Molecular Genetic Analysis of Su(z)2 Identifies Key Functional Domains
Richard B. Emmons†, Heather Genetti, Stephen Filandrinos, Jillian Lokere, and C.-ting
Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA
† Present address: Lahive & Cockfield, LLP, One Post Office Square, Boston, MA
Sequence data from this article have been deposited with the EMBL/Genbank Data
Libraries under accession nos. FJ876147-FJ876149, FJ897446-FJ897460, and FJ917397.
Genetics: Published Articles Ahead of Print, published on June 15, 2009 as 10.1534/genetics.108.097360
Running head: Functional domains of Su(z)2
Key words: Su(z)2, Psc, Bmi-1, Polycomb group, negative complementation
Department of Genetics
Harvard Medical School
NRB, Room 264
77 Avenue Louis Pasteur
Boston, MA 02115
The Su(z)2 complex contains Posterior sex combs (Psc) and Suppressor 2 of zeste
(Su(z)2), two paralogous genes that likely arose by gene duplication. Psc encodes a
Polycomb group (PcG) protein that functions as a central component of the PRC1
complex, which maintains transcriptional repression of a wide array of genes. Although
much is known about Psc, very little is known about Su(z)2, the analysis of which has
been hampered by a dearth of alleles. We have generated new alleles of Su(z)2 and
analyzed them at the genetic and molecular levels. Some of these alleles display negative
complementation in that they cause lethality when heterozygous with the gain-of-function
Su(z)21 allele but are hemizygous and, in some cases, homozygous viable. Interestingly,
alleles of this class identify protein domains within Su(z)2 that are highly conserved in
Psc and the mammalian Bmi-1 and Mel-18 proteins. We also find several domains of
intrinsic disorder in the C-terminal regions (CTRs) of both Psc and Su(z)2 and suggest
that these domains may contribute to the essential functions of both proteins.
The Su(z)2 complex contains two paralogous genes, Posterior sex combs (Psc) and
Suppressor 2 of zeste (Su(z)2). Although much is known about Psc, relatively little is
known about Su(z)2. We have generated new alleles of Su(z)2, some of which display
negative complementation. Interestingly, alleles of this class identify protein domains
within Su(z)2 that are highly conserved in Psc and the mammalian Bmi-1 and Mel-18
proteins. We also find several domains of intrinsic disorder in the C-terminal regions of
both Psc and Su(z)2 and suggest that these domains may contribute to the essential
functions of both proteins.
The Su(z)2 complex of Drosophila spans approximately 100 kb and contains two
divergently transcribed genes, Posterior sex combs (Psc) and Suppressor 2 of zeste
(Su(z)2) (ADLER et al. 1989; WU et al. 1989; WU AND HOWE 1995). Of the two, Su(z)2 is
the lesser known. It stands in stark contrast to Psc, which has been the focus of extensive
genetic, molecular, and biochemical analyses for many years. Psc is a member of the
Polycomb group (PcG) of genes, many of which function at the level of chromatin as part
of at least two PcG repressive complexes, called PRC1 and PRC2 (reviewed by BROCK
and FISHER 2005; BREILING et al. 2007; SCHUETTENGRUBER et al. 2007; SCHWARTZ and
PIRROTTA 2007; MATEOS-LANGERAK and CAVALLI 2008; SCHWARTZ and PIRROTTA
2008). PRC2 contains the Enhancer of zeste (E(z)) protein, which provides a histone
methyl transferase activity that methylates histone H3 on lysine 27 (reviewed by CAO and
ZHANG 2004). This epigenetic chromatin mark is believed to recruit PRC1 (FISCHLE et al.
2003; MIN et al. 2003; but see also KAHN et al. 2006), which then functions to maintain
target gene silencing. PRC1 contains >15 subunits (SAURIN et al. 2001) and blocks both
transcription and chromatin remodeling in vitro (SHAO et al. 1999). These inhibitory
activities can be reproduced by a minimal complex, called the PRC1 core complex
(PCC), consisting of four proteins, including Psc, Polycomb (Pc), Polyhomeotic (Ph), and
Sex combs extra (Sce) (FRANCIS et al. 2001; Sce is also known as dRing1; FRITSCH et al.
2003; GORFINKIEL et al. 2004). Psc can reproduce the inhibitory activities by itself,
suggesting that it is a central component of PCC (FRANCIS et al. 2001).
Many lines of evidence suggest that Su(z)2 is functionally related to, and even
partially redundant with, Psc. For example, overexpression of either gene leads to bristle
defects (BRUNK et al. 1991b; SHARP et al. 1994; WU and HOWE 1995) and, as detailed
below, certain alleles of either gene can act as suppressors or enhancers of an allele of the
zeste (z) gene (WU and HOWE 1995). In addition, embryos homozygous for a deficiency
that removes both genes, Su(z)21.b8, display cuticle defects that are more severe than those
of embryos lacking either Psc or Su(z)2 alone (ADLER et al. 1991; SOTO et al. 1995; WU
and HOWE 1995). Similarly, somatic clones homozygous for Su(z)21.b8 in wing imaginal
discs show derepression of homeotic genes and cellular overgrowth, whereas clones
homozygous for loss-of-function (l-o-f) alleles of either Psc or Su(z)2 do not (BEUCHLE et
al. 2001). Su(z)2 also co-localizes with Psc and Pc at many sites on polytene
chromosomes (RASTELLI et al. 1993; PLATERO et al. 1996; SHARP et al. 1997) and, very
recently, co-immunoprecipitation experiments using Drosophila and cell line extracts
suggest that Su(z)2 exits in a complex that also contains Pc, Ph, and Sce/dRing1, which
are the three non-Psc members of PCC (LO et al. 2009).
Psc and Su(z)2 also resemble each other at the structural level. First, both Psc and
Su(z)2 are large proteins, consisting of 1603 and 1365 amino acids, respectively. Second,
they are homologous over a 200 amino acid interval located in their N-terminal regions.
This interval, called the homology region (HR), contains a Ring-finger (RF) domain and
a helix-turn-helix (HTH) domain and is 37% identical between the two proteins (Figure
1) (BRUNK et al. 1991a; van LOHUIZEN et al. 1991). RF and HTH domains have been
implicated in mediating protein interactions. Finally, the two proteins are similar in the
amino acid content of the ~1,000 amino acids of their C-terminal regions (CTRs). While
the CTRs are not conserved at the level of primary amino acid sequence, both show a
high level of flexibility and are enriched in proline and serine and contain runs of one or
more of the following amino acids: asparagine, glutamine, glycine, proline, serine, and
threonine (Figure 1) (BRUNK et al. 1991a). Functional studies of Psc have confirmed that
both the HR and the CTR contribute to the activity of the protein. In particular, genetic
and molecular analyses indicate that the RF is required for Psc function in vivo, the HR is
necessary for assembly of the PCC in vitro, and the CTR, which is functionally separable
from the RF, is essential for wild-type Psc activity in vivo as well as the inhibition of
transcription and chromatin remodeling in vitro (KING et al. 2005). Importantly, Su(z)2
behaves similarly to Psc in in vitro assays; it can replace Psc in a complex with Pc, Ph,
and Sce/dRing1, its HR is essential for formation of the complex, and its CTR inhibits
chromatin remodeling (LO et al. 2009). This latter finding is consistent with studies in
mammalian cells showing that Su(z)2, either full-length or lacking the majority of its HR,
can repress activator function (BUNKER and KINGSTON 1994).
The mammalian orthologs of Psc and Su(z)2 are Bmi-1, which is involved in stem
cell maintenance and cancer (for example, see PIETERSEN et al. 2008; SANGIORGI and
CAPECCHI 2008; reviewed in SPARMANN and VAN LOHUIZEN 2006; PIETERSEN and VAN
LOHUIZEN 2008), and Mel-18 (also known as PCGF2), which has been implicated in
tumor suppression and the regulation of c-myc and bmi-1 (for example, see GUO et al.
2007a,b; WIEDERSCHAIN et al. 2007; LEE et al. 2008). Bmi-1 and Mel-18 are homologous
to Psc and Su(z)2 throughout the HR (BRUNK et al. 1991a; VAN LOHUIZEN et al. 1991;
ALKEMA et al. 1993; ISHIDA et al. 1993) and, although the CTRs of these mammalian
proteins are relatively short, they resemble the long CTRs of Psc and Su(z)2 in that they
are enriched in proline and serine.
