3371
Development 128, 3371-3379 (2001)
Printed in Great Britain © The Company of Biologists Limited 2001
DEV7901
Su(z)12, a novel Drosophila Polycomb group gene that is conserved in
vertebrates and plants
Anna Birve1, Aditya K. Sengupta2, Dirk Beuchle2, Jan Larsson1, James A. Kennison3, Åsa RasmusonLestander1 and Jürg Müller2,*
1Department of Genetics, Umeå University, S-90187 Umeå, Sweden
2Max Planck Institute for Developmental Biology, Spemannstrasse 35/III,
3Laboratory of Molecular Genetics, National Institute of Child Health and
72076, Tübingen, Germany
Human Development, National Institutes of Health,
Bethesda, MD 20892-2785, USA
*Author for correspondence (e-mail: juerg.mueller@tuebingen.mpg.de)
Accepted 22 June 2001
SUMMARY
In both Drosophila and vertebrates, spatially restricted
expression of HOX genes is controlled by the Polycomb
group (PcG) repressors. Here we characterize a novel
Drosophila PcG gene, Suppressor of zeste 12 (Su(z)12).
Su(z)12 mutants exhibit very strong homeotic
transformations and Su(z)12 function is required
throughout development to maintain the repressed state of
HOX genes. Unlike most other PcG mutations, Su(z)12
mutations are strong suppressors of position-effect
variegation (PEV), suggesting that Su(z)12 also functions
INTRODUCTION
In both flies and vertebrates, HOX genes are expressed in
spatially restricted patterns to control development of the head,
trunk and limbs (Lewis, 1963; Lewis, 1978; McGinnis and
Krumlauf, 1992). HOX genes have the potential to be active
both within and outside of their correct expression domains,
but each HOX gene is selectively silenced in cells where it must
remain inactive. This silencing depends on the function of the
Polycomb group (PcG) genes (Kennison, 1995; Simon, 1995;
Pirrotta, 1998). PcG genes were first identified in Drosophila
due to mutant phenotypes that suggested that their products
function in repressing multiple HOX genes (Lewis, 1978;
Struhl, 1981; Duncan, 1982; Ingham, 1984; Jürgens, 1985).
Subsequent studies showed that mutations in 11 PcG genes
indeed cause misexpression of HOX genes in embryos and in
larvae. These genes are Polycomb (Pc), Polycomblike (Pcl),
polyhomeotic (ph), Posterior sex combs (Psc), Sex combs on
midleg (Scm), Sex combs extra (Sce), super sex combs (sxc),
pleiohomeotic (pho), Enhancer of zeste (E(z)), extra sex combs
(esc) and Additional sex combs (Asx) (Beachy et al., 1985;
Ingham, 1985; Struhl and Akam, 1985; White and Wilcox,
1985; Cabrera et al., 1985; McKeon and Brock, 1991; Simon
et al., 1992, Fritsch et al., 1999; Beuchle et al., 2001). Most
PcG proteins are conserved in both sequence and function in
vertebrates (Brunk et al., 1991; van Lohuizen et al., 1991;
Pearce et al., 1992; van der Lugt et al., 1994; Müller et al.,
in heterochromatin-mediated repression. Furthermore,
Su(z)12 function is required for germ cell development. The
Su(z)12 protein is highly conserved in vertebrates and is
related to the Arabidopsis proteins EMF2, FIS2 and VRN2.
Notably, EMF2 is a repressor of floral homeotic genes.
These results suggest that at least some of the regulatory
machinery that controls homeotic gene expression is
conserved between animals and plants.
Key words: Su(z)12, Polycomb group, Drosophila melanogaster
1995; Schumacher et al., 1996; reviewed in van Lohuizen,
1998). However, only E(z) and esc appear to be more widely
conserved. In C. elegans, sequence homologues of E(z) and esc
function in germline development but, strikingly, they
apparently have no role in the regulation of homeotic genes
(Holdeman et al., 1998; Korf et al., 1998; Kelly and Fire,
1998). However, in Arabidopsis, the E(z)-like protein CURLY
LEAF (CLF) functions as a transcriptional repressor of floral
homeotic genes in vegetative tissues (Goodrich et al., 1997).
This conservation of Polycomb group gene function in plants
is particularly striking since HOX genes in animals and
homeotic genes in plants are structurally unrelated (McGinnis
et al., 1984; Yanofsky et al., 1990).
In Drosophila, all PcG genes are expressed in the female
germline and maternally deposited wild-type protein often
rescues homozygous mutant embryos to a considerable extent
(Struhl, 1981; Breen and Duncan, 1986; Soto et al., 1995).
