2753
Journal of Cell Science 111, 2753-2761 (1998) Printed in Great Britain © The Company of Biologists Limited 1998 JCS4579
Dynamics of the sub-nuclear distribution of Modulo and the regulation of position-effect variegation by nucleolus in Drosophila L. Perrin1,‡, O. Demakova2, L. Fanti3,4, S. Kallenbach1, S. Saingery1, N. I. Mal’ceva2, S. Pimpinelli4, I. Zhimulev2 and J. Pradel1,* 1Laboratoire
de Génétique et de Physiologie du Développement, Institut de Biologie du Développement de Marseille, CNRS/INSERM/Université de la Méditerranée/AP de Marseille, Campus de Luminy Case 907. 13288 Marseille cedex 9, France 2Institute of Cytology and Genetics, Russian Academy of Sciences, Acad. Lavrentiev Ave, 10, 630090 Novosibirsk, Russia 3Istituo di Genetica, Univ. di Bari, V. Amendola 165/A, 70126 Bari, Italy 4Dip. Genetica e Biologia Molecolare, Univ. La Sapienza, P. Aldo Moro, 500185 Roma, Italy ‡Present
address: Institut de Génétique Humaine, UPR 1142 du CNRS, 141 rue de la Cardonille, 34396 Montpellier Cedex 5, France *Author for correspondence (e-mail:
[email protected])
Accepted 8 July; published on WWW 27 August 1998
SUMMARY modulo belongs to the class of Drosophila genes named ‘suppressor of position-effect variegation’, suggesting the involvement of the encoded protein in chromatin compaction/relaxation processes. Using complementary procedures of cell fractionation, immunolocalisation on mitotic and polytene chromosomes and crosslinking/immunoprecipitation of genomic DNA targets, we have analysed the sub-nuclear distribution of Modulo. While actually associated to condensed chromatin and heterochromatin sites, the protein is also abundantly found at nucleolus. From a comparison of Modulo pattern on chromosomes of different cell types and mutant lines, we propose a model in which the nucleolus balances the Modulo protein available for chromatin compaction and PEV modification. At a molecular level, repetitive elements
instead of rDNA constitute Modulo DNA targets, indicating that the protein directly contacts DNA in heterochromatin but not at the nucleolus. Consistent with a role for Modulo in nucleolus activity and protein synthesis capacity, somatic clones homozygous for a null mutation express a cellautonomous phenotype consisting of growth alteration and short slender bristles, characteristic traits of Minute mutations, which are known to affect ribosome biogenesis. The results provide evidence suggesting that Modulo participates in distinct molecular networks in the nucleolus and heterochromatin and has distinct functions in the two compartments.
INTRODUCTION
A number of second-site mutations and chromosomal rearrangements that modify variegation have been studied, demonstrating that PEV is influenced by cis- and trans-acting factors. On one side, PEV is subjected to the dosage of heterochromatic DNA, which mostly consists of repetitive sequences (Gatti and Pimpinelli, 1992). For example, tandemly repeated rDNA cistrons influence PEV in a dose-dependent manner (Hilliker and Appels, 1982; Spofford and DeSalle, 1991), since rDNA deletion or amplification induces PEV enhancement or suppression, respectively. On the other side, PEV is regulated by trans-acting factors. More than one hundred modifier mutations has been identified in Drosophila (Reuter and Spierer, 1992; Zhimulev, 1998). Although only a few of the corresponding transcription units are identified to date, the nature of encoded products, chromosomal proteins or modifiers of chromosomal proteins, are consistent with a role in chromatin structure modulation (Elgin, 1996). The modifier of PEV modulo (mod) is essential for development and several recessive lethal amorphic mutations in this gene have been isolated that display a dominant
Position-effect variegation (PEV) in Drosophila is an example of how chromatin compaction/relaxation processes modulate gene activity (for recent reviews, see Elgin, 1996; Henikoff, 1996; Wakimoto, 1998; Zhimulev, 1998). PEV is associated with chromosomal rearrangements where genes juxtaposed to new euchromatin/heterochromatin boundaries are randomly turned on or off, depending on whether the environment is relaxed (euchromatic) or condensed (heterochromatic). As the decision to express the gene or not is clonally inherited during development, the phenomenon gives rise to adult structures containing a mix of phenotypically distinct groups of cells (Reuter and Spierer, 1992). Two series of data have reinforced the link between PEV and chromatin structure. First, the chromosomal domain subjected to PEV becomes cytogenetically distinguishable as a dense block of chromatin, (Zhimulev et al., 1989). Second, variegating inserts exhibit reduced accessibility to restriction enzymes (Wallrath and Elgin, 1995).