Despite the many similarities between Psc and Su(z)2, there are also differences
between the two. For example, Psc and Su(z)2 alleles differ with respect to their lethal
phases and cuticular phenotypes (JÜRGENS 1985; ADLER et al. 1989; WU et al. 1989;
ADLER et al. 1991; WU and HOWE 1995) as well as their interactions with trithorax group
(trxG) genes, which act in opposition to PcG genes (reviewed by BROCK and FISHER
2005; BREILING et al. 2007; SCHUETTENGRUBER et al. 2007; SCHWARTZ and PIRROTTA
2007; SCHWARTZ and PIRROTTA 2008). Given the structural similarities and partial
functional redundancy between Psc and Su(z)2, these differences suggest that Su(z)2 has
roles beyond those associated with prototypical PcG genes.
Su(z)2 was first identified by the Su(z)21 allele (KALISCH and RASMUSON 1974).
This allele was isolated as a gain-of-function (g-o-f) dominant suppressor of an allele of
the X-linked zeste gene, called z1, which represses expression from the white+ (w+) eye
color gene in a manner that is sensitive to whether the w+ gene is paired with another w+
gene (JACK and JUDD 1979; reviewed by WU and GOLDBERG 1989; PIRROTTA 1991;
KASSIS 2002). For example, the eye color of z1 w+/z1 w+ females is yellow instead of
wild-type red because the somatic homolog pairing that occurs in Drosophila (STEVENS
1907; STEVENS 1908; METZ 1916; LEWIS 1954; reviewed by MCKEE 2004) brings the
two w+ genes together, making them subject to silencing by z1. Strikingly, Su(z)21
suppresses the z1 eye color phenotype in a dominant antimorphic fashion, such that z1
w+/z1 w+ ; Su(z)21/+ females have eyes that are red, rather than yellow (KALISCH and
RASMUSON 1974; WU and HOWE 1995). A dominant allele of Psc, called Psc1, is also a
strong suppressor of the z1 phenotype. Interestingly, Psc1 displays second site non-
complementation (SSNC) (reviewed by HAWLEY and GILLILAND 2006) with Su(z)21 such
that Psc1 +/+ Su(z)21 heterozygotes are not viable (WU 1984; ADLER et al. 1989; WU et
al. 1989; WU and HOWE 1995). This SSNC suggests that the gene products of Su(z)2 and
Psc may interact, consistent with their co-localization at many sites on polytene
chromosomes (RASTELLI et al. 1993; PLATERO et al. 1996; SHARP et al. 1997).
How Su(z)21 and Psc1 suppress z1 is unclear, although much has been learned
about Zeste, which is found in PRC1 (SAURIN et al. 2001; MULHOLLAND et al. 2003) and
known to bind DNA (reviewed by Pirrotta 1991; also see MOHRMANN et al. 2002), self-
associate or aggregate (reviewed by Pirrotta 1991; also see CHEN and PIRROTTA 1993;
ROSEN et al. 1998), and participate in both gene activation and gene repression (BIGGIN
et al. 1988; LANEY and BIGGIN 1992; KAL et al. 2000; HUR et al. 2002; MULHOLLAND et
al. 2003; DEJARDIN and CAVALLI 2004). Of the mechanisms being considered, several
suggest that Su(z)2 and Psc interact with Zeste directly or within the context of a larger
complex, the form or occurrence of such interactions being contingent on the mutant or
wild-type state of the proteins (MANSUKHANI et al. 1988; WU and GOLDBERG 1989; WU
et al. 1989; CHEN and PIRROTTA 1993; RASTELLI et al. 1993; ROSEN et al. 1998; SAURIN
et al. 2001; MULHOLLAND et al. 2003). For example, the z1 protein may silence w+ by
drawing Su(z)2 and/or Psc to the locus or, if Su(z)2 and/or Psc are normally present at the
target, may induce them to silence w+ to an abnormal degree. If so, Su(z)21 and Psc1 may
suppress z1 by antagonizing that silencing. Alternatively, as Zeste has been implicated in
silencing, it is possible that Zeste1 is hypermorphic for that activity and that Su(z)21 and
Psc1 suppress z1 by antagonizing Zeste1 directly.
Our studies have focused on extant as well as newly isolated alleles of Su(z)2 and
identified a special class that display negative complementation with Su(z)21. Consistent
with the implication that instances of negative complementation result from protein-
protein interactions (FINCHAM 1966), we find that three of the alleles that display
negative complementation with Su(z)21 contain missense mutations in the RF, a protein
motif known to mediate such interactions. Two other alleles that display negative
complementation identify two domains in the HR that lie outside the RF and HTH and
are conserved in both Bmi-1 and Mel-18. Finally, we have looked more closely at the
CTRs of Su(z)2 and Psc and find that they both contain many regions of intrinsic protein
disorder, which may speak further to the functional similarities between these two
MATERIALS AND METHODS
Culture conditions and stocks
All crosses were conducted at 25ºC on standard Drosophila cornmeal, yeast,
sugar, and agar medium with p-hydroxybenzoic acid methyl ester added as a mold
inhibitor. In general, crosses were carried out with ~3 females and ~3 males in vials and
brooded daily to prevent crowding. All chromosomes carrying Su(z)2 mutations were
isogenic and kept in stock heterozygous with the CyO-19 GFP-bearing balancer
chromosome (Bloomington Stock Center). Su(z)24 is an unstable allele, as we have
identified two isolates with distinct molecular signatures: both isolates contain an 8 bp
deletion in exon 5, but one contains an ~9 kb insertion in exon 1 while the other does not.
As we cannot state with certainty that either isolate corresponds to the original Su(z)24
mutation, we have renamed the insert-bearing allele Su(z)24-34, in recognition of its
recovery from stock 34abl.1$, and the allele lacking the insert Su(z)24-31, in recognition of
its recovery from stock 31ar.1$. Note that stocks 34ab1.1$ and 31ar.1$ are related by
lineage to a single originating stock of Su(z)24. Su(z)2sM was discovered in a series of
control crosses designed to confirm the full viability of Su(z)21 in trans to wild-type
second chromosomes derived from a variety of standard laboratory strains. To our great
surprise, we discovered that one of our Canton-S stocks displayed nearly complete
lethality when crossed to Su(z)21. Single chromosomes extracted from this stock
displayed similar lethality when heterozygous with Su(z)21, indicating that this Canton-S
stock was homozygous for a mutation on the second chromosome that was lethal when
heterozygous Su(z)21. This spontaneous mutation was subsequently called Su(z)2sM. The
stock of dp cn bw; + that was used in the mutagenesis is isogenic for chromosomes II and
The Su(z)2 complex, including Psc and Su(z)2, is located on chromosome II at
meiotic map position 67.3 and polytene position 49E (WU and HOWE 1995). In our
mutageneses designed to recover Su(z)21 interacting mutations (SIMs), males of the
genotype dp cn bw ; + were fed EMS as previously described (WU and HOWE 1995) and
crossed to T(2;3)apXa/CyO virgin females. Approximately 17,500 F1 males heterozygous
for T(2;3)apXa and mutagenized (*) chromosomes II* and III* were then individually
mated in vials with three Su(z)21/CyO virgin females. F2 progeny were scored for the
absence of flies with normal wings (i.e. Cy+ and ap+), indicating that chromosome II*
and/or chromosome III* carried a SIM mutation that was lethal in a Su(z)21 background.
To verify that such lethality was due to Su(z)21, and not an extraneous mutation
on the Su(z)21 chromosome, we assessed the linkage of the capacity of Su(z)21 to
suppress z1 to that of the lethal interaction between Su(z)21 and the SIMs. This analysis
was applied to six (s14, s15, s20, s21, s36, and s84) of the seven SIMs; the s95 allele was
not tested because it suppresses z1 on its own. We crossed z1 wis; Su(z)21/+ virgin females
to putative SIM/CyO males and looked for z1 wis/Y; putative SIM/Su(z)21 recombinant F1
males, which would be predicted to be viable with red eyes and straight wings if the
lesion on the Su(z)21 chromosome that was responsible for the lethal interaction with the
SIMs were separable from Su(z)21. No z1 wis/Y; putative SIM/Su(z)21males were observed
for s20 (0 recombinants/195 total flies scored), s36 (0/343), and s84 (0/361), while few
were observed for s15 (1/257) and s21 (5/147). The frequency of red-eyed straight-
winged males carrying s15 and s21 can be explained by the low but significant viability
of s15/Su(z)21 and s21/Su(z)21 flies (Table 1). A few recombinants were also recovered in
the analysis of s14, consistent with other data indicating it to be an allele of Psc.
