A dominant mutation due to the insertion of a P-element at 93E on the third chromosome of Drosophila melanogaster enhances position-effect variegation. The corresponding gene was cloned by transposon tagging and the sequence of the transcript revealed that it corresponds to the gene encoding the transcriptional activator and cell cycle regulator dE2F. The transposon-tagged allele is homozygous viable, and the insertion of the transposon in an intron correlates with a strong reduction in the amount of transcript. A homozygous lethal null allele was found to behave as a strong enhancer when heterozygous. Overexpression of the gene in transgenic flies has the opposite effect of suppressing variegation. A link is established here, and discussed, between the dose of a transcriptional activator, which controls the cell cycle, and epigenetic silencing of chromosomal domains in Drosophila.

whitemottled 4 is a chromosomal inversion resulting from a break of the X chromosome within centromeric heterochromatin, and another within euchromatin. In this rearrangement, the euchromatic gene white responsible for the red color of the eye, is relocated at the vicinity of heterochromatin and exhibits variegated expression. It is silenced in some cells and not in others, thus resulting in a mosaic phenotype. The phenomenon, reported for many chromosomal rearrangements and affecting many genes, is called position-effect variegation (PEV). The most popular model proposes that the condensed and inactive conformation of heterochromatin spreads over the breakpoint of the rearrangement and occasionnally encompasses and inactivates neighbouring euchromatic genes (Eissenberg, 1989; Tartof et al., 1989; Henikoff, 1990; Reuter and Spierer, 1992). The proportion of cells in which inactivation occurs for a given gene in a given rearrangement is regulated by dominant and recessive genetic modifiers acting in trans. Overall, an estimated 100-200 genes either enhance or suppress variegation (Reuter and Wolff, 1981; Tartof et al., 1989; Sinclair et al., 1989; Wustmann et al., 1989). It has been proposed that the products of many of these genes encode chromatin components or modifiers of chromatin components. They are recognized by having a dose-dependent effect on variegation. Molecular studies show that some suppressor mutations indeed identify heterochromatin constituents, with dose-dependent opposite effects (Eissenberg et al., 1990, 1992; Reuter et al., 1990; Garzino et al., 1992; Tschiersch et al., 1994). Enhancer mutations, in contrast, have identified transcriptional activators, or candidates for such a function (Dorn et al., 1993a; Farkas et al., 1994). Whether these factors act directly on chromatin, as ‘architectural transcription factors’, or by indirect effects through their target genes remains to be determined.

There is an interesting structural and functional overlap between the modifiers of PEV and the regulators of the bithorax complex of homeotic genes. Two classes of dominant trans-regulators affect the expression of these homeotic genes, which are clustered in large chromosomal domains. On the one hand, the Polycomb-group of genes are seen as repressors of homeotic genes. They act in a dose-dependent mode on the maintenance of the repressed state of homeotic genes (Moehrle and Paro, 1994). On the other hand, the trithorax-group of genes are general activators of homeotic genes. Structural and functional overlap was found between suppressors of variegation and Polycomb-group genes (Paro and Hogness, 1991; Moehrle and Paro, 1994; Fauvarque and Dura, 1993), and between enhancers of variegation and trithorax-group genes (Tamkun et al., 1992; Dorn et al., 1993a; Farkas et al., 1994; Reuter et al., unpublished data). Therefore, the study of modifiers of position-effect variegation may shed light on general mechanisms of genetic control of activity of chromosomal domains. Other epigenetic silencing phenomena such as telomere and mating-type silencing in yeast, or X-inactivation and parental imprinting in mammals may use similar mechanisms to those used in PEV.

A large effort is underway to identify dominant enhancers of position-effect variegation in Drosophila (Dorn et al., 1993b). We report here the genetic and molecular characterization of one of them, E(var)3-93E164, which corresponds to the Drosophila cell cycle controlling transcriptional activator dE2F (Ohtani and Nevins, 1994; Dynlacht et al., 1994; Duronio et al., 1995; Duronio and O’Farrell, 1995).

Genetic analysis

All fly stocks were maintained under standard conditions. A description of chromosomes and mutations can be found in Lindsley and Zimm (1992). The allele of E(var)3-93E induced by insertion of the P{pUChsneo ry+} transposon described by Dorn et al. (1993a) is designated E(var)3-93E164.