Key words: Drosophila, Position-effect variegation, Heterochromatin, Nucleolus, Modulo target, Clonal analysis
2754 L. Perrin and others suppressor effect on PEV (Garzino et al., 1992). The encoded protein (Krejci et al., 1989) contains two basic domains thought to be responsible for DNA binding, a large acidic region that is presumably involved in protein/protein interactions and a central part composed of a repetition of four RNA recognition motifs (Birney et al., 1993). In vitro, the protein actually binds DNA without nucleotide specificity and recognises a specific RNA motif (our unpublished data). To gain further insight on mod in PEV, we have analysed the subnuclear distribution of the protein using complementary procedures of cell fractionation, immunolocalisation on mitotic and polytene chromosomes and crosslinking/immunoprecipitation with anti-Mod protein antibodies of genomic DNA targets. The data indicate that the nucleolus balances the amount of Mod available for chromatin compaction and PEV modification. We also report that clones of somatic cells mutant for mod express a Minute-like phenotype, consistent with a function in protein synthesis capacity at nucleolus. The results provide evidence to suggest that the Mod protein participates in distinct molecular networks at the nucleolus and in heterochromatin and has distinct functions in the two compartments.
MATERIALS AND METHODS Flies Oregon-R and Canton-S were used as wild-type standards. Null alleles of mod, A4-4L8 and A4-4L6 are described in Garzino et al. (1992). otu7 and y cv otu11 v f are described in Lindsley and Zimm (1992). rib 7 (1A) line was kindly provided by G. Karpen (Karpen et al., 1988). Stocks used for the generation of somatic clones were from the Bloomington stock centre: w1118, P(ry+, hs-neo, FRT)82B; yw1118 HS FLP; yw1118; P(ry+, hs-neoFRT)82B, P(w+, cmyc)87E, Sb63, P(y+, ry+)96E/TM6B, ry. Clonal analysis A chromosome containing P(ry+, hs-neo, FRT) at 82B and the null mod allele A4-4L8 at 100F was obtained by recombination and checked for rescue by a mod transgene (Garzino et al., 1992). Generation of somatic clones was performed as described in Golic (1991). For adult cuticle preparations, animals were treated for 1 minute in boiling 10% KOH and then dehydrated in 100% ethanol. Dissected thoraces were mounted in GMM (Ashburner, 1989).
Fig. 1. Subcellular distribution of Mod. Immunoblot of embryo homogenates depleted of nuclei (lane 1), purified nuclei (lane 2) and five sub-nuclear fractions (lanes 3-7). Lanes 3 and 4: supernatants from high speed centrifugations of nuclei extracted with 0.15 M NaCl and then by increasing salt concentration from 0.15 M to 0.3 M, respectively. Lanes 5 and 6: corresponding pellets. Lane 7: soluble fraction obtained from sonicated nuclei after further increasing NaCl from 0.3 to 0.45 M. Each lane contains 7.5 µg of protein. Mod is mainly detected in the supernatant (lane 3) and slightly in the pellet (lane 5) of the 0.15 M NaCl extract; it is absent from the 0.15/0.3 M NaCl extract (lanes 4, 6), and found again in chromatin fragments solubilised after sonication in 0.45 M NaCl (lane 7). The positions of molecular mass markers are shown.
foetal calf serum (FCS), washed three times in PBS, incubated for 30 minutes at room temperature (RT) with secondary antibody (FITCanti-mouse in PBS, 1% FCS), washed three times in PBS, stained with DAPI (0.01 g/ml) and mounted in anti-fading medium. Chromosome preparations were analysed using a computer-controlled Zeiss Axioplan epifluorescence microscope equipped with a cooled CCD camera (Photometrics). The fluorescent signals, recorded separately as grey-scale digital images, were pseudocolored and merged using the Adobe Photoshop program. The fluorescence intensity quantification was performed by the same program.
