associated protein of Drosophila.
Proc. Natl. Acad. Sci. USA
88
,
263
267
Paro
R.
,
Zink
B.
(
1993
)
The Polycomb gene is differentially regulatedduring oogenesis and embryogenesis of Drosophila melanogaster.
Mech. Dev
40
,
37
46
Paroush
Z.
,
Finley
R. L.
,
Kidd
T.
,
Wainwrirht
S. M.
,
Ingham
P. W.
,
Brent
R.
,
Ish-Horowicz
D.
(
1994
)
Groucho is required for Drosophila neurogenesis, segmentation and sex determination and interacts directly with Hairy-related bHLH proteins.
Cell
79
,
805
815
Pelegri
F.
,
Lehmann
R.
(
1994
)
A role of Polycomb Group genes in the regulation of gap gene expression in Drosophila.
Genetics
136
,
1341
1353
Peterson
A. J.
,
Kyba
M.
,
Bornemann
D.
,
Morgan
K.
,
Brock
H. W.
,
Simon
J.
(
1997
)
A domain shared by the Polycomb group proteins Scm and ph mediates heterotypic and homotypic interactions.
Mol. Cell. Biol
17
,
6683
6692
Phillips
M. D.
,
Shearn
A.
(
1990
)
Mutations in polycombeotic, a Drosophila Polycomb-group gene, cause a wide range of maternal and zygotic phenotypes.
Genetics
125
,
91
101
Pirrotta
V.
(
1997
)
PcG complexes and chromatin silencing.
Curr. Op. Gen. Dev
7
,
249
258
Pirrotta
V.
,
Chan
C.-S.
,
McCabe
D.
,
Qian
S.
(
1995
)
Distinct parasegmental and imaginal enhancers and the establishment of the expression pattern of the Ubx gene.
Genetics
141
,
1439
1450
Pirrotta
V.
,
Rastelli
L.
(
1994
)
white gene expression, repressive chromatin domains, and homeotic gene regulation in Drosophila.
BioEssays
16
,
549
556
Platero
J. S.
,
Csink
A. K.
,
Quintanilla
A.
,
Henikoff
S.
(
1998
)
Changes in chromosomal localization of heterochromatin-binding proteins during the cell cycle in Drosophila.
J. Cell Biol
140
,
1297
1306
Platero
J. S.
,
Sharp
E. J.
,
Adler
P. N.
,
Eissenberg
J. C.
(
1996
)
In vivo assay for protein-protein interactions using Drosophila chromosomes.
Chromosoma
104
,
393
404
Poux
S.
,
Kostic
C.
,
Pirrotta
V.
(
1996
)
Hunchback-independent silencing of late Ubx enhancers by a Polycomb group response element.
EMBO J
15
,
4713
4722
Rastelli
L.
,
Chan
C. S.
,
Pirrotta
V.
(
1993
)
Related chromosome binding sites for zeste, suppressors of zeste and Polycomb group proteins in Drosophila and their dependence on Enhancer of zeste function.
EMBO J
12
,
1513
1522
Rost
B.
,
Sander
C.
(
1993
)
Improved prediction of protein secondary structure by use of sequence profiles and neural networks.
Proc. Natl. Acad. Sci. USA
90
,
7558
7562
Rost
B.
,
Sander
C.
(
1993
)
Prediction of protein structure at better than 70% accuracy.
J. Mol. Biol
232
,
584
599
Rost
B.
,
Sander
C.
(
1994
)
Combining evolutionary information and neural networks to predict protein secondary structure.
Proteins
19
,
55
72
Sathe
S. S.
,
Harte
P. J.
(
1995
)
The Drosophila Extra sex combs protein contains WD repeats essential for its function as a repressor of homeotic genes.
Mech. Dev
52
,
77
87
Sathe
S. S.
,
Harte
P. J.
(
1995
)
The Extra sex combs protein is highly conserved between Drosophila virilis and Drosophila melanogaster.
Mech. Dev
52
,
225
232
Satijn
D. P.
,
Gunster
M. J.
,
van der Vlag
J.
,
Hamer
K. M.
,
Schul
W.
,
Alkema
M. J.
,
Saurin
A. J.
,
Freemont
P. S.
,
van Driel
R.
,
P
O. A.
(
1997
)
RING1 is associated with the Polycomb Group protein complex and acts as a transcriptional repressor.
Mol. Cell Biol
17
,
4105
4113
Schumacher
A.
,
Faust
C.
,
Magnuson
T.
(
1996
)
Positional cloning of a global regulator of anterior-posterior patterning in mice.
Nature
383
,
250
253
Shearn
A.
,
Hersperger
G.
,
Hersperger
E.
(
1978
)
Genetic analysis of two allelic temperature-sensitive mutants of Drosophila melanogaster, both of which are zygotic and maternal-effect lethals.
Genetics
89
,
341
353
Shearn
A.
,
Hersperger
G.
,
Hersperger
E.
,
Pentz
E. S.
,
Denker
P.
(
1978
)
Multiple allele approach to the study of genes in Drosophila melanogaster that are involved in imaginal disc development.
Genetics
89
,
355
370
Shearn
A.
,
Rice
T.
,
Garen
A.
,
Gehring
(
1971
)
Imaginal disc abnormalities in lethal mutants of Drosophila.