Embryos that are doubly homozygous for mutations in two
different PcG genes typically show strongly enhanced
homeotic transformations, and the phenotype of such embryos
is often similar to the null phenotype of the corresponding
single mutants (i.e., lacking both maternal and zygotic gene
function). Jürgens (1985) used this striking property and
generated embryos that were doubly homozygous for PcG
mutations and large chromosomal deficiencies. From these
tests he estimated that the total number of PcG genes in the
Drosophila genome would be in the range of 30 to 40 genes
3372 A. Birve and others
(Jürgens, 1985). Although this number is frequently cited, only
two Drosophila genes with bona fide PcG mutant phenotypes
have been described since Jürgens’ original proposal 15 years
ago. These are multi-sex combs (mxc; Santamaria and
Randsholt, 1995) and cramped (crm; Yamamoto et al., 1997).
We report the mutant phenotypes and molecular analysis of
a new PcG member, Suppressor of zeste 12 (Su(z)12). Su(z)12
mutants show very strong homeotic phenotypes caused by
widespread misexpression of HOX genes. The phenotypes of
Su(z)12 mutants are comparable to those of the strongest PcG
mutants. However, our analyses of Su(z)12 also reveal some
striking properties that distinguish this gene from most other
PcG genes; Su(z)12 function is needed for the development of
germ cells and Su(z)12 loss-of-function mutations suppress
PEV. Moreover, Su(z)12 is not only conserved in vertebrates,
but is also related to Arabidopsis proteins that function as
regulators of floral homeotic genes and other developmental
processes.
MATERIALS AND METHODS
Drosophila strains
The four EMS-induced Su(z)12 alleles, Su(z)123, Su(z)122, Su(z)124
and Su(z)125 were originally called l(3)76BDo1, l(3)76BDo2,
l(3)76BDo4 and l(3)76BDo5, respectively, and their isolation as
mutations that fail to complement Df(3L)kto2 has been described
(Kehle et al., 1998). The Su(z)121 allele was isolated in a P-element
mutagenesis screen for mutations that modify the eye color of z1
mutants. Mutagenized males were mated to z1 Dp(1;1)w z+R61e19
virgin females and the male offspring were screened for suppression
or enhancement of the yellow eye color. One red-eyed male was
isolated and subsequently crossed with balancers to establish a stock.
Of several revertants of Su(z)121, obtained by mobilizing the P
element, we isolated five that were viable and fertile in trans to
Su(z)121.
Genetic analyses
All Su(z)12 alleles were recombined onto an FRT2A chromosome to
obtain the following strains:
w; Su(z)121 FRT2A/ TM6C, cu Sb e Tb ca
w; Su(z)122 FRT2A/ TM6C, cu Sb e Tb ca
w; Su(z)123 FRT2A/ TM6C, cu Sb e Tb ca
w; Su(z)124 FRT2A/ TM6C, cu Sb e Tb ca
w; Su(z)125 FRT2A/ TM6C, cu Sb e Tb ca
Germline clones of each mutant were generated using the standard
ovoD technique. No eggs from germ-line clones were obtained in the
case of Su(z)123 and Su(z)124. To ‘clean’ the left arm of the Su(z)123
and Su(z)124 chromosomes from other potential lethal mutations, we
substituted most of the chromosome arm distal to the Su(z)12 locus
with DNA from a homozygous viable ru h th st cu sr e ca
chromosome. Four independent ru h th Su(z)123 FRT2A and six
independent ru h th Su(z)124 FRT2A recombinant chromosomes were
isolated and tested for production of germline clones but no eggs were
obtained in either case.
Imaginal disc clones were generated by crossing the appropriate
Su(z)12 FRT2A mutant strains with either yw flp122; hs-nGFP
FRT2A or yw flp122; M(3)i55hs-nGFP FRT2A/TM6B flies and heatshocking the F1 larvae. Heat shock treatment to induce clones was
done in vials for 1 hour in a 37°C water bath, and the larvae were
then allowed to develop for the appropriate time at 25°C. Prior to
dissection, larvae were subjected to another 1 hour heat shock
followed by a 1 hour recovery period to induce expression of the GFP
marker protein.
Effects on PEV were analysed by crossing Su(z)12 mutant males
to In(1)wm4 females and comparing the eye phenotypes of the
In(1)wm4; Su(z)12/+ and In(1)wm4; Balancer/+ male progeny.
Antibody staining
Antibody staining of embryos with antibody against Ubx protein was
done following standard protocols. Imaginal discs were stained with
antibodies against Ubx or Abd-B and GFP proteins as described
(Beuchle et al., 2001).
Cloning of Su(z)12
A LAMBDA library of EcoRI digested genomic DNA was generated
from Su(z)121 heterozygotes and screened using P-element sequences
as probe. A subclone containing a 2.2 kb insert was isolated; this insert
contained P-element sequences and 1.6 kb of flanking genomic DNA.