Immunostaining of polytene chromosomes Salivary glands or ovaries were dissected in Ephrussi-Beadle solution containing 0.5% NP-40, transferred to formaldehyde fixative (salivary glands for 10 minute; ovaries for 15-20 minutes), then to a drop of 45% acetic acid (salivary glands for 1 minute and ovaries for 2-2.5 minutes) and squashed. Immunostaining procedures were done as described in Clark et al. (1991). The secondary antibody was FITCanti-mouse. Photo-microscopy was done using a −IV microscope with an epifluorescence attachment.
Immunodetection on whole mount mAb LA9 or two rat-polyclonal antibodies raised against GST-Mod fusion protein were used. Polyclonal antisera were pre-adsorbed at 1/10 dilution in 10% FCS, 0.1% Triton, PBS on Canton-S embryos (0-18H) and used at 1/100 (final dilution). Monoclonal antibody AJ1 was kindly provided by H. Saumweber (Frasch et al., 1986). Embryos were dechorioned in 50% bleach, fixed in PBS, 8% formaldehyde for 15 minutes at room temperature (RT), devitelinised with methanol and progressively rehydrated in ethanol/PBS, 0.1% Triton X-100. Larvae were dissected in PBS and tissues fixed as the embryos. Samples were saturated in 10% FCS, 0.1% Triton, PBS for 1 hour at RT, incubated with primary antibodies overnight at 4°C, washed five times in PBS at RT, incubated for 1 hour at RT with secondary antibodies (FITC-anti-rat, TRITC-anti-mouse) in 10% FCS, washed five times in PBS, mounted in permafluor (Immunotech) and the slides analysed under a Zeiss confocal microscope.
Immunostaining of mitotic chromosomes Brains were dissected from third instar larvae in physiological saline solution, transferred in a drop of fixative (45% acetic acid, 5% formaldehyde) for 8 minutes and squashed. Slides were frozen in liquid nitrogen, washed once in PBS after flipping off the coverslip, immersed in PBS, 1% Triton for 10 minutes and then in PBS with dried non-fat milk for 30 minutes. Preparations were incubated overnight at 4°C with anti-Mod monoclonal antibody LA9 (mAb LA9; Krejci et al., 1989) at 20 µg/ml final concentration in PBS, 1%
Sub-nuclear fractionation and western blot analysis Fractionation of nuclei was carried out as previously described (Garzino et al., 1987). Briefly, purified nuclei were successively extracted with 0.15 M and 0.3 M NaCl, and chromatin fragments were solubilised upon sonication in 0.45 M NaCl. Proteins (7.5 µg) were separated by 7.5% SDS-PAGE and electrophoretically transferred to nitrocellulose. Mod protein was detected after sequential incubations with mAb LA9, alkaline phosphatase-anti-mouse antibody and both NBT-BCIP as substrates (Promega).
Modulo, nucleolus and PEV 2755
Fig. 2. Immunolocalisation of Mod (green) and AJ1 (red) antigens in embryonic and larval cells. (A) Anterior part of an embryo during germ band extension. Mod is detected in the entire nucleolus. Note completely overlapping stainings (yellow) for Mod and AJ1 in the magnified view of insert (B). (C,D) Ectoderm and endoderm cells of a stage-16 embryo, respectively. Mod is detected at the nucleolus periphery coincident with AJ1 (yellow rings), and also in the remainder of the nucleus. (E) (single staining for Mod) and insert (F) (double staining): nucleolar localisation of Mod in wing disc cells. (G) Salivary gland cells. Mod is detected at nucleolus periphery; the green ribbons likely correspond to polytene chromosomes. Mod target library Conditions for UV irradiation, immunoprecipitation of protein-DNA adducts and cloning the resulting DNA fragments (1 kb average size) into λGT10 are detailed in Graba et al. (1992). Samples of Mod and Ultrabithorax (Ubx) target libraries were plated at a density of 104 plaques on 150 mm plates, and replicas (hybondC, Amersham) were hybridised with following probes: 4.5 kb HindIII fragment from
repetitive element f; 2.9 kb EcoRI fragment from mdg1; 1.7 kb HindIII-PstI fragment from copia; 4.7 kb BglII fragment from doc; 1.5 kb EcoRI-HindIII fragment from jockey. Probe for rDNA covers the entire rDNA locus but a HindIII fragment of 900 bp within the 28S region.
