Department of Zoology and Animal Biology, University of Geneva, 30 quai Ernest-Ansermet, CH-1211 Geneva 4, Switzerland
*Author for correspondence (e-mail: pierre.spierer{at}zoo.unige.ch)
Accepted 23 May 2002
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SUMMARY |
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Key words: Drosophila melanogaster, Position-effect variegation, Heterochromatin
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INTRODUCTION |
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Another component of constitutive heterochromatin in Drosophila is the protein HP1 encoded by the Su(var)2-5 gene (Eissenberg et al., 1990; Eissenberg et al., 1992
). On polytene chromosomes, and as Su(var)3-7, HP1 is associated with pericentric heterochromatin, telomeres and some euchromatic sites (Fanti et al., 1998
; James et al., 1989
; Kellum and Alberts, 1995
). HP1 contains two main domains, the chromodomain and the chromoshadow domain. These domains are conserved in the HP1 orthologues reported in mammals (Saunders et al., 1993
; Singh et al., 1991
; Wreggett et al., 1994
) and in the two paralogues recently found in Drosophila melanogaster (Smothers and Henikoff, 2001
). The chromoshadow domain of mouse HP1 orthologues is required for protein-protein interactions and dimerisation (Brasher et al., 2000
; Jones et al., 2000
; Nielsen et al., 2001
). Dimerisation has also been seen for the HP1 homologue Swi6 of yeast (Cowieson et al., 2000
). In mammals, HP1 is found associated with Suvar39H1, the orthologue of the Drosophila modifier of PEV Su(var)3-9 (Tschiersch et al., 1994
; Aagaard et al., 1999
). Suvar39H1 contains a SET domain adjacent to a cystein-rich region carrying a histone methyl transferase activity (Rea et al., 2000
). Suvar39H1 specifically methylates the lysine 9 of histone H3, and HP1 recognises and associates with this methylated histone (Bannister et al., 2001
; Lachner et al., 2001
; Jacobs et al., 2001
). Drosophila HP1 also interacts with Su(var)3-7, as shown by the two hybrid assay in yeast (Delattre et al., 2000
), by co-immunoprecipitation from nuclear extract, and by recruitment of Su(var)3-7 by delocalised HP1 (Cléard et al., 1997
; Delattre et al., 2000
). These data suggest that HP1, Su(var)3-9 and Su(var)3-7 are associated in heterochromatin.
To investigate in vivo the function of the different Su(var)3-7 domains, we have expressed tagged fragments of Su(var)3-7 in the fly. We have analysed the chromosomal localisation of these fragments, their effect on position-effect variegation, and their ability to modify endogenous HP1 localisation. The data obtained lead us to propose a model for the function of Su(var)3-7 in the genomic silencing of position-effect variegation.
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MATERIALS AND METHODS |
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Western blot analysis
Ten males and ten females from each transgenic line were heat-shocked for 30 minutes. After 1 hour of recovery, flies were homogenised in 200 µl of protein homogenisation buffer (8 M urea, 100 mM NaH2PO4, 10 mM Tris-HCl pH 6.4). 200 µl of sample buffer (4% SDS, 17.5% glycerol 120 mM Tri-HCl pH 6.8 and 0.01% bromophenol blue) were added and the samples were boiled for 5 minutes before western analysis. Proteins were stained with an anti-HA monoclonal antibody diluted 1:100, and detected with an anti-mouse IgG-alkaline phosphatase-conjugated antibody diluted 1:2000.
Two-hybrid interaction trap in yeast
Screens and tests were performed as described by Delattre et al. (Delattre et al., 2000).
Immunostaining of polytene chromosomes
Third instar larvae were heat shocked 15 minutes at 37°C, unless specified otherwise, and allowed to recover at room temperature for 1 hour before squashing. Procedures for immunostaining were as described previously (Platero et al., 1995). Anti-HA and anti-Su(var)3-7 antibodies were used at a dilution of 1:100, and anti-HP1 (CI49, a gift from Sarah Elgin) at 1:400.
Effect of HA:Su(var)3-7 mutant proteins on variegation
To test the effect of the heat-shock induction of HA:Su(var)3-7 proteins on the wm4h and Heidi lines (Seum et al., 2000), females bearing the variegated alleles were crossed with males homozygous for the HA-Su(var)3-7 transgene, and with yw males as control. Heat shock was carried out by incubating embryos at 30°C until the beginning of the third instar larval stage, and then by administrating three 30-minute heat shocks at 37°C per day until adult emergence. Eye pigment measurements of males were made according to the method of Sun and coworkers (Sun et al., 2000
).