This genomic fragment was used as a probe to isolate a larger genomic
fragment from an EMBL 4 library and, with that as a probe, cDNAs
for three different transcription units were isolated from an embryonic
cDNA library (Clontech). Northern blot analysis revealed that one of
these transcripts showed an altered pattern in Su(z)121 mutants. Two
EST clones with 5′ sequences identical to this cDNA were obtained
from the Berkeley Drosophila Genome Project (LD13365 and
LD02025). LD02025 was sequenced and LD13365 was partially
sequenced. Introns were mapped by use of internal primers, PCR
amplification and sequencing. The Su(z)12 gene was mapped to 76D
using a digoxigenin-labeled probe for in situ hybridisation on polytene
chromosomes from salivary glands.
Sequencing of the EMS-induced Su(z)12 alleles was done as
follows. Genomic DNA was isolated from Su(z)123 or Su(z)124
heterozygotes or, in the case of Su(z)122, from Su(z)122 homozygous
larvae that were identified by the red marker mutation on the mutant
chromosome. In each case, the genomic DNA spanning the Su(z)12
open reading frame was amplified by PCR. Three overlapping
subfragments covering this interval were amplified, subcloned into
bluescript and several independent clones were sequenced. In each
mutant, only a single base change was found in several independent
clones (Fig. 5). For each mutant allele the identified base changes
were confirmed by sequencing clones obtained from a second,
independent PCR amplification.
RESULTS
Mutations in the Su(z)12 locus cause misexpression
of HOX genes
In a screen for zygotic-lethal mutations in the 76D region
(Kehle et al., 1998), we previously identified a lethal
complementation group, l(3)76BDo, that turned out to be
allelic to Su(z)121, a P-element-induced mutation that was
isolated in a screen for modifiers of the zeste-white interaction
(see Materials and Methods). Since Su(z)121 fails to
complement any of the four EMS-induced l(3)76BDo alleles
we have renamed l(3)76BDo1, l(3)76BDo2, l(3)76BDo4 and
l(3)76BDo5 (Kehle et al., 1998) as Su(z)123, Su(z)122, Su(z)124
and Su(z)125, respectively.
Animals that are homozygous or hemizygous for Su(z)121,
Su(z)122, Su(z)123 or Su(z)124 die during the first or second
larval instar, whereas several transheterozygous combinations
with Su(z)125 develop into pharate adults with strong
posteriorly directed homeotic transformations (Fig. 1). These
homeotic transformations are consistent with inappropriate
activation of several HOX genes in the Antennapedia and
bithorax complexes. For example, the additional sex combs on
meso- and metathoracic legs suggests misexpression of Sex
combs reduced (Scr) in these primordia (Pattatucci and
Su(z)12, a novel Drosophila PcG gene 3373
Kaufman, 1991), whereas the antenna to leg transformation is
consistent with inappropriate activation of Antennapedia
(Antp) in the eye-antennal disc (Struhl, 1981) and the wing to
haltere transformations most likely reflects misexpression of
BXC genes in the wing disc (Cabrera et al., 1985; Fig. 1). This
suggests that Su(z)12 acts as a repressor of several homeotic
genes and is a member of the PcG.
Since Su(z)121, Su(z)122, Su(z)123 and Su(z)124
Fig. 1. Homeotic transformations in a Su(z)12 mutant pharate adult
male. Homeotic transformations are evident in several body
segments. Sex combs, a structure normally only present on the first
leg, are present on the first tarsal segments of all meso- and
metathoracic legs (arrowheads); the antennae are partially
transformed into legs (asterisk) and wings are much smaller and
partially transformed into haltere-like structures (arrows). These
homeotic transformations are consistent with inappropriate activation
of several ANTC and BXC genes in the imaginal disc primordia of
these structures (see text). The genotype of the animal shown is
Su(z)125/Su(z)123 but similar homeotic phenotypes are observed in
Su(z)125/Su(z)124 pharate adults; these mutant combinations die as
pharate adults and never eclose from the pupal case.
Fig. 2. Misexpression of homeotic genes and homeotic
transformations in Su(z)12 mutant embryos. (A,B) Embryos at stage
16 (A) and stage 11 (B) stained with antibody against Ubx protein. In
wild-type (wt) embryos, Ubx is expressed from parasegments
(ps) 5 to ps 13 (anterior margin of ps 5 in all cases marked by an
arrowhead) and in five midline cells in ps 4 (visible in A).
(A) Su(z)124 homozygous embryos (zyg−) show misexpression of
Ubx anterior to ps 5 but only in a few cells in the CNS and in the
brain (asterisk); more extensive misexpression of Ubx is seen in ps 14 of Su(z)121 homozygotes. (B) Su(z)122/Df(3L)kto2 embryos
derived from Su(z)122 germline clones show strong expression of
Ubx from ps 1 to ps 14 already at this earlier stage owing to the lack
of both maternal and zygotic (mat− zyg−) wild-type Su(z)12 protein.