RESULTS
Fig. 3. Somatic clones produced in the thorax epidermis by mitotic recombination. Clones were induced at the first instar and identified from loss of a yellow transgene and Stubble bristle marker. White lines delineate the approximate limits of clones homozygous for either wild-type allele of mod (A) or A4-4 L8 null mutation (B). Arrows point to bristles that normally differentiate as machrochaetes (Pdc, Posterior dorsocentral; Adc, Anterior dorsocentral). The mod mutation systematically causes a strong reduction of the clone size and a short slender bristle phenotype.
Western blot analysis of Mod sub-cellular distribution We previously reported a procedure that combines step-wise salt extraction of purified nuclei with low and high speed centrifugations, to isolate distinct sub-nuclear fractions from Drosophila embryos (Garzino et al., 1987). A comparative western analysis, performed in the same study, demonstrated that nucleosoluble proteins (including hnRNPs) and components firmly bound to chromatin, like histone 2B, were recovered in distinct sub-fractions; the former by extraction at low salt concentration (0.15 M and 0.3M NaCl) and the latter by sonication in 0.45 M NaCl (Garzino et al., 1987). Data with mAb LA9, a monoclonal antibody which specifically recognises Mod, are shown in Fig. 1. They indicate (1) that the protein is strictly nuclear (detected in nuclei but not in cytoplasmic fractions), and (2) that it principally localises in sub-nuclear fractions 3 and 7, which correspond to nucleosoluble (0.15 M NaCl extract) and chromatin-bound components (solubilised by sonication in 0.45 M NaCl), respectively. It is noteworthy that Mod is not detected in the nuclear material released by raising the salt concentration from 0.15 to 0.30 M (fraction 4), indicating that such an increase in ionic strength does not solubilise chromatin-associated protein. We therefore conclude that Mod localises in two sub-nuclear
2756 L. Perrin and others compartments. On one side it is associated to chromatin and on the other side it is either soluble in the nucleoplasm or solubilised from a nuclear organelle during fractionation. We roughly estimated, by comparing western signals obtained from serial dilutions of fractions 3 and 7 (not shown), that 90% of Mod is recovered within the nucleoplasmic fraction and 10% of the protein remains chromatin-bound. Immunolocalisation reveals a preferential accumulation in the nucleolus The cytological distribution of Mod was analysed using confocal microscopy. Double staining experiments for Mod and nucleolar antigen AJ1 (Frasch et al., 1986) revealed that Mod is always preferentially associated to the nucleolus. In interphasic nuclei of embryos at the onset of germ band extension, Mod is essentially nucleolar (Fig. 2A,B). It perfectly co-localises with AJ1, while remaining parts of the nucleus appear weakly stained. The magnified view of Fig. 2B (insert) indicates that the staining for Mod and AJ1 is found all over nucleolus at that stage. This pattern has significantly changed by the end of embryogenesis. In cells from the ectoderm and gut endoderm of a stage-16 embryo (Fig. 2C,D), Mod is no longer detected in the whole nucleolus. Rather, it is excluded from the centre (red staining by AJ1) but remains co-localised with AJ1 at the periphery of the organelle (yellow ring). At that time, Mod accumulates in a number of distinct foci within the nucleus. This nuclear pattern is clearly distinguishable from nucleolar labelling. We also analysed Mod sub-cellular distribution in larvae. In all adult primordia examined (brain and imaginal discs) that are mitotically active in larvae, Mod and Aj1 perfectly co-localise, and, as in the early embryo, occupy the whole nucleolus (Fig.