All crosses to test viability were conducted in the following way: w- ; mutant
allele 1/CyO males or females were crossed to w- ; mutant allele 2/CyO females or males,
respectively. We defined viability as the number of Cy+ flies/ total progeny. Under ideal
conditions, viability should equal 33% when mutant allele 1/mutant allele 2
heterozygotes are 100% viable and transmission rates of all chromosomes are equal. We
have avoided calculating viability in terms of expected viability (i.e., the relative
percentage of 33%) because the mutant allele 1/CyO and mutant allele 2/CyO classes
could not be distinguished in the majority of our crosses, precluding our ability to
determine the relative transmission rates for the two mutant alleles.
Molecular analysis of mutant alleles
Southern analysis was performed on DNA extracted from flies heterozygous for a
mutant allele and the Cy0-19 balancer. Thirty flies of each genotype were frozen at –80ºC
overnight, and their DNA was extracted using the BDGP crude fly protocol (SPRADLING
et al. 1999). Aliquots of DNA corresponding to 2.5 flies were then digested with EcoRI,
NotI, and EcoRI/NotI, separated on an agarose gel, transferred to nylon filters via
standard Southern blot protocols, and probed with 32P-labeled Su(z)2 cDNA.
Sequence analysis was conducted on embryos homozygous for a mutant allele as
previously described (KING et al. 2005), using primer sets specific for all Su(z)2 exons.
Double strand sequence was obtained for all exons of all alleles, except the three
structurally rearranged alleles: Su(z)21.b7, for which only exons 4 and 5 were sequenced,
Su(z)2s95, for which only exons 1, 2, and 5 were sequenced, and Su(z)24-34 for which only
exons 1-5 were sequenced. Note that our sequencing strategy for Su(z)21.b7 did not allow
us to confirm the presence of the L120F missense mutation found in Su(z)21, from which
Su(z)21.b7 was derived. The key molecular lesions associated with Su(z)21, Su(z)24-31,
Su(z)24-34, Su(z)2h29, Su(z)2s20, Su(z)2s21, Su(z)2s36, Su(z)2s84, Su(z)2sM, Su(z)31, and
Su(z)2Deos were verified by obtaining genomic DNA from flies heterozygous for a mutant
allele and CyO-19, amplifying the relevant region by PCR, and sequencing the resulting
fragment. We found the Su(z)2 locus to be highly polymorphic between mutant and wild-
type laboratory strains from different backgrounds. In fact, the high frequency of strain
specific polymorphisms required the use of strain specific primer sets. Polymorphic
changes within the exonic regions are noted in Table S1.
Identification of the roo element insert in s95 resulted from our inability to
amplify either exon 3 or 4 of this allele. As the primer sets for these two exons overlap,
we anticipated that s95 would contain foreign sequence that had either inserted between
the sites homologous to the upstream primer for exon 4 and the downstream primer for
exon 3 or had disrupted one of these two sites. This localized the putative insertion to a
196 bp region that spanned the third intron. We then used inverse PCR to identify the
distal breakpoint of the insertion, followed by sequence analysis to identify sequences
homologous to a roo element LTR. Primers internal to the roo element were then used
with the upstream primer for exon 3 and the downstream primer for exon 4 to amplify the
two ends of the insertion, producing amplicons of the expected size and sequence.
Additional analysis suggested the insertion may not be a full length wild-type roo
The Su(z)21.b7 deficiency breakpoints were amplified by PCR from Su(z)21.b7/CyO
genomic DNA using the upstream primer, (95delus) 5’-TGTTCGGTCCCAAAGAAGC-
3’, and downstream primer, (95delds4) 5’-TGATCAAGGAAAATGTGTATTTTAGC-
3’. While these primers are predicted to generate a 5,262 bp PCR product from wild-type
DNA, they instead amplified a 1.5 kb fragment, consistent with the results of our
Southern analyses of Su(z)21.b7. This amplicon was subcloned into the TOPO-TA vector
(Invitrogen), and ten independent clones were end-sequenced with the M13 forward and
reverse primers to identify the sequence at the junction of the deficiency breakpoints. The
sequence, 5’-CCAAGGTTCTTAGTTCT-3’, contains a 4 bp insertion at the junction
Sequence data for Su(z)2 mutations have been deposited in GenBank and
correspond to accession numbers FJ897446-FJ897460. The roo element/genomic DNA
junction sequences for Su(z)2s95 and the breakpoint sequence for Su(z)21.b7 correspond to
GenBank accession numbers FJ876147-FJ876149.
The s14 mutation is caused by a G to A mutation in Psc that abolishes the exon 5
splice site. This mutation is predicted to truncate the Psc protein and may result in a
protein that is similar in size to that encoded by Psc1, which is also lethal in trans to
Su(z)21. The Pscs14 sequence data have been given accession number FJ917397.
Our studies began with five extant alleles: Su(z)21, Su(z)21.b7, Su(z)24-31, Su(z)24-34,
and Su(z)2h29 (GELBART 1971; KALISCH and RASMUSON 1974; WU and HOWE 1995).
Su(z)21, described above, was induced by EMS, suppresses z1, and shows SSNC with
Psc1. Su(z)21.b7 was recovered as an X-ray-induced l-o-f derivative of Su(z)21 and neither
suppresses z1 nor shows SSNC with Psc1 (WU 1984; ADLER et al. 1991; WU and HOWE
1995). Su(z)24-31 and Su(z)24-34 represent distinct isolates derived from our stock of
Su(z)24, which appears to be an unstable allele (MATERIALS AND METHODS). Su(z)24 had
been induced by X-rays and behaved as a g-o-f allele that was lethal in trans to Su(z)21. It
also suppressed z1 and showed SSNC with Psc1 although, in both cases, its phenotype
was weaker than that of Su(z)21 (GELBART 1971; WU et al. 1989; WU and HOWE 1995).
The Su(z)24-31 isolate remains a suppressor of z1 but shows a degree of SSNC with Psc1
that exceeds that observed with Su(z)24 (data not shown); whereas Psc1 +/+ Su(z)24
animals had a viability of ~33% as compared to wild type, Psc1 +/+ Su(z)24-31 animals
are not viable. The second isolate, Su(z)24-34, differs from Su(z)24-31 in that it is a weaker
suppressor of z1 and shows only weak, if any, SSNC with Psc1 (data not shown). Finally,
Su(z)2h29 is an EMS-induced l-o-f allele that is also lethal when heterozygous with
Su(z)21 but neither suppresses z1 nor exhibits SSNC with Psc1 (WU and HOWE 1995).
A genetic screen for new alleles of Su(z)2: We undertook a mutagenesis to
generate additional Su(z)2 alleles, anticipating that the molecular genetic analysis of such
alleles would identify important protein domains and elucidate how the structure of
Su(z)2 contributes to its function. Previous attempts to generate Su(z)2 alleles by
screening for mutations that failed to complement deficiencies deleting both Psc and
Su(z)2 were largely unsuccessful (WU and HOWE 1995). These screens tested over 17,000
mutagenized second chromosomes and recovered eight alleles of Psc but only one of
Su(z)2, indicating a large bias against the recovery of Su(z)2 alleles. To shift this bias
towards Su(z)2, we conducted an F2 screen for EMS-induced mutations that are lethal
when heterozygous with Su(z)21 (Figure 2; MATERIALS AND METHODS). Since Su(z)21 is
lethal when heterozygous with deficiencies of the locus, the l-o-f Su(z)21.b7 allele, both
isolates of the g-o-f Su(z)24 allele, and the l-o-f Su(z)2h29 allele, we reasoned that this
strategy would allow recovery of both l-o-f and g-o-f alleles of Su(z)2. Furthermore, since
Su(z)21 displays SSNC with Psc1, this strategy also had the potential of recovering
extragenic mutations in genes, such as Psc, whose products may interact with the Su(z)2
We screened over 14,000 mutagenized dp cn bw second chromosomes and
identified seven Su(z)21 interacting mutations (SIMs): s14, s15, s20, s21, s36, s84, and
s95. In addition, we independently identified a spontaneous mutation (sM) in our Canton
S wild-type stock that behaved like a SIM (MATERIALS AND METHODS). Taken together,
the eight mutations showed a range of reduced viability when heterozygous with Su(z)21
(Table 1; data for s14 are not shown). Note that we calculate the viability of flies
heterozygous for two alleles of Su(z)2 as the percentage of such flies emerging from a
cross in which females heterozygous for one allele are crossed to males heterozygous for
the other allele. Under ideal conditions, wild-type viability is expected to give a score of
33% with this mating scheme (MATERIALS AND METHODS, legend to Table 1).