(C) Cuticles of a wild-type and a Su(z)12 mutant embryo of the same
genotype as in B. In the Su(z)12 mutant embryo all abdominal,
thoracic and several head segments (not all visible in this focal plane)
are homeotically transformed into copies of the eighth abdominal
segment owing to misexpression of the Abd-B gene in every segment
(the eighth abdominal segment is marked by an arrow in the wildtype embryo and the first abdominal segment is marked by a white
arrowhead). Su(z)125/Df(3L)kto2 embryos derived from
Su(z)125germline clones show similar misexpression of Ubx and
cuticle phenotypes like the embryos shown in B and C (data not
shown).
homozygotes die as larvae without any obvious Pc-like
phenotypes, we next analyzed Su(z)12 mutant embryos for
misexpression of HOX genes (Fig. 2). Embryos that are
homozygous for any of the four EMS-induced Su(z)12 alleles
(Su(z)122−5) show very subtle misexpression of Ubx; similar
misexpression is observed in embryos that are homozygous for
3374 A. Birve and others
Df(3L)kto2, a deficiency that deletes the Su(z)12 locus (Fig. 2
and data not shown). Interestingly, Su(z)121 homozygotes
show substantially more misexpression than embryos that are
homozygous for the deficiency or for any of the other alleles
(Fig. 2; see below). A likely explanation for these subtle Pclike phenotypes is that maternally deposited wild-type Su(z)12
product rescues these Su(z)12 mutant embryos.
We therefore attempted to generate embryos from Su(z)12
mutant germ cells. Females with Su(z)12 mutant germ cells
were crossed to males heterozygous for Df(3L)kto2. We found
that Su(z)12 hemizygous embryos derived from Su(z)122 or
Su(z)125 germ cells showed very extensive misexpression of
Ubx already at the extended germ band stage (Fig. 2 and data
not shown). These animals showed severe homeotic phenotypes
with all abdominal, thoracic and several head segments
transformed into copies of the eight abdominal segment. This
phenotype is consistent with Abd-B being misexpressed in all
segments (Fig. 2 and data not shown). The strong PcG
phenotype of these Su(z)12 mutant embryos is comparable to
that of embryos lacking esc or Pc function (Struhl, 1981;
Lawrence et al., 1983). We find that zygotically provided
Su(z)12 function is sufficient to prevent the inappropriate
activation of HOX genes; Su(z)122/+ heterozygotes obtained as
the progeny of Su(z)12 mutant germ cells and a wild-type sperm
develop into wild-type-looking adults.
In contrast to Su(z)122 or Su(z)125, we found that germ cells
mutant for any of the other three Su(z)12 alleles failed to develop
(Su(z)123 and Su(z)124) or developed into highly abnormal eggs
(Su(z)121). Two observations suggest that the failure to obtain
embryos in the case of Su(z)121 and Su(z)124 is not caused by
second-site mutations on the Su(z)12 mutant chromosomes but
can be attributed to a requirement for Su(z)12 function in germcell development. First, we found that revertants obtained by
excision of the P element in the Su(z)121 allele are viable and
fertile. Second, Su(z)124 mutant germ cells still failed to develop
even after “cleaning” the chromosome from other potentially
Fig. 3. Su(z)12 function is required throughout development to
repress HOX genes. Wing imaginal discs with clones of cells that
are homozygous for the indicated Su(z)12 allele were stained with
antibodies against GFP (green) and Ubx or Abd-B protein (red) as
indicated. Neither Ubx nor Abd-B proteins are normally expressed
in the wing imaginal disc. In each case, homozygous Su(z)12 mutant
cells are marked by the absence of GFP protein (green) and in all
experiments the Minute technique was used (see text). (A) Clones
were induced 96 hours before analysis. Strong misexpression of
Ubx and Abd-B protein is detected in clones that are homozygous
for the strong alleles Su(z)121 and Su(z)124; note that both homeotic
genes are derepressed in almost all clones, throughout the disc. In
clones homozygous for the hypomorphic allele Su(z)122,
misexpression of Ubx occurs only in clones in central regions of the
wing pouch and in the hinge region; Abd-B is not derepressed in
these clones. No derepression of Ubx or Abd-B is detected in
Su(z)125 mutant clones, consistent with the genetic data that this
allele is a weaker hypomorph than Su(z)122. (B) Kinetics of
derepression of Ubx and Abd-B in Su(z)121 and Su(z)124 mutant
clones. 48 hours after clone induction Ubx is still repressed in
almost all Su(z)124 mutant clones but Su(z)121 mutant clones in the
wing pouch already show strong misexpression of Ubx. 72 hours
after clone induction, most Su(z)121 mutant clones show strong
misexpression of Ubx, whereas only Su(z)124 mutant clones in the
pouch show strong Ubx signal and Ubx is apparently still repressed
in other regions of the disc.
lethal mutations by replacing the chromosomal DNA flanking
this Su(z)12 allele with unmutagenized wild-type DNA (see
Materials and Methods). Hence, these results suggest that
Su(z)12 function is essential for the development of germ cells.