2E,F). Mod intracellular distribution in larval tissues, which are composed of post-mitotic cells, rather resembles that seen in old embryos. In salivary gland cells, for example, the protein is detected at the periphery but not the inner nucleolus (Fig. 2G). Fig. 2G in addition shows a clear staining of polytene chromosomes, which become visible as large green ribbons. Clonal analysis of mod mutation in adult To analyse the consequences of mod loss of function in mitotically active cells of the imaginal disc, clones of cells homozygous for the null allele A4-4L8 were generated by FLPmediated recombination. In these experiments, clones were identified on the adult epidermis of mosaic animals by the loss of yellow and Stubble bristle markers. mod-deficient clones were found in adult flies on head, thorax, legs and abdomen. They present three main features. First, they are systematically of reduced size when compared to controls (wild-type clones induced at the same stage). Second, mutant thoracic cells produce short slender bristles, which are best seen when the clone encompasses a macrochaete (arrows in Fig. 3B). Third, the effect of the mod mutation appears to be cell-autonomous, as we never saw any alteration in cell or bristle morphology in wild-type cells surrounding the clone. These features are reminiscent of the dominant phenotypes of Minute mutations (Morata and Ripoll, 1975; Lindsley and Zimm, 1992), which are believed to affect ribosomal protein genes (see SaeboeLarssen et al., 1998 and references therein). Immunofluorescent localisation of Mod on polytene chromosomes The general immunofluorescence pattern observed with antiMod mAb LA9 on salivary glands chromosomes is illustrated
Fig. 4. Immunolocalisation of Mod on salivary gland polytene chromosomes. (A,B) DAPI staining and Mod immunofluorescence pattern on salivary gland squashes. The prominent label is at the nucleolus (n). Pericentric heterochromatin (Ch) and the majority of bands along chromosome arms are also stained. (C,D) Phase contrast and Mod immunodetection on chromosomes from rib 7(1A) homozygous larvae. The strong additional signal in 1A (see text; arrow in C,D) reveals Mod association to the mini-nucleolus that results from the insertion of a rDNA cistron at that position. (E,F) Phase contrast and Mod immunodetection on Df(3R)A4-4 L8/TM6 larvae. Note the bright and very slight staining of nucleolus (n) and chromosome arms, respectively.
Modulo, nucleolus and PEV 2757 in Fig. 4B. The most prominent label is found at the nucleolus, while the chromocenter demonstrates significantly slighter staining. As for chromosome arms, Mod is immunodetected at all or almost all bands but never at puffs and interbands. To confirm the protein’s ability to bind nucleolar structures, we analysed the staining pattern on salivary gland polytene chromosomes from rib 7 (1A) larvae. This strain contains an extra ribosomal transcription unit inserted at euchromatin position 1A and develops an ectopic mini-nucleolus from this site (Karpen et al., 1988). mAb LA9 consistently reveals a strong additional signal at position 1A on rib 7 (1A) polytene chromosomes (arrow in Fig. 4D). We further asked whether the Mod pattern on polytene chromosomes changed depending on the mod gene dosage. Note that the nucleolar staining was found to be less sensitive than the chromosomal staining to a reduction of available Mod. Indeed, in heterozygous animals carrying only one copy of the gene, the protein is still abundantly found at the nucleolus, but surprisingly is no longer detected along chromosome arms at sites corresponding to condensed regions (Fig. 4E,F). Wildtype pattern is restored (not shown) on polytene chromosomes from transgenic animals heterozygous for endogenous mod on the third chromosome and containing an extra copy of mod genomic fragment on the second chromosome (Garzino et al., 1992). This provides confirmation that the strong reduction of immunostaining of heterozygous chromosomes specifically results from the mutation of one copy of mod. Moreover, no staining at all is seen in third larval instar escapers homozygous for the null mutation A4-4L8 (not shown), which rules out nonspecific antibody reaction with an unrelated nucleolar component. To compare the Mod pattern on polytene chromosomes from different tissues, the female sterile mutation otu was used. The particular combination of otu7 and otu1 alleles has the property
to be fertile, but to retain the polyteny of nurse cell chromosomes through egg chamber formation, in contrast to wild type where chromosomes are transiently polytenised at stage 4/5 of oogenesis (King, 1970). Fig. 5 shows Mod immunostaining on polytene chromosomes from mutant nurse cells (called pseudonurse cells, PNCs; Storto and King, 1988) and larval salivary gland cells. Mod distributions on salivary gland chromosomes from otu7/otu11 and wild-type animals are similar (Fig. 5A,B). PNC chromosome staining is very weak (Fig. 5C-F) and varies on the same slide from nucleus to nucleus, from being practically absent to being slight with minor sites (arrow) visible in the favourable nuclei (Fig. 5F). These sites correspond to some dense euchromatic bands, but it is not possible to correlate this pattern to that of salivary gland chromosomes. Notice that nucleoli in PNCs remain intensively stained. Immunofluorescent localisation of Mod on mitotic chromosomes The nucleolus disassembles during mitosis. At metaphasis, some nucleolar proteins remain associated to nucleolus organiser (NO), some decorate the whole chromosomes and others spread throughout the cell (Warner, 1990). We have analysed the immunolocalisation pattern of Mod in mitotic cells from larval brains. Staining at interphase confirms the protein association with the nucleolus. On mitotic chromosomes, Mod appears concentrated at some heterochromatic regions of all chromosomes except the fourth (Fig. 6). Heterochromatic sites correspond to H11-13 and H3 regions of chromosome Y and pericentric heterochromatin of chromosomes X, 2 and 3. Intriguingly, it has been shown that these heterochromatic regions mainly comprise different families of repetitive mobile elements (Pimpinelli et al., 1995). Mod is also detected in a domain on chromosome Y that
Fig. 5. Immunolocalisation of Mod on polytene chromosomes of PNCs and salivary gland cells from otu7/otu11 animals. (A,B) Phase contrast and Mod immunodetection on salivary gland chromosomes. The Mod pattern appears similar to that of wild-type control. (C,D) Phase contrast and Mod immunodetection on ovarian preparations. Note the intensive staining of nucleoli in PNCs and follicular cells, contrasting with the poor label of PNC chromosomes. (E,F) Focus on slight immunodetection of Mod at rare dense euchromatin bands (arrows). n, nucleolus.