To characterize these mutations further, we crossed each to the Su(z)21.b8 deletion
that removes both Psc and Su(z)2, as well as to Su(z)21.b7, Su(z)24-31, Su(z)24-34, and
Su(z)2h29 (Table 1). These crosses revealed that we had identified putative mutations in
both Psc and Su(z)2, as predicted. These are exemplified by s14, s84, and s95, which are
all lethal when heterozygous with Su(z)21.b8. s14 proved to be a new allele of Psc, as it
fails to complement l-o-f alleles of Psc but is viable in trans to Su(z)21.b7, Su(z)24-31,
Su(z)24-34, and Su(z)2h29 (data not shown; MATERIALS AND METHODS). In contrast, s84
and s95 fail to, or only minimally, complement Su(z)21.b7 (Table 1), suggesting they are
new alleles of Su(z)2. Consistent with this, both mutations fail to complement one
another (Table 2), as well as Su(z)24-31, Su(z)24-34, and Su(z)2h29 (Table 1). The recovery
of these three alleles, one identifying Psc and two identifying Su(z)2, validated the
efficacy of our mutagenesis and suggested that the remaining mutations would be
The behavior of the remaining five mutations (s15, s20, s21, s36, and sM) was
notable. First, although they all show reduced viability when heterozygous with Su(z)21,
they differ in the strength of their lethal interaction: s20 and s36 are completely lethal,
s21 and sM are weakly viable, and s15 shows significant viability (Table 1). Second, their
behavior in trans to Su(z)24-31 demonstrates that these alleles do not represent a simple
allelic series; whereas s15 proved to be the most viable of the five when heterozygous
with Su(z)21, it is not among the four (s20, s21, s36, and sM) that show significantly
increased viability in trans to Su(z)24-31. Third, all heterozygous combinations of these
five alleles show some degree of viability, s15 again distinguishing itself as the least able
among the five to promote viability (Table 2). Note that these alleles are also viable in
trans to s84 and s95 (Table 2) and that s36 is homozygous viable (data not shown).
Fourth, and perhaps most surprisingly, each is markedly viable in trans to the l-o-f alleles
Su(z)21.b8, Su(z)21.b7, and Su(z)21.h29 (Table 1). Taken together, these data made it difficult
to assign these mutations unambiguously to Su(z)2. However, as described below,
molecular analysis revealed that all except s15 either grossly disrupted the structure of
Su(z)2 or contained lesions within the exons of the gene.
Molecular analysis of Su(z)2 alleles: We carried out a molecular analysis of the
Su(z)2 locus for Su(z)21, Su(z)21.b7, Su(z)24-31, Su(z)24-34, Su(z)2h29, and the seven SIMs
that we believed would prove to be alleles of Su(z)2 (s15, s20, s21, s36, s84, s95, and
sM). Southern analyses revealed that Su(z)21.b7, Su(z)24-34, and s95 contain gross
structural changes. Except for s15, all of the remaining eight alleles were found to be
structurally normal by Southern analysis but to contain discrete lesions within Su(z)2 as
identified by sequencing of the exons (below; see MATERIALS AND METHODS and Table
S1 for additional details). We have therefore formalized the nomenclature for s20, s21,
s36, s84, s95, and sM by giving them a base name of Su(z)2: Su(z)2s20, Su(z)2s21,
Su(z)2s36, Su(z)2s84, Su(z)2s95, and Su(z)2sM. Although we were unable to find any change
associated with s15, we have tentatively named this SIM Su(z)2s15 based on its behavior
in complementation analyses. Below we describe the lesion associated with Su(z)21, the
founding allele of the locus, after which we detail the structure of the three grossly
rearranged alleles and then the six alleles resulting from point mutations.
Su(z)21 contains an L120F missense mutation as well as a K284* nonsense
mutation, which occurs shortly after the helix-turn-helix (HTH) domain (Figure 3) and is
expected to produce a truncated protein. The L120F missense mutation is located in a
region between the RF and HTH domains that will be discussed further below. The
Su(z)21.b7 derivative of Su(z)21 carries the K284* mutation of Su(z)21 as well as a ~3.5 kb
deletion of the first two exons and a four bp insertion (TTCT) at the site of the deletion
(Figure 3; MATERIALS AND METHODS; see Table S1 for sequence data regarding regions
of Su(z)21 and Su(z)21.b7 lying C-terminal to K284*). This finding differs from that of a
previous study, which reported a deletion of only ~2 kb (BRUNK et al. 1991b). Further
analysis of Su(z)21.b7 (MATERIALS AND METHODS) identified a putative TATA box
promoter sequence 8 bp upstream of the Su(z)21.b7 breakpoint. The presence of this
putative promoter sequence is consistent with observations that Su(z)21.b7 is competent
for transcription (ALI and BENDER 2004).
Southern and sequence analyses revealed that Su(z)24-34 and Su(z)2s95 are complex
mutations. Su(z)2s95 contains a roo or roo-like element inserted in the third intron, as well
as a Q370* nonsense mutation in exon 5 (Figure 3, MATERIALS AND METHODS).
Similarly, Su(z)24-34 contains an ~9 kb insertion in the first intron as well as an 8 bp
deletion in exon 5, that results in a three amino acid frameshift followed by a nonsense
codon (E235K, Q236K, T237R, and K238*) (Figure 3). The other Su(z)24 isolate,
Su(z)24-31, retains the 8 bp deletion but does not carry the insertion (MATERIALS AND
METHODS; see Table S1 for sequence data regarding regions of Su(z)24-31 and Su(z)24-34
lying C-terminal to K238*). Consistent with this structural difference between the two
alleles, Su(z)24-31 displays a genetic behavior that differs from that of Su(z)24-34. In
particular, Su(z)24-31 complements Su(z)2s36, while Su(z)24-34 does not (Table 1) and, as
mentioned earlier, is a stronger suppressor of z1 and shows a stronger interaction with
The genetic behavior of Su(z)2s84 and Su(z)2h29 indicated that they would have
alterations in Su(z)2, and this proved to be true. The Su(z)2s84 allele is caused by a Q124*
nonsense mutation just after the RF (Figure 3). The small size of the predicted Su(z)2s84
protein suggests that its phenotype should be severe, consistent with observations that its
capacity to complement other alleles is poor relative to that of several other alleles (Table
1). Su(z)2h29 results from a G to A transition that abolishes the 5’ splice acceptor site for
exon 4. If exon 3 is able to splice over exon 4 to exon 5, this allele is predicted to cause a
frameshift that extends from amino acid 139 to 186 after which a nonsense codon is
encountered (Figure 3).
The four remaining alleles (Su(z)2s20, Su(z)2s21, Su(z)2s36, and Su(z)2sM) did not at
first appear to be alleles of Su(z)2 because they complement Su(z)21.b8, Su(z)21.b7, and
Su(z)2h29. However, as recombination analyses placed all four in the vicinity of the Su(z)2
complex (data not shown), we proceeded with sequence analyses and discovered that all
four contain missense mutations in the HR of Su(z)2 (Figure 3). The lesions associated
with Su(z)2s36 and Su(z)2sM alter the structure of the RF. The Su(z)2s36 allele contains a
S58N missense mutation located within the first Zn++ coordinating domain of the RF,
while Su(z)2sM contains a D49H missense mutation in the loop between the two Zn++
coordinating domains. Although each of these mutations is predicted to de-stabilize the
RF, both are hemizygous viable (Table 1). The mutations associated with Su(z)2s20 and
Su(z)2s21 are located between the RF and HTH domains. The Su(z)2s20 allele contains a
P101S change, while the Su(z)2s21 allele contains a E136K change.