Furthermore, Su(z)121, Su(z)123 and Su(z)124 are strong alleles
and Su(z)122 and Su(z)125 are weaker alleles (see below). The
Su(z)12, a novel Drosophila PcG gene 3375
fact that Su(z)121 homozygous embryos show more severe
misexpression than Df(3L)kto2 homozygotes suggests that
Su(z)121 is not a simple loss-of-function allele but is an
antimorphic allele that encodes a product that interferes with the
function of maternally deposited, wild-type Su(z)12 protein. We
note that Su(z)121/+ embryos show no misexpression of
homeotic genes in the embryo (not shown).
We next tested the requirement for Su(z)12 at later
developmental stages by generating Su(z)12 mutant clones in
Fig. 4. Su(z)12 mutations suppress position-effect variegation. Heads
of adult flies that are hemizygous for the wm4 rearrangement. In wildtype flies (left), pigmentation in eyes is drastically reduced owing to
silencing of the white (w) gene in most ommatidia. In animals that
are heterozygous for any of the Su(z)12 alleles (right), there is a
partial release from silencing and the w gene is expressed in most
ommatidia; the loss of silencing is stronger in case of the EMSinduced Su(z)12 alleles. In each case, the wild-type (+/+) control
flies on the left carry the TM3 balancer chromosome and are the
siblings of the corresponding Su(z)12 mutants.
imaginal discs. We assayed for HOX gene silencing in such
clones by monitoring the expression of the HOX genes Ubx
and Abd-B in the imaginal wing disc (where they are normally
stably repressed) using antisera against their protein products.
In these experiments, the Su(z)12 mutant cells were identified
by the absence of a GFP-expressing marker gene (see Materials
and Methods). In addition, we used the Minute technique to
generate Su(z)12−/Su(z)12−clones that carry two copies of a
wild-type Minute allele (i.e., Su(z)12− M+/ Su(z)12− M+),
which gives them a growth advantage relative to their Su(z)12−
M+/ Su(z)12+ M− neighbours.
In a first set of experiments, we analyzed cell clones of the
different Su(z)12 alleles 96 hours after clone induction. We
found that Su(z)121 and Su(z)124 mutant clones showed strong
misexpression of both Ubx and Abd-B in most mutant cells (Fig.
3A). Su(z)122 mutant clones also showed misexpression of Ubx
96 hours after clone induction but misexpression is confined to
the pouch and hinge region in the posterior compartment of the
wing disc (Fig. 3A). No misexpression of Abd-B was detected
in Su(z)12 2 mutant clones and neither Ubx nor Abd-B were
misexpressed in Su(z)125 mutant clones (Fig. 3A). We also
found no misexpression in Su(z)123 mutant clones but we found
that these clones were much smaller than those obtained with
the other Su(z)12 alleles (data not shown). We do not know
whether the cell proliferation/survival defect associated with the
Su(z)123 chromosome is caused by a second mutation in a
closely linked gene (see Materials and Methods) or is a unique
property of this particular allele. In summary, the PcG
phenotypes observed with several Su(z)12 alleles suggest that
Su(z)12 is needed throughout development to keep HOX genes
repressed. Moreover, these results support the allele
classification obtained by the analysis of germ-line clones;
namely, that Su(z)122 and Su(z)125 are hypomorphic alleles
whereas Su(z)121 and Su(z)124 appear to be stronger alleles.
We next examined the kinetics of HOX gene derepression in
Su(z)121 and Su(z)124 mutant clones by analyzing Ubx
expression 24, 48 and 72 hours after clone induction. We again
used the Minute technique in these experiments. 24 hours after
clone induction, Ubx is still tightly repressed. 48 hours after
clone induction, Su(z)121 mutant clones show misexpression
of Ubx protein in the wing pouch but Ubx is still stably silenced
in other parts of the wing disc (Fig. 3B). In Su(z)124 mutant
clones, Ubx is still stably silenced 48 hours after clone
induction except in a few clones in the center of the pouch
where we detect weak Ubx signal (Fig. 3B). Finally, 72 hours
after clone induction, repression of Ubx is lost in most Su(z)121
and Su(z)124 mutant clones in the pouch, in the latter case Ubx
is still silenced in some parts of the disc (Fig. 3B). This slow
and gradual loss of silencing is comparable to the kinetics of
HOX gene derepression in Pc, Pcl, Scm or Sce mutant clones
(Beuchle et al., 2001).