2758 L. Perrin and others
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Fig. 6. Immunolocalisation of Mod on mitotic chromosomes of larval neuroblasts. (A) DAPI coloration; (B) Mod immunodetection; (C) merged image. The antibody weakly decorates euchromatic arms while the Mod protein shows a strong accumulation in the nucleolus at interphase (arrowheads) and in specific heterochromatic regions of metaphasic chromosomes (arrows), with the exception of chromosome four. Chromosome Y shows a clear enrichment in Mod at NO, at h11-13 and at h3 of the long arm. This region is mainly made by many transposable elements families. Note that Mod reveals a nucleolus remnant in mitotic cells (nucl). C, pericentric heterochromatin.
topologically coincides with NO, which is composed of tandemly repeated rDNA genes. These results suggest that Mod binds both repetitive elements and rDNA sequences on chromosome Y. Mod association to repetitive mobile elements We previously reported a library of genomic DNA fragments associated with the homeotic protein Ubx in the developing embryo (Graba et al., 1992). The method is based on the generation of DNA-protein adducts by UV irradiation of intact nuclei, immunoselection with relevant antibodies and cloning of resulting DNA pieces. The results from this procedure are different from those obtained by the formaldehyde crosslinking/immunoprecipitation procedure (Orlando and Paro, 1993), since under UV irradiation only those proteins that directly contact DNA can form covalent adducts. We applied the UV method using mAb LA9 to isolate and clone direct DNA targets of Mod. The resulting library, together with the library of Ubx targets, used here as a control, was screened for the presence of rDNA and several repetitive elements. The results are given in Table 1. Four repetitive elements are found at high frequency in the Mod target library. Consistent with a direct association to Mod, they are absent from the control (Ubx target) library. Elements f, mdg1 and doc are likely candidates to promote Mod binding to heterochromatic sites. jockey is not heterochromatic. Instead, it shows a dispersed distribution along chromosome arms (not shown), suggesting that binding to jockey depends on the interaction between Mod and this element in euchromatic regions. Not immunoprecipitated by Mod, copia is highly enriched in the Ubx target library, which is consistent with previous report of copia-like element regulation by homeotic proteins (Brookman et al., 1992). rDNA is poorly enriched in the Mod target library, indicating that direct binding of Mod to rDNA cannot account for the very strong immunofluorescence signals observed in situ. Instead the result suggests that Mod does not bind rDNA at the nucleolus of interphasic cells, and that rDNA clones in
Table 1. Relative abundance of repetitive sequences in Mod and Ubx target libraries Repetitive element f mdg1 copia doc jockey rDNA
Mod targets
Ubx targets
n
A
B
A
50 25 60 40 50 200/250
249 130 10 160 139 38
498 520 17 400 278 9
0 0 71 0 2 0
B
118 4
A, number of positives over 104 screened clones. B, data normalised to 100 copies of repetitive elements per haploid genome. n, number of copies per haploid genome.