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RESULTS |
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One explanation for the suppressor effect on PEV of the C-terminal half of Su(var)3-7 could be its ability to form an inactive complex with endogenous Su(var)3-7 or a protein interacting with it. We have used the yeast two-hybrid protein interaction trap system (Fields and Song, 1989) to search for proteins interacting with Su(var)3-7. A segment of Su(var)3-7 extending from the seventh zinc finger to the C-end of the protein, namely amino acids 736-1169, was used as a bait to screen a cDNA library derived from Drosophila embryos (Materials and Methods). About three million yeast transformants were screened, and 50 positive clones isolated. One of them, named
21, encodes a fragment of Su(var)3-7 itself, corresponding to amino acids 808 to 991. To delimit the domain of Su(var)3-7 involved in its self-association, we performed a deletion analysis illustrated in Fig. 3. The zinc finger-containing region was found not to be involved in this interaction. Only fragments containing the C-terminal part of Su(var)3-7 interact with
21. The smallest construct tested that was capable of promoting self-association of Su(var)3-7 contained amino acids 845 to 971. This construct also interacts with HP1 (Delattre et al., 2000
), but fails to interact with unrelated proteins, or with the empty pJG4-5 vector (data not shown). As the fragment promoting self-association contains the BESS motif (amino-acids 906-945), we surmise that this motive is responsible for Su(var)3-7 dimerisation.
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We then tested whether, reciprocally, the over-expression of wild-type Su(var)3-7 delocalises the C terminus fragment, namely 7-Ct. To do so, we have combined it, under heat shock, with the construct over-expressing the wild-type Su(var)3-7 (Cléard et al., 1995
). After heat shock, the over-expressed Su(var)3-7 protein covers the entire chromosome (Fig. 5) (Delattre et al., 2000
). In these conditions, the HA:
7-Ct protein was found predominantly at the chromocenter, but it also stained weakly, but significantly, the whole of the chromosome arms (Fig. 5). In the wild-type background, its localisation was restricted to the chromocenter (Fig. 5). To allow for comparison of intensity of staining in Fig. 5, glands from both genotypes were placed on the same slide. Therefore, the two nuclei shown went through the same procedures. Finally, the pericentric spots of HA:
7-Ct seen in a wild-type background are replaced by a strong, smooth staining when Su(var)3-7 is over-expressed. We conclude that endogenous Su(var)3-7 seems able to recruit the C-terminal construct HA:
7-Ct and vice versa.
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Finally, we examined the localisation of endogenous HP1 on polytene chromosomes from larvae expressing fragments of Su(var)3-7. None of the nine Su(var)3-7-tagged fragments we made was able to recruit HP1 away from its normal pattern, as illustrated for one case in Fig. 7. This figure shows a total depletion of endogenous Su(var)3-7 from pericentric heterochromatin when HA:7-B is expressed. Even in these conditions, HP1 localisation is not modified: staining of pericentric heterochromatin, telomeres and euchromatic bands decorated by HP1 is still visible. Depletion of Su(var)3-7 from the chromocenter does not disturb HP1 localisation.
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DISCUSSION |
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The region of Su(var)3-7 promoting self-association contains the motif BESS (Altschul et al., 1997). Interestingly, among members of the family, the BESS motif is also found in the part of the BEAF protein implicated in the oligomerisation of the protein (Hart et al., 1997
). Moreover, and although Su(var)3-7 seems to be a fast evolving protein, the BESS motif is one of the best conserved region in the Su(var)3-7 proteins of Drosophila melanogaster and Drosophila virilis (our unpublished work). This leads us to propose that the BESS motif is an important domain of Su(var)3-7, ensuring self-association.
The functional significance of this self-association is not known, but it provides a means of forming multimeric complexes promoting heterochromatin formation and associated silencing. Indeed, mammalian HP1 proteins homo- and heteromerise in vivo and in vitro (Le Douarin et al., 1996; Ye et al., 1997
; Nielsen et al., 2001
). Furthermore, recent in vitro studies have shown that HP1 is required as a dimer for interaction with CAF1 and TIFß in mice (Brasher et al., 2000
).
The C-terminal part of Su(var)3-7 is required for specific binding to heterochromatin
When over-expressed, not only does Su(var)3-7 bind strongly to heterochromatin, but staining expands through euchromatin. We have shown here that the full-length tagged version of Su(var)3-7 (HA:FL) behaves in an analogous manner. The banding pattern is neither identical nor complementary to DNA staining. HA:FL does not specifically bind all bands or interbands, but it has a yet unknown specificity for a particular DNA or chromatin landscape. In contrast, HA:1-6, the construct containing the first six zinc fingers and lacking the C-terminal half of Su(var)3-7, covers complete polytene chromosomes, without preference for heterochromatin, with a pattern similar to DNA staining. In vitro, fragments containing two or more Su(var)3-7 zinc fingers motifs have a general affinity for DNA, with a preference for some satellite DNA sequences (Cléard and Spierer, 2001). However, the apparent absence of specificity of HA:1-6 association with polytene chromosomes leads us to conclude that in vivo the heterochromatin specificity of Su(var)3-7 is not given by its zinc fingers. Possible preference for satellite DNA in vivo is difficult to assess because of vast underreplication of these sequences in polytene chromosomes.