Proc. Natl. Acad. Sci. USA
68
,
2695
2698
Simon
J.
,
Bornemann
D.
,
Lunde
K.
,
Schwartz
C.
(
1995
)
The extrasex combs product contains WD40 repeats and its time of action implies a role distinct from other Polycomb group products.
Mech. Dev
53
,
197
208
Simon
J.
,
Chiang
A.
,
Bender
W.
(
1992
)
Ten different Polycomb group genes are required for spatial control of the abd-A and Abd-B homeotic products.
Development
114
,
493
505
Simon
J.
,
Peifer
M.
,
Bender
W.
,
O'Connor
M.
(
1990
)
Regulatory elements of the bithorax complex that control expression along the anterior-posterior axis.
EMBO J
9
,
3945
3956
Sinclair
D.
,
Milne
T.
,
Hodgson
J.
,
Shellard
J.
,
Salinas
C.
,
Kyba
M.
,
Randazzo
F.
,
Brock
H.
(
1998
)
The Additional sex combs gene of Drosophila encodes a chromatin protein that binds to shared and unique Polycomb group sites on polytene chromosomes.
Development
125
,
1207
1216
Sinclair
D. A.
,
Campbell
R. B.
,
Nicholls
F.
,
Slade
E.
,
Brock
H. W.
(
1992
)
Genetic analysis of the Additional sex combs locus of Drosophila melanogaster.
Genetics
130
,
817
825
Sinclair
D. A.
,
Clegg
N. J.
,
Antonchuk
J.
,
Milne
T. A.
,
Stankunas
K.
,
Ruse
C.
,
Grigliatti
T. A.
,
Kassis
J. A.
,
Brock
H. W.
(
1998
)
Enhancer of Polycomb is a suppressor of position-effect variegation in Drosophila melanogaster.
Genetics
148
,
211
220
Sondek
J.
,
Bohm
A.
,
Lambright
D. G.
,
Hamm
H. E.
,
Sigler
P. B.
(
1996
).
Crystal structure of a GAproteindimer at 2.1 Å resolution.
Science
379
,
369
374
Soto
M. C.
,
Chou
T. B.
,
Bender
W.
(
1995
)
Comparison of germline mosaics of genes in the Polycomb group of Drosophila melanogaster.
Genetics
140
,
231
243
Struhl
G.
(
1981
)
A gene product required for correct initiation of segmental determination in Drosophila.
Nature
293
,
36
41
Struhl
G.
(
1983
)
Role of the esc+ gene product in ensuring the selective expression of segment-specific homeotic genes in Drosophila.
J. Embryol. Exp. Morphol
76
,
297
331
Struhl
G.
,
Akam
M.
(
1985
)
Altered distributions of Ultrabithorax transcripts in extra sex combs mutant embryos of Drosophila.
EMBO J
4
,
3259
3264
Struhl
G.
,
Brower
D.
(
1982
)
Early role of the esc+ gene product in the determination of segments in Drosophila.
Cell
31
,
285
292
Strutt
H.
,
Cavalli
G.
,
Paro
R.
(
1997
)
Co-localization of Polycomb protein and GAGA factor on regulatory elements responsible for the maintenance of homeotic gene expression.
EMBO J
16
,
3621
3632
Strutt
H.
,
Paro
R.
(
1997
)
The Polycomb group protein complex of Drosophila melanogaster has different compositions at different target genes.
Mol. Cell. Biol
17
,
6773
6783
Tokunaga
C.
,
Stern
C.
(
1965
)
The developmental autonomy of extra sex combs in Drosophila melanogaster.
Dev. Biol
11
,
50
81
Wall
M. A.
,
Coleman
D. E.
,
Lee
E.
,
Iñiguez-Lluhi
J. A.
,
Posner
B. A.
,
Gilman
A. G.
,
Sprang
S. R.
(
1995
)
The structure of the G protein heterotrimer Gi1 12.
Cell
83
,
1047
1058
Wedeen
C.
,
Harding
K.
,
Levine
M.
(
1986
)
Spatial regulation of Antennapedia and bithorax gene expression by the Polycomb locus in Drosophila.
Cell
44
,
739
748
Zink
D.
,
Paro
R.
(
1995
)
Drosophila Polycomb-group regulated chromatin inhibits the accessibility of a trans-activator to its target DNA.
EMBO J
14
,
5660
5671
Sewalt
R. G. A. B.
,
van der Vlag
J.
,
Gunster
M. J.
,
Hamer
K. M.
,
den Blaauwen
J. L.
,
Satijn
D. P. E.
,
Hendrix
T.
,
van Driel
R.
,
Otte
A. P.
(
1998
)
Characterization of interactions between the mammalian polycomb-group proteins Enx1/EZH2 and EED suggests the existence of different mammalian polycomb-group protein complexes.
Mol. Cell. Biol
18
,
3586
3595
van Lohuizen
M.
,
Tijms
M.
,
Voncken
J. W.
,
Schumacher
A.
,
Magnuson
T.
,
Wientjens
E.
(
1998
)
Interaction of mouse polycomb-group (Pc-G) proteins enx1 and enx2 with eed: indication for separate Pc-G complexes.
Mol. Cell. Biol
18
,
3572
3579
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