the library originate from a transient association of the protein with NO at mitosis. DISCUSSION Mod is a component of condensed chromatin domains Based on the suppression of PEV caused by mod and on the DNA-binding activity of its product, it has been proposed that Mod serves to anchor multimeric complexes promoting chromatin compaction and silencing (Garzino et al., 1992). The combined use of polytene and mitotic chromosomes has revealed distinct but consistent features, and allows a clear picture of Mod at chromatin. The prominent conclusion from immunolocalisation on polytene chromosomes is the association to the chromocenter and almost all dense bands on chromosome arms. Binding to heterochromatin is best seen in mitotic cells, at the pericentric heterochromatin of all chromosomes (except the fourth) and specific sites on chromosome Y. The data thus provide direct evidence of Mod binding to condensed chromosomal regions. Consistent with a
Modulo, nucleolus and PEV 2759 role in chromatin structure, mutation of one gene copy results in PEV suppression and a strong decrease of Mod at pericentric heterochromatin and chromosome bands. Cross-linking experiments demonstrate that Mod interacts with several repetitive elements, which is in general agreement with its association to heterochromatin and the PEV suppression phenotype of mod mutant animals. Several points, however, indicate that these interactions do not direct the protein to specific heterochromatic sites. First, it was never found associated to chromosome four, which is essentially heterochromatic, on mitotic cell preparations. Thus, Mod is not a general component of heterochromatin. Second, the Mod pattern does not strictly overlap the distribution of the cognate repetitive elements that show a wider distribution spectrum on mitotic chromosomes (Pimpinelli et al., 1995). Third, while directly contacting DNA (Garzino et al., 1992), Mod has no canonical DNA binding domain and does not recognise a specific DNA motif in vitro (our unpublished observations). This suggests that Mod might associate with unknown factors that provide DNA binding specificity and direct the complex towards particular sites, at which interaction of Mod with repetitive elements could favour and stabilise the formation of condensed heterochromatin. Chromatin and nucleolus compete for Mod binding Only a small fraction of Mod, about 10%, remains bound to chromatin during step-wise salt extraction of embryonic nuclei, while 90% is recovered with the nucleoplasmic fraction. Mod thus appears either to be essentially free in the nucleoplasm or loosely bound to a nuclear organelle and released during the experiment. Double staining experiments for Mod and the nucleolar antigen AJ1 on a variety of embryonic or larval tissues unambiguously demonstrate that the most intensive staining is always associated with nucleolus. We therefore conclude that in vivo the major part of Mod is nucleolar, and it becomes released in the nucleoplasm during cell fractionation. In the tentative model of Fig. 7, we propose that the nucleolus titrates the bulk of Mod available for chromatin compaction and PEV modification. Competition for Mod between chromatin and nucleolus is supported by three lines of evidence. (1) In salivary glands cells, the nucleolar staining is far less sensitive to a reduction of mod gene dosage than is chromatin staining. (2) Mod does associate to the ectopic mininucleolus that results from rDNA insertion in euchromatin. No significant reduction of chromosomal association of Mod has been observed in this experiment, which is actually not surprising as the rib7(1A) line contains a single extra rDNA unit in addition to about 200 units normally found in the Drosophila genome (Kay and Jacobs-Lorena, 1987). (3) In PNCs Mod is mainly found at the nucleolus and barely detectable on chromosomes. The difference in pattern between PNCs and salivary glands is best understood by considering the relative amount of rDNA in the two cell types. While rDNA is heavily under-replicated during salivary gland development, it endoreplicates at almost the same rate as the rest of the genome in nurse cells (Hammond and Laird, 1985). The nucleolus is therefore large (Klug et al., 1970), a process related to the production of protein and rRNA for the oocyte. Competition for Mod between this large and active nucleolus and chromatin sites explains the poor chromosomal staining of PNCs. In
A
NO NO
mod PEV enhancement
B
Salivary gland cell
PEV suppression Pseudo nurse cell
Nucleolar specific proteins
NO : nucleolus organiser
Common proteins (ex: Mod)
increase
Chromosomal specific proteins
decrease
Fig. 7. Model for PEV regulation by the nucleolus. The model is based on competition between heterochromatin and the nucleolus for common protein factors. (A) Deletion of NO sequences leads to a small-sized nucleolus, preferential binding of common factors by heterochromatin and PEV enhancement; conversely, NO amplification results in common factor titration by the nucleolus and PEV suppression (also obtained by a reduced synthesis of any common protein factor, such as Mod). (B) The relationship between the reduced potential of PNCs to modulate PEV with regard to salivary gland cells and titration of common proteins by the large PNC nucleolus.