We did not find any change associated with the Su(z)2s15 allele. This allele may
contain an alteration outside the coding sequence of Su(z)2 that affects either the
regulation of the gene or the stability of its mRNA. Both of these possibilities would be
consistent with the genetic behavior of this allele. Alternatively, Su(z)2s15 may represent a
mutation in a gene that interacts with Su(z)2. Unfortunately, the semi-lethality of this
mutation complicates an accurate mapping of its location.
Su(z)2D mutations are alleles of Su(z)2: The discovery that Su(z)2s20, Su(z)2s21,
Su(z)2s36, and Su(z)2sM are alleles of Su(z)2 prompted us to reconsider our prior genetic
analyses of three alleles that had been previously proposed to represent a third
complementation group of the Su(z)2 complex (WU and HOWE 1995). The existence of
this third complementation group, called Su(z)2D, had been suggested primarily by the
behavior of Su(z)25, which suppresses z1 in a dominant fashion despite the fact that it
deletes both Psc and Su(z)2. Additional support for the existence of Su(z)2D came from
the complementation patterns of Su(z)2De26, Su(z)2Deos, and Su(z)31, all three of which
were believed to represent Su(z)2D (WU and HOWE 1995). Interestingly, the behavior of
these three alleles is reminiscent of the SIMs. Our current findings show that, in fact,
Su(z)2De26, Su(z)2Deos, and Su(z)31 all carry mutations within Su(z)2.
First, we noted that the genetic behavior of Su(z)2De26 strongly resembles that of
Su(z)2s36 (WU and HOWE 1995; RBE and CtW, unpublished): Su(z)2De26 is lethal when
heterozygous with either Su(z)21 or Su(z)24-34, but shows significant viability when
homozygous or heterozygous with Su(z)21.b8 or Su(z)21.b7. Remarkably, we found that
Su(z)2De26 is similar to Su(z)2s36 at the molecular level as well, containing a missense
mutation (H53Y) in the RF (Figure 3). As His53 is required to form the second Zn++
coordinating domain in the RF, this mutation would be expected to severely disrupt the
RF and compromise Su(z)2 function. Indeed, mutations disrupting the Zn++ coordinating
domains within the RF of Bmi-1 disrupt the ability of Bmi-1 to interact with other
proteins as well as localize to subnuclear regions (ALKEMA et al. 1997; HEMENWAY et al.
1998). Interestingly, the Su(z)2De26 mutation predicts a protein that would be structurally
similar to that produced by Psce23, which contains a C268Y change expected to disrupt
the RF of Psc. However, unlike Psce23, which is homozygous and hemizygous lethal,
Su(z)2De26 shows significant homozygous and hemizygous viability. This finding
suggests that the RF is not required for Su(z)2 function, which is in stark contrast to the
requirement of the RF for wild-type Psc function, or that the mutated RF of the
Su(z)2De26 protein retains some wild-type function.
Su(z)2Deos and Su(z)31 also display some similarities with the SIMs; like Su(z)2s20,
Su(z)2s21, and Su(z)2sM, they show reduced viability in trans to Su(z)21 and higher
viability in trans to Su(z)21.b7 and Su(z)24-34. However, they differ from these three SIM
alleles in that they display complete or nearly complete lethality in trans to Su(z)21.b8
(WU and HOWE 1995; RBE and CtW, unpublished), with separate studies suggesting
Su(z)2Deos to be the more severe of the two (WU and HOWE 1995). We found that both
Su(z)2Deos and Su(z)31 contain mutations predicted to truncate Su(z)2 after the HR and
more C-terminal to the K284* nonsense mutation of Su(z)21 (Figure 3). Su(z)2Deos
contains a Q448* nonsense mutation while Su(z)31 has a G inserted after nucleotide
position 1873, resulting in a seven amino acid frameshift followed by a nonsense codon
(E577R, E578G, A579G, R580A, S581E, I582Y, N583Q, S584*)(Figure 3; MATERIALS
AND METHODS; see Table S1 for sequence data regarding regions of Su(z)31 lying C-
terminal to S584*). That Su(z)31, Su(z)2Deos, and Su(z)21 are predicted to produce
increasingly shorter proteins as well as increasingly more severe phenotypes (Tables 1
and 2; WU and HOWE 1995; RBE and CtW, unpublished) suggests that the lethality
associated with Su(z)2Deos and Su(z)31 stems at least in part from the loss of critical
functions encoded by the CTR sequences, perhaps specifically by amino acids 285 - 576.
Furthermore, the viability of Su(z)2Deos and Su(z)31 in trans to Su(z)21.b7 may indicate a
capacity of Su(z)21.b7 to provide some function and/or reflect the contribution Psc, which
remains intact upstream of Su(z)21.b7 but is lacking from the Su(z)21.b8 deletion.
Su(z)2s20, Su(z)2s21, Su(z)2s36, Su(z)2sM, and Su(z)2De26 display negative
complementation with Su(z)21, Su(z)24-31, and/or Su(z)24-34: Our molecular
confirmation that Su(z)2s20, Su(z)2s21, Su(z)2s36, Su(z)2sM, and Su(z)2De26 contain missense
mutations within the HR of Su(z)2 was interesting given their genetic behavior. While all
five are quite viable when hemizygous in trans to Su(z)21.b8, three of the alleles, Su(z)2s20,
Su(z)2s21, and Su(z)2sM, are lethal or semi-lethal when in trans to Su(z)21, and the
remaining two, Su(z)2s36 and Su(z)2De26, are lethal in trans to both Su(z)21 and Su(z)24-34,
with Su(z)2De26 also being lethal in trans to Su(z)24-31 (RBE and C-tW, unpublished).
Taken together, the genetic behavior of these alleles is consistent with negative
complementation, a type of interallelic interaction in which the activity of one allele is
specifically poisoned by another (FINCHAM 1966). Typically, negative complementation
is seen when two alleles, m1 and m2, display a phenotype that is stronger when they are
heterozygous with each other than when either is homozygous (i.e. m1/m2 is worse than
m1/m1 and m2/m2) (FINCHAM 1966), or when m1 and/or m2 is heterozygous with a
deficiency (i.e. m1/m2 is worse than m1/Df and/or m2/Df) (BICKEL et al. 1996). This
latter situation mirrors the behavior of Su(z)2s20, Su(z)2s21, Su(z)2s36, Su(z)2sM, and
Su(z)2De26 with respect to Su(z)21. For example, we find complete lethality when
Su(z)2s36 is heterozygous with Su(z)21 even though Su(z)2s36 is viable when hemizygous
in trans to Su(z)21.b8. This negative complementation cannot be attributed to an
interaction with another mutation on the Su(z)21 chromosome because Su(z)21.b8, which
does not display negative complementation with Su(z)2s36, is a derivative of Su(z)21. Note
the additional levels of negative complementation associated with Su(z)2s36 and
Su(z)2De26 (Table 1; RBE and CtW, unpublished); whether these additional levels of
negative complementation stem from the lesions of Su(z)2s36 and Su(z)2De26 falling
directly in the first and second, respectively, Zn++ coordinating domains of the RF is as
Su(z)2s20 and Su(z)2s21 identify conserved subregions within the HR: The
mutations associated with Su(z)2s20 and Su(z)2s21 are interesting because they are located
in the HR, but do not affect either the RF or HTH domains. To better understand this
region, we generated ClustalX alignments (THOMPSON et al. 1997; CHENNA et al. 2003)
between the predicted protein products of Su(z)2, Psc, a third Drosophila homolog called
lethal(3)73Ah (l(3)73Ah; IRMINGER-FINGER and NOTHIGER 1995), as well as human and
mouse bmi-1 and mel-18. Although it is not clear whether l(3)73Ah is a PcG gene, it
contains the HR, but not the CTR (Figure 1). We found that the lesions associated with
Su(z)2s20 and Su(z)2s21 fall within two highly conserved subregions located between the
RF and HTH domains (Figure 4). We will refer to these conserved subregions as CSR1
CSR1 contains a core sequence of Y K L V P G L that is conserved in all seven
proteins examined and mutated in the Su(z)2s20 protein, where the proline is replaced by a
serine (Figure 4). Database searches (ELM, ProSite, Pfam, SMART) did not identify any
known protein motifs within this region and, while a number of potential sites for post-
translational modification are present, there is currently no evidence for post-translational
modification of this region. CSR2 is less well defined than is CSR1, and contains a core
sequence of [E/D] Ψ Ψ S L S [I/L] [E/Q] [F/Y]. Database searches (ELM, ProSite, Pfam,
SMART) did not identify any protein motif, but did reveal a putative CK2
phosphorylation site that is conserved in Bmi-1, whose localization to chromatin
fluctuates throughout the cell cycle in a phosphorylation dependent manner (VONCKEN et
al. 1999). The lesion associated with Su(z)2s21 substitutes a lysine for the glutamic acid in
this putative phosphorylation site, which is also the first glutamic acid in the core
It is interesting to note that the L120F missense mutation associated with Su(z)21
falls in the region between CSR1 and CSR2 (Figure 4). Although this mutation does not
appear to identify a region of strong conservation, it does alter a leucine that is conserved
in both human and mouse mel-18 (Figure 4). Therefore, although our consideration of the
structural basis for the Su(z)21 phenotype has centered on the K284* nonsense mutation
and the CTR truncation that it predicts, it remains possible that the L120F missense
mutation also contributes to the severity of Su(z)21.