HA:7-Ct, the construct containing the C-terminal half, specifically binds to pericentric heterochromatin. We cannot exclude that this specific localisation to heterochromatin depends at least in part on protein interaction with endogenous Su(var)3-7. Nonetheless, the in vivo analysis clearly shows that the domain of Su(var)3-7 self-association is not sufficient for the targeting to heterochromatin. HA:
7-B, a short C-terminal segment, contains the dimerisation domain mapped in yeast, does not bind heterochromatin, and depletes pericentric heterochromatin from endogenous Su(var)3-7.
The specificity for pericentric heterochromatin located in the C-terminal half is probably mediated by interaction with another heterochromatic partner. The domain of Su(var)3-7 interacting with this partner should lie around the BstEII restriction site separating the constructs HA:7-B and HA:B-Ct (Fig. 1). These constructs do not bind to chromatin, whereas HA:
7-Ct, from which they originate by cleavage with BstEII, does bind.
Interactions between Su(var)3-7 and HP1
We believe that HP1, although known to bind to Su(var)3-7, is not the partner necessary for the targeting of Su(var)3-7 to heterochromatin through the BstEII site region of the C-terminal part. First, none of the three domains of HP1 interaction mapped in yeast corresponds to this BstEII region. Second, the localisation of the HA:7-Ct construct of Su(var)3-7 as spots in the chromocenter has no effect on HP1 localisation. Reciprocally, ectopic HP1, as a HP1/Pc chimera, does not recruit HA:
7-Ct, the C-terminal half of Su(var)3-7, while it does recruit the full-length protein. We infer that the targeting of HA:
7-Ct to the chromocenter is due to a yet unidentified partner. And although HP1 does recruit the full-length Su(var)3-7 (Delattre et al., 2000
), the constructs containing one or two HP1-binding domains are not delocalised by the HP1/Pc chimera. We therefore propose that the Su(var)3-7-HP1 interaction requires the presence of the three interaction domains together. Finally, when endogenous Su(var)3-7 is totally depleted from the chromocenter by the presence of the HA:
7-B construct, HP1 localisation is not at all modified. The evidence above indicates that Su(var)3-7 does not recruit HP1 to heterochromatin.
A model for the role of Su(var)3-7 in heterochromatin
Over-expression of the full-length Su(var)3-7 protein dramatically enhances variegation. In contrast, over-expression of HA:1-6, the construct with six zinc fingers, has no effect on PEV. This suggests that to be functional in heterochromatin, the zinc fingers domains of Su(var)3-7 need the dimerisation of the protein and/or interaction with partners. Besides, the constructs containing one to four zinc fingers plus the complete C-terminal part do not enhance variegation as the full-length protein does. This means that the number of zinc fingers is also crucial for the function of the protein. The zinc fingers of Su(var)3-7 are not classical C2H2 type zinc fingers because of their non-canonical sequence, an upstream tryptophan motif and of the rather long inter-digital space (Cléard et al., 1995). We propose that the dispersion of Su(var)3-7 zinc fingers in the N-terminal half of the protein allows them to make contact with DNA at a distance. It provides the flexibility required to pack the dispersed bound DNA sequences in a more compact conformation, thus contributing to the condensed heterochromatic conformation and to the repressed state. An analogous function in altering the DNA conformation has been described for the 12 zinc finger proteins encoded by the Suppressor of Hairy-wing gene. These zinc fingers directly contact DNA and increase its flexibility together with the C-terminal of the protein, itself not required for DNA binding (Shen et al., 1994
).
The Su(var)3-7 protein consisits of two complementary domains: the N-terminal zinc fingers moiety and the C-terminal part. The zinc fingers contribute to the repression function as a DNA compacting tool, while the C-terminal domain promotes dimerisation through the BESS motif and interaction with partners to ensure heterochromatin recognition and association. In position-effect variegation, expansion of the repressive complexes, or sequestration of heterochromatin-like sequences at distance, are accompanied through yet unknown steps by methylation of lysine 9 of histone H3 by the modifier of PEV Su(var)3-9 (Schotta et al., 2002). This would lead to the recruitment of HP1, which in turn recruits Su(var)3-7. Then compaction of chromatin by Su(var)3-7 imposes silence.
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ACKNOWLEDGMENTS |
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