comparison the reduced size and activity of salivary gland nucleoli improves the amount of protein available for chromatin and accordingly allows immunodetection at pericentric heterochromatin and bands. NO, which is composed of tandem repeats of rDNA cistrons, has long been implicated in modification of variegation, as a decrease in its dosage leads to PEV enhancement and an increase in PEV suppression (Hilliker and Appels, 1982; Spofford and DeSalle, 1991). In a similar way, the reduced potential of PNCs to modulate PEV (Mal’ceva et al., 1997) is presumably due to already mentioned unusually large nucleolus and rDNA over-representation. The simplest explanation for this rDNA effect is that the nucleolus and pericentric heterochromatin compete for common protein factors. Mod is the first candidate playing a role in such a mechanism to be identified (Fig. 7). In animals heterozygous for mod, displacement of the protein from dense DNA regions is amplified by the competitive effect of the nucleolus, thus leading to the expression of the PEV suppression phenotype. Distinct functions for Mod at chromatin and nucleolus? Recent developments in silencing effects in yeast established that the nucleolus is a compartment for Silent Information Regulator proteins (Gotta and Cockell, 1997; Sherman and Pillus, 1997). These proteins are involved in silencing at telomeres and mating-type loci. At the nucleolus they are
2760 L. Perrin and others associated to rDNA and are thought to function in the extension of life span. Since PEV in Drosophila and silencing in yeast are likely related phenomena, one can hypothesise that Mod also directly binds rDNA. Several lines of evidence actually argue against this hypothesis. On the one hand, the low abundance of rDNA sequences in the library of Mod DNA targets makes it unlikely that the preferential accumulation of the protein in the nucleolus results from an interaction with rDNA. On another hand, nucleolar Mod is loosely bound to the organelle and is released into nucleoplasm at low ionic strength. Lastly, unpublished work has shown (1) that the protein purified from nucleoplasm is highly phosphorylated and unable to bind DNA in vitro, and (2) that it is associated to an RNA molecule and migrates as a riboprotein complex on native gel electrophoresis. We therefore conclude that Mod does not contact rDNA at the nucleolus. The strong ectopic signal of Mod labelling on polytene chromosomes of the rib7(1A) transgenic line does not imply that Mod binds the extra rDNA unit. As the transgene is active in transcription and nucleolus formation (Karpen et al., 1988), this result simply indicates that Mod is associated to the mini-nucleolus that develops from the rDNA insertion site. It is therefore tempting to assume that nucleolar localisation of Mod depends on interaction with a RNA molecule involved in nucleolus biology, either rRNA or small nucleolar RNA. The involvement of Mod in different molecular networks in heterochromatin and the nucleolus suggests that it has distinct functions in the two compartments. It is noteworthy that the intranucleolar distribution of the protein changes during development, from a homogeneous distribution in the entire nucleolus of actively dividing cells (young embryos, imaginal discs) to a restricted accumulation at the organelle periphery in post-mitotic cells (late embryos, salivary glands). This change in pattern in mitotic versus quiescent cells could tentatively be related to a function for Mod at the nucleolus. In support of this, somatic clonal analysis revealed that mod loss of function is cell-autonomous and results in growth alteration and in short slender bristle morphology. These phenotypes are characteristic traits of a large class of dominant mutations, the Minute class (Lindsley and Zimm, 1992), widely believed to affect ribosomal protein genes (see Saeboe-Larssen et al., 1998 and references therein). Two additional classes of mutations, mini and bobbed, which affect rRNA genes, also alter protein synthesis and actually present similar phenotypes (Shermoen and Kiefer, 1975; Procunier and Dunn, 1978; see Kay and Jacobs Lorena, 1987 for a review). The Minute-like phenotypes of mod somatic clones point to a lesion in the protein synthesis capacity of progenitor cells from which the clones derive. As FLP-mediated recombination was induced at the first larval instar, progenitors correspond to actively dividing imaginal disc cells, which accumulate high levels of Mod at the nucleolus. When considered together, these data strongly suggest that Mod, in addition to its function in chromatin compaction processes, has a role in the regulation of nucleolus activity. We acknowledge G. Karpen and H. Saumweber for reagents and fly stocks. This work was supported by the CNRS, grants from l’Association pour la Recherche contre le Cancer and la Ligue Nationale Contre le Cancer (LNCC) to J. Pradel and doctoral fellowships from le Ministère de la Recherche et de la Technologie
and LNCC to L. Perrin. This work has also been partially supported by a contribution of the Istituto Pasteur-Fondazione Cenci Bolognetti, Università di Roma.
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