The CTR of Su(z)2 is intrinsically disordered: The CTRs of Su(z)2 and Psc are
important domains as they are essential for the function of these proteins in vivo (this
report and KING et al. 2005) as well as in vitro (KING et al. 2005; LO et al. 2009).
However, consistent with other studies (BRUNK et al. 1991a; VAN LOHUIZEN et al. 1991;
LO et al. 2009), we were unable to identify significant regions of homology or conserved
functional domains, although both CTRs contain a myriad of sites for potential post-
translational modification. Using SMART analysis, however, we discovered that each
CTR is predicted to contain high levels of intrinsic protein disorder. Figure 5 shows a
disorder probability plot using the default parameters of DisEMBL (Figure 5A) as well as
the regions of the Su(z)2 CTR that are predicted to be disordered by all three parameters
(Loops/coil, Remark-465, and Hot-loops) (Figure 5B)(LINDING et al. 2003). Disordered
regions were merged in instances where peak distances were less than 20 amino acids
apart for one of the predictors. Note that these predictions are conservative, as they
require a statistically significant score from all three parameters, and the level of
predicted disorder in the CTR increases dramatically if only two out of the three
parameters are considered. We obtained similar results for Psc (Figure 6).
Of the many PcG genes known, several belong to gene pairs: Psc and Su(z)2
(ADLER et al. 1989; WU et al. 1989; WU AND HOWE 1995), ph-p and ph-d (DURA et al.
1987), pho and phol (BROWN et al. 2003), and esc and escl (WANG et al. 2006). These
gene pairs show some degree of redundancy or similarity between the two members of a
pair, and are generally typified by double mutant combinations in which the phenotype of
flies carrying a mutation in each gene is worse than that of flies carrying a mutation in
only one of the genes. Here we have focused on the Su(z)2 gene of the Psc-Su(z)2 gene
pair. In particular, we have carried out a molecular genetic analysis of fourteen Su(z)2
alleles, seven of which (Su(z)2s15, Su(z)2s20, Su(z)2s21, Su(z)2s36, Su(z)2s84, Su(z)2s95, and
Su(z)2sM) were newly generated for this study and three of which (Su(z)2De26, Su(z)2Deos,
and Su(z)31) were previously thought to represent a third region of the Su(z)2 complex
called Su(z)2D. Here, we discuss negative complementation at the locus and then
compare the structure of Su(z)2 to Psc in the context of the CTR and its disordered
Negative complementation at the Su(z)2 locus: The allele specific non-
complementation of Su(z)2s20, Su(z)2s21, Su(z)2s36, Su(z)2sM, and Su(z)2De26 with Su(z)21, Su(z)24-
31, and/or Su(z)24-34 represents a rare type of genetic interaction called negative complementation
(BICKEL et al. 1996; FINCHAM 1966). In Drosophila, negative complementation has been
described at Notch (FOSTER 1975; PORTIN 1975), dEGFR (RAZ et al. 1991; CLIFFORD and
SCHUPBACH 1994), ord (BICKEL et al. 1996; BICKEL et al. 1997), α-tubulin84B (MATTHEWS and
KAUFMAN 1987), and Mos1 (LOHE et al. 1996). All of the proteins encoded by these genes
require protein-protein interactions for wild-type function.
Negative complementation at Su(z)2 highlights the multidomain structure of the Su(z)2
protein, as it occurs between alleles that contain missense mutations in the HR and either Su(z)21,
Su(z)24-31, or Su(z)24-34, all three of which are predicted to generate proteins lacking nearly all of
the CTR. As this interaction is not associated with other Su(z)2 alleles predicted to delete CTR
sequences, negative complementation at the locus may be specific for Su(z)21, Su(z)24-31, and
Su(z)24-34 and not a general consequence of partial or complete CTR loss. Alternatively, CTR
sequences including and lying C-terminal to lysine 284, which is the point of truncation in the
longest of these three truncation alleles, may antagonize the capacity of longer proteins, such as
those predicted by Su(z)2Deos and Su(z)31, to effect negative complementation, reminiscent of
proposals of intramolecular regulation for Psc and Su(z)2 (SHARP et al. 1994; WU and HOWE
1995; PLATERO et al. 1996; KING et al. 2005). Regardless, as the Su(z)21 and Su(z)24-31 proteins
are predicted to contain little more than the HR, these observations suggests that much, if not all,
of the HR (including the RF, CSR1, CSR2, and the HTH) can function independently of the rest
of the protein. This interpretation likely applies also to Su(z)24-34 because the negative
complementation observed between this allele and Su(z)2e26 argues that it produces a product
even though it carries a large insertion. These observations are consistent with the in vitro assays
of truncated Su(z)2 proteins (LO et al. 2009) and reminiscent of the structural organization of
Psc, which also consists of more than one functional domain (WU and HOWE 1995; KING et al.
Although the potential participation of Su(z)2 in a larger complex can complicate models
explaining negative complementation, one interpretation is that an antimorphic nature (WU and
HOWE 1995) of the Su(z)21, Su(z)24-31, and Su(z)24-34 proteins compromises or poisons the
function encoded by the missense alleles which, however, are able to support wild-type or nearly
wild-type viability on their own (Table 1). In light of models for negative complementation that
invoke protein-protein interactions, it may be that the amino acid substitutions within the HR that
are encoded by the missense alleles may compromise the ability of the resulting mutant Su(z)2
protein to interact properly with itself or other factors, protein or otherwise, either transiently or
as part of a more stable complex. For example, the RF in the Su(z)2De26 protein may be
compromised such that it cannot compete effectively against the Su(z)21 protein in a
Su(z)21/Su(z)2De26 heterozygote, resulting in complexes that are nonfunctional or abnormal,
simultaneously reducing the amount of functional Su(z)2De26-containing complexes. The
scenario in which Su(z)2 interacts with another protein is supported by the behavior of Psc,
which interacts with Ph, Pc, and Sce (KYBA and BROCK 1998; FRANCIS et al. 2001), and in vitro
observations that the HR of Psc and Su(z)2 is important for complex formation (KING et al.
2005; LO et al. 2009). Furthermore, co-localization of Su(z)2 with Psc and Pc in polytene
chromosomes suggests that Su(z)2 can associate with PRC1 or another PcG complex in vivo
(RASTELLI et al. 1993; PLATERO et al. 1996; SHARP et al. 1997).
If Su(z)2 functions as a homodimer, our observations would be consistent with the
missense mutations causing the affinity of the resulting proteins for themselves to be less than
their affinity for Su(z)21, thereby creating inactive Su(z)21-containing dimers. Su(z)21 could
compromise this dimer in many ways, including acting in a prion-like fashion to inactivate
Su(z)2De26, mislocalizing Su(z)2De26 to a subcellular region that does not support Su(z)2
activity, or creating a dimer with abnormal activity. On the other hand, if Su(z)2 functions as a
heterodimer, our findings would be consistent with the missense mutations causing the affinity of
the resulting Su(z)2 protein for its partner to be less than that of Su(z)21. In this scenario,
dimerization would favor the inclusion of Su(z)21, which would, again, compromise the dimer.
Both scenarios assume that the missense mutations decrease the capacity of the resulting proteins
for intermolecular interactions, consistent with their location in the RF.