We note that the loss of silencing occurs more rapidly in
Su(z)121 clones than in Su(z)124 clones (Fig. 3). The molecular
characterization of Su(z)124 suggests that this is most likely a
null allele (see below). Our analysis of Su(z)121 homozygous
embryos suggested that Su(z)121 is not a simple loss-of-function
allele but is an antimorphic allele (see above). It is possible that
the more rapid loss of silencing in Su(z)121 mutant clones again
reflects an interference of the mutant Su(z)121 product with
wild-type Su(z)12 molecules (i.e., during the depletion of
persisting wild-type Su(z)12 protein after clone induction).
3376 A. Birve and others
Fig. 5. Lesions in mutant Su(z)12
alleles and comparison between
Su(z)12 and related proteins in
humans and in Arabidopsis. (Top)
Schematic representation of the
Drosophila malnogaster Su(z)12
(Dm Su(z)12), Homo sapiens
Su(z)12 (Hs SU(Z)12, KIAA0160)
and Arabidopsis thaliana (At) EMF2
(N. Yoshida, personal
communication), VRN2 (A. Gendall,
personal communication) and FIS2
(Luo et al., 1999) proteins (white
rectangles) with zinc finger (black
box) and VEFS box (stippled box).
The locations of the molecular
lesions in four different Su(z)12
alleles are indicated (see text).
Alignment of the zinc fingers (top
right) and the VEFS box (below).
Identical amino acids that are found
in all five proteins are boxed in red
and listed below the alignments,
similarities are boxed in colors using
the code listed below. Bottom:
alignment of Drosophila Su(z)12 and
human SU(Z)12 amino acid
sequences. Note that the two proteins
are conserved over the whole length
of the protein. The conserved Gly
mutated in Su(z)122 is indicated by
an arrowhead.
Su(z)12, a novel Drosophila PcG gene 3377
Su(z)12 mutations suppress position-effect
variegation
To test whether Su(z)12 may also participate in other processes
of transcriptional silencing, we tested whether Su(z)12
mutations suppress position-effect variegation (PEV). PEV is
observed in chromosomal rearrangements in which a
euchromatic gene is placed near heterochromatin. The
translocated gene may then become inactivated in a fraction of
cells, presumably because transcription of the gene is silenced
by heterochromatin-associated proteins. A number of
mutations have been identified that suppress or enhance PEV
in a dosage-dependent fashion (reviewed by Wakimoto, 1998).
Mutations that suppress PEV are generally referred to as
Su(var)s; some Su(var) gene products have indeed been shown
to be components of heterochromatin (Eissenberg et al., 1990).
One well-studied reporter for PEV is wm4, a chromosomal
inversion juxtaposing the white gene to centromeric
heterochromatin (Muller, 1930). As illustrated in Fig. 4,
mutations in Su(z)12 strongly suppress PEV at the wm4 locus;
in animals that are heterozygous for any of the five Su(z)12
alleles, the white locus is transcriptionally active in a higher
proportion of ommatidia than in control animals. We note that
suppression of PEV was observed with four different EMSinduced Su(z)12 alleles but not with most other mutations that
were isolated in the same EMS-mutagenesis experiments,
suggesting that suppression of PEV is indeed due to the
mutations at the Su(z)12 locus and not due to other PEV
modifiers on the mutagenized chromosomes (data not shown).
These results suggest that Su(z)12 can be classified as a
suppressor of PEV.
The Su(z)12 protein is conserved in vertebrates and
plants
To identify the Su(z)12 gene we isolated and cloned the
chromosomal DNA flanking the P-element insertion in the
Su(z)121 allele (see Materials and Methods). Using this
genomic DNA as probe we isolated several cDNAs by
screening a cDNA library and by searching an EST database
for cDNAs that match this genomic DNA. Sequence analysis
of the longest cDNA (LD 02025) revealed a single open
reading frame of 900 amino acids. The P-element in Su(z)121
is inserted into codon 564 of this open reading frame and would
therefore result in a C-terminal truncation of the predicted
protein (Fig. 5). Further proof that the identified open reading
frame encodes the Su(z)12 protein was obtained by sequencing
the coding region of three EMS-induced Su(z)12 alleles.
Su(z)122, Su(z)123 and Su(z)124 each show single base changes
in this open reading frame; Su(z)123 and Su(z)124 both
show base substitutions that result in predicted premature
termination codons after codon 218 and codon 298,
respectively, and in Su(z)122, a base substitution changes the
codon for Gly274 into a codon for Asp (Fig. 5; this glycine is
conserved in the human Su(z)12 homologue described below).
The molecular characterizations of these lesions prove that we
identified the Su(z)12 open reading frame. Furthermore, these
lesions support our classification of the different alleles based
on phenotypic criteria; Su(z)122 and Su(z)124 both have stop
codons in the N-terminal third of the protein and therefore may
represent null (or at least strong loss-of-function) alleles
whereas the amino-acid substitution in Su(z)122 is consistent
with the idea that this is a hypomorphic allele.
Database searches show that the Su(z)12 protein is highly
conserved in vertebrates and, strikingly, that Su(z)12-related
proteins also exist in plants (Fig. 5). In contrast, the worm and
yeast genomes do not seem to encode Su(z)12-related proteins.
The function of the highly conserved human homologue of
Su(z)12, HsSU(Z)12 (Fig. 5), is not known but EMF2, FIS2
and VRN2, the three Su(z)12-related proteins in Arabidopsis,
have been identified as regulators in plant development
(Yang et al., 1995; Luo et al., 1999; N. Yoshida, personal
communication; A. R. Gendall, personal communication). One
characteristic feature of all these proteins is a single classical
C2H2 zinc finger similar to the fingers found in sequencespecific DNA-binding proteins (Fig. 5). Attempts to show any
DNA-binding activity of a polypeptide containing the Su(z)12
zinc finger have failed so far (A. K. S. and J. M., unpublished).
A second stretch of amino acids that is conserved between
Su(z)12, HsSU(Z)12, EMF2, VRN2 and FIS2 is located Cterminal to the zinc finger (Fig. 5). We term this part of the
protein VEFS box (VRN2-EMF2-FIS2-Su(z)12 box). We note
that the predicted protein products encoded by Su(z)123 and
Su(z)124 lack both the zinc finger and the VEFS box, whereas
the protein encoded by Su(z)121 would contain the zinc finger
but lack the VEFS box.
DISCUSSION
We report the mutant phenotypes and cloning of Su(z)12, a
novel Drosophila gene. The most notable phenotypes of
Su(z)12 mutants are strong homeotic transformations caused
by the widespread misexpression of several HOX genes in
embryos and in larvae. These phenotypes clearly classify
Su(z)12 as a PcG gene and our clonal analyses show that
Su(z)12 plays an essential role in HOX gene silencing
throughout development. However, a number of properties
distinguish Su(z)12 from most other Drosophila PcG genes and
we shall discuss these in turn.
Our genetic and molecular analyses suggest that Su(z)123
and Su(z)124 are most likely null or at least strong loss-offunction alleles, whereas Su(z)122 and Su(z)125 appear to be
hypomorphic alleles. Su(z)121 is also a strong loss-of-function
allele but in addition, it also shows properties of an antimorphic
allele. Since Su(z)123 may contain a second, cell-lethal
mutation, we will omit this allele for discussion of the Su(z)12
mutant phenotype and presume that the phenotype of Su(z)124
represents the Su(z)12 null phenotype. The analysis of Su(z)121
and Su(z)124 germline clones suggests that Su(z)12 function is
essential for germ-cell development and only germ cells
carrying hypomorphic Su(z)12 mutations develop into
embryos. By contrast, germ cells mutant for most other PcG
members complete oogenesis (Struhl, 1981; Lawrence et al.,
1983; Breen and Duncan, 1986; Soto et al., 1995) and only
E(z), crm and mxc seem to be required for germ cell
development (Phillips and Shearn, 1990; Yamamoto et al.,
1997; Saget et al., 1998). Although we do not know which
processes in germ-cell development require Su(z)12 function,
the requirement for Su(z)12 in the germline clearly
distinguishes Su(z)12 from most other PcG genes.
A second distinction between Su(z)12 and most other PcG
mutants is suggested by the suppression of PEV in Su(z)12
mutants. Heterochromatin-mediated silencing has often been
3378 A. Birve and others
compared to HOX gene silencing (e.g. Paro, 1990; Pirrotta and
Rastelli, 1994). Although the two processes may use similar
molecular mechanisms, they require two distinct sets of
proteins; Su(var) mutants show no PcG phenotypes and most
PcG mutations do not suppress wm4 variegation (Kennison,
1995; Sinclair et al., 1998). Among the exceptions (besides
Su(z)12), mutations in the PcG gene crm suppress wm4
variegation (Yamamoto et al., 1997). E(Pc) mutations also
suppress wm4 variegation (Kennison, 1995; Sinclair et al.,
1998), but it is not clear whether E(Pc) is a PcG gene (Soto et
al., 1995; Sinclair et al., 1998). Finally, E(z) mutations have
been reported to weakly suppress wm4 variegation (Laible et
al., 1997) or to enhance it (Sinclair et al., 1998). Although we
favour the interpretation that Su(z)12 protein functions directly
in heterochromatin-mediated gene silencing, (e.g. as a
component of heterochromatin), we cannot exclude the
possibility that the effect on PEV is indirect.