Su(z)2s20 and Su(z)2s21 are especially noteworthy in that the causative lesions of these two
alleles fall within the HR but outside of the RF and HTH motifs, reminiscent of studies
suggesting that sequences just C-terminal to the RF of Bmi-1 are important for Bmi-1 function
(HEMENWAY et al. 1998; SATIJN and OTTE 1999). Su(z)2s20 is predicted to alter a proline residue
in the CSR1 core sequence YKLVPGL, which is completely conserved in Bmi-1 and Mel-18.
This change, in conjunction with the negative complementation observed with Su(z)2s20, suggests
that CSR1 mediates protein-protein interactions. This interpretation is supported by the crystal
structure of the Bmi-1/Ring1B heterodimer, which reveals that the region we designate as CSR1
lies at the interface between these two proteins. Specifically, the proline residue appears to
establish the three dimensional geometry of two alpha helical regions in Bmi-1, α3 and α4, that
contain residues which form salt bridges with Ring1B as well as residues that stabilize these
interactions (Figure 4; BUCHWALD et al. 2006; LI et al. 2006). Based on this, we believe that a
substitution of a serine for this proline would alter the capacity of Su(z)2 to interact with other
Su(z)2s21 substitutes a lysine for the first glutamic acid of the [E/D] Ψ Ψ S L S [I/L]
[E/Q] [F/Y] motif in a region we refer to as CSR2. The conservation of CSR2 is not as
prominent as that of CSR1 and, perhaps consistent with this, the negative complementation of
Su(z)2s21 with Su(z)21 is not as strong as that of Su(z)2s20 (Table 1). As the region of Bmi-1 that is
orthologous to CSR2 was not included in the crystal structures mentioned above (BUCHWALD et
al. 2006; LI et al. 2006), we cannot postulate how the amino acid change directed by Su(z)2s21
would affect the specificity and/or avidity of any potential interaction between the Su(z)2 protein
and other factors. However, as this change resides within a putative CK2 phosphorylation site,
and the activity of Bmi-1 is modulated by phosphorylation (VONCKEN et al. 1999), our findings
suggest that such modulation could function by mediating the regulation of interactions between
Bmi-1 and other factors.
At first glance, the negative complementation of Su(z)2 alleles would appear to be in
stark contrast to the genetic behavior of structurally similar alleles of Psc. Psce23 predicts a
C268Y missense mutation in the RF that is analogous to that of Su(z)2De26, yet it displays
intragenic complementation with Psch28, Psch30, and Psce22, all three of which delete significant
portions of the CTR (WU and HOWE 1995; KING et al. 2005). In fact, it is this complementation
and subsequent biochemical and molecular analyses which indicated that Psc contains multiple
domains that are functionally separable. Psce23 does not, however, complement Psc1 (WU and
HOWE 1995), which encodes the truncation of Psc that is most similar in structure to the
truncation of Su(z)2 encoded by Su(z)21 (KING et al. 2005). Our current analysis suggests this
failure could be due to negative complementation.
The CTRs of Su(z)2 and Psc: Our prediction that several recessive lethal Su(z)2 alleles
truncate Su(z)2 within the CTR recalls our earlier report that truncations removing ~40% or
more of the CTR of Psc reduce viability (KING et al. 2005). These findings argue for the in vivo
importance of the CTR of both proteins and provide further support that the function of the CTR
of Psc and Su(z)2 may be conserved despite differences in their primary amino acid sequences.
We have also found that ≥ 45% of the CTRs of both Su(z)2 and Psc are contained within
domains of predicted intrinsic disorder scattered throughout the CTR (Figures 5 and 6). As such,
the CTRs are reminiscent of intrinsically disordered proteins (IDPs), which are proteins
containing regions that do not possess a defined conformation under native conditions but adopt
specific conformations when they interact with ligands, DNA, protein, or other factors, or when
they self-associate, as is seen with prions (reviewed by DYSON and WRIGHT 2005; HANSEN et al.
2006). IDPs are generally enriched for particular amino acids, such as arginine, glutamine,
glutamic acid, lysine, proline, serine, and occasionally alanine and glycine, and their tendency
for disorder can be computationally predicted with a high degree of accuracy (VUCETIC et al.
2003; reviewed by DUNKER et al. 2001; DUNKER et al. 2002). Indeed, based on the amino acid
composition of the CTRs of Psc and Su(z)2, LO et al. (2009) also recently hypothesized the
potential of these two proteins to contain regions of disorder. Importantly, analyses of IDPs show
that intrinsic disorder in and of itself can be sufficient for function. For example, the long C-
terminal regions of linker histones are essential for their functions even though they are
intrinsically disordered and functionally interchangeable among evolutionarily diverged species
of linker histones (reviewed by HANSEN et al. 2006). In light of these features of IDPs, it may be
that the role of the CTRs of Su(z)2 and Psc in vivo and their capacity to inhibit transcription
and/or chromatin remodeling in vitro rests on regions of disorder and the capacity of such
regions to transition to an ordered state (also see LO et al. 2009).
The structural nature of the CTRs may also pertain to the capacity of mutations in Su(z)2
and Psc to suppress the effect of z1 on white gene expression. Of the alleles that truncate the
protein within the CTR, all suppress z1 and, of these, the strongest, Su(z)21, is predicted to delete
nearly all of the CTR and, hence, nearly all of the blocks of intrinsic protein disorder. In this
way, Su(z)21 resembles Psc1, which is the strongest suppressor of z1 at Psc and also leads to a
severe truncation of the CTR. Although we cannot assess the involvement of the L120F missense
mutation of Su(z)21 in suppression, the two simple truncation alleles, Su(z)2Deos and Su(z)31, rule
out any requirement of L120F for z1 suppression even as they emphasize the importance of the
CTR. Further support for a role of the CTR in the z1 phenotype comes from three truncation
alleles of Psc that, curiously, enhance z1 (WU and HOWE 1995; KING et al. 2005). These
observations may be particularly relevant, as the zeste protein also contains regions of disorder
(RBE, unpublished), has runs of glutamine and alanine in its CTR, and displays a strong
tendency to self-associate or aggregate (reviewed by Pirrotta 1991; also see CHEN and PIRROTTA
1993; ROSEN et al. 1998). The positions of the lesions of z1 and two z1-like alleles are clustered
within this CTR (PIRROTTA et al. 1987; ROSEN et al. 1998), further implicating the CTR in Zeste
function. These findings raise the possibility that cooperative and/or competitive interactions
between the disordered regions of Su(z)2, Psc, and/or Zeste may underlie the ability of z1 to
repress white and the capacity of Su(z)2 and Psc mutations to modify the z1 phenotype. Finally,
we have found that the short CTRs of both Bmi-1 and Mel-18 also contain regions that are likely
to be intrinsically disordered (data not shown), suggesting that the long CTRs of Psc and Su(z)2
may be closely related in structure and function to the minimal CTRs of their mammalian
homologs despite their very different lengths.