A third, striking feature of Su(z)12 is its conservation not only
in vertebrates but also in plants. Most Drosophila PcG proteins
have vertebrate homologues and studies on PcG mutant mice
showed that these proteins are needed to repress HOX gene
transcription outside of the normal HOX expression domains
(reviewed by van Lohuizen, 1998). The Su(z)12 protein is highly
conserved in humans and hence, it seems likely that vertebrate
Su(z)12 homologues are also needed for silencing of HOX
genes. Of the other Drosophila PcG genes, only E(z) and esc are
also conserved in plants and previous studies showed that the
E(z) homologue CURLY LEAF (CLF) is needed for repression
of floral homeotic genes in leaves (Goodrich et al., 1997).
Su(z)12 shows sequence similarity with three Arabidopsis
proteins; FIS2, VRN2 and EMF2. Each of these proteins
functions as a regulator to suppress a particular developmental
process during plant development. FIS2 is needed to repress seed
development in the absence of fertilization, a process that also
requires the E(z)- and esc-related proteins FIS1/MEA and
FIS3/FIE (Grossniklaus et al., 1998; Luo et al., 1999). VRN2 is
needed for the stable repression of FLC, a key regulator that
controls flowering (Sheldon et al., 2000, A. R. Gendall, personal
communication). Particularly intriguing is the similarity between
Su(z)12 and EMF2 (N. Yoshida personal communication).
EMF2 acts as a floral repressor by suppressing the onset of
reproductive development; EMF2 mutants show misexpression
of the floral homeotic genes APETALA1 (AP1) and AGAMOUS
(AG) in germinating seedlings (Chen et al., 1997). Thus, it
appears that repression of HOX genes in Drosophila and
repression of floral homeotic genes in Arabidopsis both depend
on a conserved set of PcG proteins, Su(z)12 and E(z) in flies and
EMF2 and CLF in plants.
The hallmarks of Su(z)12, EMF2, FIS2 and VRN2 are a
single C2H2 zinc finger and a conserved stretch of amino acids
that we named the VEFS-box. In all four genes, the VEFS-box
is located C-terminal to the zinc finger. In DNA-binding assays,
we have found no evidence that the Su(z)12 zinc finger by itself
binds to DNA (A. K. S. and J. M., unpublished data). However,
most other PcG proteins also do not bind to DNA directly but
bind to chromatin as multiprotein complexes that contain
different PcG members (Franke et al., 1992; Strutt and Paro,
1997; Shao et al., 1999; Ng et al., 2000; Tie et al., 2001). It is
possible that the Su(z)12 protein also functions in a chromatinbinding protein complex and that in the context of such a
complex, the zinc finger is needed for making DNA or protein
contacts. As discussed in the following, the comparison of
Su(z)124 and Su(z)121 mutant phenotypes suggests that the zinc
finger and the VEFS box are probably two distinct functional
domains. In embryos, Su(z)121 homozygotes show more
extensive misexpression of HOX genes than Su(z)124 or
Df(3L)kto2 homozygotes, and in imaginal discs, Su(z)121
mutant clones show a more rapid loss of HOX gene silencing
than Su(z)124 mutant clones. As already discussed, the stronger
phenotype of Su(z)121 mutants may be attributed to the
interference of an aberrant Su(z)121 product with persisting
Su(z)12+ protein molecules. The lesion in Su(z)121 may result
in the expression of a truncated polypeptide that contains the
zinc finger but lacks the VEFS box, whereas the short
polypeptide encoded by the Su(z)124 allele lacks both the zinc
finger and the VEFS box. One possible molecular explanation
for the stronger phenotype of Su(z)121 mutants would therefore
be that the truncated Su(z)121 protein, containing the C2H2 zinc
finger, competes with wild-type Su(z)12 protein for binding to
its natural target (i.e., a DNA sequence or another protein) but
is not functional since it lacks the VEFS box and the C terminus.
It is possible that the VEFS box is needed for interaction with
other (PcG) proteins or, alternatively, that it is a catalytic
domain providing an enzymatic activity needed for silencing.
We are grateful to Nobumasa Yoshida and Anthony Gendall for
communicating the EMF2 and VRN sequences prior to publication.
We thank Christiane Nüsslein-Volhard for continuous encouragment,
support and critical reading of the manuscript. The work of A. B., J.
L. and A. R.-L. was supported by grants from the Swedish Natural
Science Research Council, the Royal Physiographic Society and the
Lars Hiertas Fund.
Note added in proof
A very recent study (Koontz et al., 2001) reports that
endometrial stromal tumors in humans show chromosomal
rearrangements in which the human homologue of Su(z)12,
HsSU(Z)12, is fused to a zinc-finger protein.
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