We would like to thank N. Francis and members of her laboratory, in particular S. Lo, for
sharing unpublished data and many thought provoking discussions. We would also like to thank
M. Ashburner, W. Bender, S. Hawley, J. Kennison, and E. Weischaus for insightful discussions,
with special thanks to S. Hawley regarding negative complementation. Finally, we would like to
acknowledge the anonymous reviewers of our manuscript for key comments, and members of the
Wu laboratory for many helpful conversations and technical assistance, and D. Schwartz for
input on protein sequence alignment. This work was supported by a Kirschstein National
Research Service Award to R.B.E., NIH grant RO1GM61936 to C.-t.W. and Harvard Medical
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Genetic analysis of SIMs
P 14 (353) 0 (339) 2 (261) 0 (202) 0 (200) 0 (259) 8 (371)
M 16 (223) 0 (182) 3 (184) 0 (135) 0 (138) 0 (154) 8 (195)
10 (123) 31 (196) 26 (165) 25 (134) 0 (218) 0 (113) 34 (149)
M 25 (186) 30 (174) 30 (252) 35 (135) 0 (108) 0 (213) 32 (249)
P 26 (136) 23 (251) 24 (187) 16 (216) 0 (182) 4 (253) 25 (177)
M 30 (128)
32 (253) 34 (207) 35 (224) 0 (157) 3 (272) 29 (214)
P 18 (257) 29 (270) 28 (183) 33 (238) 0 (132) 0 (219) 34 (270)
M 21 (260) 26 (253) 33 (229) 33 (262) 0 (127) 0 (186) 30 (287)
P 13 (111) 35 (210) 36 (199) 0 (238) 0 (144) 0 (174) 32 (176)
M 10 (201) 27 (205) 31 (285) 0 (205) 0 (248) 0 (145) 30 (114)
P 15 (185) 27 (210) 23 (337) 23 (157) 0 (107) 0 (221) 33 (166)
M 34 (144)
33 (196) 39 (163) 32 (117) 0 (123) 0 (144) 29 (100)
Table 1. Genetic analysis of SIMs. The first column lists the allele contributed by one
parent, while the first row lists the allele contributed by the other. The paternal (P) or
maternal (M) origin of the allele in the first column is indicated. For each cross, the
viability of the mutant class heterozygous for the allele in column one and the allele in
row one is indicated as a percentage, followed by the total number of flies scored in
parentheses. Two alleles that are completely viable when heterozygous with one another
are expected to have a viability equal to 33% under ideal conditions (see MATERIALS AND
Inter se crosses of SIMs
Su(z)2s15 Su(z)2s20 Su(z)2s21 Su(z)2s36 Su(z)2s84 Su(z)2s95
Su(z)2s20 P 5 (341)
M 8 (350)
Su(z)2s21 P 11 (152) 18 (179)
M 10 (230) 17 (212)
Su(z)2s36 P 5 (383) 17 (327) 15 (259)
M 3 (466)
10 (325) 21 (195)
Su(z)2s84 P 11 (425) 16 (524) 19 (258) 17 (256)
M 11 (303) 20 (480) 22 (224) 20 (435)
Su(z)2s95 P 11 (401) 17 (364) 22 (221) 19 (621) 0 (329)
M 6 (494) 19 (407) 26 (235) 20 (615) 0 (404)
P 18 (231) 27 (206) 23 (222) 28 (251) 24 (156) 30 (280)
18 (152) 32 (106) 29 (350) 30 (162) 33 (214) 37 (135)
Table 2. Inter se crosses of SIMs. See Table 1 for explanation of format.
Genetic analysis of Su(z)2D alleles
Su(z)2s15 Su(z)2s20 Su(z)2s21 Su(z)2s36 Su(z)2s84 Su(z)2s95
Su(z)2De26 P 9 (102) 33 (250) 36 (214) 38 (193) 21 (165) 27 (240) 36 (118)
M 15 (156) 33 (115) 31 (318) 35 (226) 20 (183) 25 (199) 33 (222)
P 14 (160) 33 (334) 27 (142) 27 (327) 20 (282) 16 (190) 37 (159)
M 10 (175) 29 (241) 34 (218) 39 (157) 23 (167) 24 (173) 33 (130)
P 15 (193) 27 (171) 30 (332) 22 (149) 19 (156) 25 (207) 33 (190)
23 (100) 25 (194) 29 (117) 37 (111) 26 (188) 23 (332) 39 (152)
Table 3. Genetic analysis of Su(z)2D alleles. See Table 1 for explanation of format.
Figure 1. Comparison of Drosophila Su(z)2, Psc, and l(3)73Ah with human Bmi-1 and
Mel-18. The homology region (HR) and C-terminal region (CTR) are labeled. The RF is
noted in red and the helix-turn-helix (HTH) is noted in blue. Regions enriched for
specific amino acids are shown in black and labeled with the relevant residue. The figure
is drawn to scale.
Figure 2. Screen for Su(z)21 interacting mutations (SIMs). dp cn bw/dp cn bw ; III/III
males, isogenic for chromosomes II, marked with dp cn and bw, and III, were fed EMS
and mated to T(2;3)apXa/II; Cbx Ubx gl3 virgin females. Single T(2;3)apXa/(dp cn bw)*;
III * F1 males bearing mutagenized (*) autosomes were then mated in vials to
Su(z)21/CyO; III/III virgin females. The vials were subsequently scored for the absence
Su(z)21/(dp cn bw)*; III/III* F2 progeny, indicating that at least one of the mutagenized
autosomes may carry a SIM. As T(2;3)apXa causes a dominant notched wing phenotype,
and the CyO balancer causes a dominant curly wing phenotype, vials lacking Su(z)21/(dp
cn bw)*; III/III* F2 progeny were identified by the absence of flies with normal (non-
notched, straight) wings. Note that use of T(2;3)apXa allowed for the simultaneous testing
of both autosomes because it is a translocation between chromosomes II and III.
Figure 3. Analysis of Su(z)2 alleles. A. Structure of the wild-type Su(z)2 locus and
insertions and deletions associated with Su(z)2 mutations. Psc (not shown) is located to
the left. Exons 1-6 are shown as numbered rectangles. The RF is shown in red, and the
HTH is shown in blue (Exon 4/5 junction). Noncoding sequence is shown in gray. The
TATA notation shown upstream of Exon 1 is the putative TATA box identified 8 bp
upstream of the Su(z)21.b7 breakpoint. R=EcoRI and N=NotI. B. Frameshift, nonsense,
and missense mutations associated with Su(z)2 mutations. Nonsense mutations are shown
above the protein and are divided into two classes: those associated with a frameshift
followed by a stop codon (FS*, top level) and those associated with only a stop codon (*,
bottom level). Missense mutations are shown below the protein. The RF is shown in an
exploded view, with bases that are altered by Su(z)2 mutations shown in black. Zn++
coordinating residues are indicated. Su(z)21 (green), Su(z)24-34 (orange), Su(z)2h29
(purple), and Su(z)2s95 (blue) are complex and have been color coded to highlight the
multiple mutations they contain. Su(z)21 contains an L120F missense mutation as well as
a K284* nonsense mutation. Su(z)24-31 and Su(z)24-34 both contain an 8 bp deletion in
exon 5 (not shown) that generates a three amino acid frameshift ending in a K238*
nonsense codon (B). Su(z)24-34 differs from Su(z)24-31 in that it also contains an insertion
(A) that has been localized to a 1.6 kb ClaI/BamHI fragment in the distal half of intron 1.
Su(z)2h29 contains a G to A transition that disrupts a 5' acceptor site for exon 4 (A) and is
predicted to result in a frameshift ending in a T186* nonsense codon (B). Su(z)2s95
contains a roo or roo-like (A) as well as a Q370* nonsense codon (B). *, Su(z)21.b7, a
derivative of Su(z)21, carries a deletion (A), a four bp insertion (TTCT, not shown) at the
site of the deletion, as well as the K284* nonsense codon that is also present in Su(z)21
(B). We have not determined whether Su(z)21.b7 also contains the L120F missense
mutation that is found in Su(z)21. Both A and B are drawn to scale.
Figure 4. ClustalX alignment of Su(z)2, Psc, and L(3)73Ah from Drosophila with Bmi-1
and Mel-18 from both H. sapiens and M. musculus. The alignment generated by ClustalX
is focused on the region of the proteins located between the RF and HTH domains, and
begins with the last conserved cysteine residue in the RF. Amino acid positions are
indicated on the left. Amino acids highlighted in red are identical (equivalent to * in
ClustalX) among the proteins indicated, while strongly conserved amino acids
(equivalent to : in ClustalX) are highlighted in yellow. More weakly conserved amino
acids (equivalent to . in ClustalX) are not highlighted. The CSR1 core sequence is
underlined twice, with the Su(z)2s20 mutation indicated above. The Su(z)21 missense
mutation is shown to the right of CSR1. Residues that correspond to alpha helical regions
3 and 4 in the Bmi-1/Ring1B structure are shown. The CSR2 core sequence is underlined
once, with the Su(z)2s21 mutation indicated above.
Figure 5. Disorder analysis of Su(z)2. A. DisEMBL analysis of Su(z)2 showing the
predictions for loops or coil (blue), remark-465 (green), and hot-loops (red). B. Su(z)2
protein is shown at a scale matching that of the plot above. The RF is shown in red, and
the HTH is shown in blue. Regions of protein disorder predicted by all three methods are
shown in orange. The predicted points of truncation of the truncation alleles is shown
Figure 6. Disorder analysis of Psc. See Figure 5 for explanation of format.
II; Cbx Ubx
dp cn bw
dp cn bw ; II
(dp cn bw)*; III*
Screen for absence of flies with normal wings.