Autosomal transposition of P{pUChsneo ry+} was selected after a cross of wm4h; Su(var)2-101/CyRoi; ry506 females with wm4h P{pUChsneo ry+}/Y; +/+; TM3,ryRKSb e P{ry+(Δ2-3)}/ry506 males. The TM3,ryRKSb e P{ry+(Δ2-3)} chromosome is an efficient source of P transposase (Reuter et al., 1993). After purine treatment (Finnerty et al., 1970) wm4h/Y; Su(var)2-101/+; ry506and wm4h/Y; CyRoi/+; ry506 males will only survive if the P{pUChsneo ry+} element transposon is present. Exceptional males with an enhanced white-mottled phenotype were selected for further analysis and were crossed to wm4h; Cy/T(2;3)apXaSu(var)2-101/Sb females. T(2;3)apXaSu(var)2-101is abbreviated below as XaSu. The offspring wm4h/Y; +/XaSu/+ males with an enhancer phenotype (variegated instead of a suppressed red eye phenotype) were backcrossed again to wm4h; Cy/XaSu/Sb females. The putative enhancer can be localized to the different chromosomes with the help of aneuploid segregants of the apXa translocation (Reuter and Wolff, 1981). Segregation of P{pUChsneo ry+} was monitored by dot blot hybridization with a pUC18 probe using single flies from each of the 5 different genotypes (+/XaSu/ry506, +/XaSu/Sb, Cy/XaSu/ry506, Cy/XaSu/Sb and Cy/+; Sb/ry506). Balanced stocks were constructed after crosses with w+; CyO/Sco; ry506 and w+; TM3,ryRKSb e/ry506 females. The enhancer effect of the mutations on wm4h variegation were quantified by red eye pigment measurements in a XaSu background after a cross of wm4h; Cy/XaSu/Sb females to wm4h; +/XaSu/ry506E(var)3-93E males. The +/XaSu/ry506E(var)3-93E offspring males were compared to the +/XaSu/Sb and Cy/XaSu/Sb control genotypes.

Excisions of the P{pUChsneo ry+} element were induced by crossing homozygotes for the mutation with a TM6,Ubx/Sb P{ry+ (Δ2-3)}strain. The dysgenic males were then crossed to female balancers TM3, Sb ryRKe/TM6b, Tb e and exceptional ry− male progeny were used to establish independent lines and tested for precise excision of the transposon on genomic Southern blots.

Effects on PEV were tested by crosses with wm4h; Cy/XaSu/Sb females. Crosses of these females with Canton S males were used as control.

Molecular biology

Plasmid rescues were obtained by following the protocol of Pirrotta (1986). RNA in situ hybridization on whole mounts were prepared as described by Tautz and Pfeifle (1989) modified by Cléard et al. (1995). Northern blot analyses were performed with the protocol of Cléard et al. (1995), except for the probe, which was a DNA probe and for the hybridization mixture which was the following: 5× Denhardt’s, 6× SSPE, 8% dextran sulfate, 36% urea (Merck no. 8487) 100 μg/ml denatured sonicated salmon sperm DNA. Primer extension were done according to the protocol of Mason et al. (1993) with primers noted on the dE2F promoter region (see Fig. 4).

Germline transformation

The plasmid construct to be injected was prepared by inserting an EcoRI full length cDNA fragment of 4.2 kb from the plasmid pNB1.4 into the plasmid pHSS7 derived from pHSS6 (Seifert et al., 1986) containing NotI sites at each extremity of the polylinker. The NotI insertion was then cloned downstream of the Hsp70 promoter in the NotI site of the pNHT4 transformation vector (Schneuwly et al., 1986).

Injections into embryos were done according to the method of Spradling (1986). ry+ flies were selected, characterized by in situ hybridization and crossed to the wm4h; XaSu/Sb strain at 29°C.

A new dominant enhancer of position-effect variegation maps at 93E

Insertional mutations with an enhancer of PEV phenotype have been successfully isolated after mobilization of the P{pUChsneo ry+} transposon (Dorn et al., 1993b). Genomic DNA fragments flanking the insertion site can be isolated via plasmid rescue and afterwards used as an entry point for cloning of the corresponding gene. A new genetic screen has been applied, which ensures efficient mutant isolation by detection of a P{pUChsneo ry+} transposition as well as the dominant enhancer effect in individual offspring males. After remobilization of an X chromosomal P{pUChsneo ry+} insert in parental males, transpositions of the modified P-transposon into autosomes were selected as males homozygous for ry506 that survived purine treatment. Purine efficiently kills ry flies (Finnerty et al., 1970) and the surviving males must have received a ry+ allele through insertion of P{pUChsneo ry+} into an autosomal site. Because all flies are wm4h, either in absence or in presence of the strong suppressor Su(var)2-101 (Reuter et al., 1982), an enhancer effect of the insertions can be monitored in the surviving F1 males by an increase in white mottled mutant phenotype. In about 250,700 offspring, 32 (1.3×10−4) enhancer mutations have been isolated. The mutations were genetically mapped and localized by in situ hybridization (see Materials and Methods). An enhancer mutation, E(var)3-93E164, was further analyzed.

Mutations induced by a transposable element can be readily mapped on larval salivary gland polytene chromosomes by in situ hybridization using transposon sequences as a probe. A single signal was found at 93E on the right arm of the third chromosome (not shown), thus suggesting that there is a single insertion of the P-transposon. This was confirmed by genomic Southern blot hybridization (not shown). Effects of mutations on PEV can be detected by observing their effects on the mosaic phenotype seen on the compound eye of flies with a variegating rearrangement of the white gene. Inactivation of white results in clones of white ommatidia in a wild-type red background. Fig. 1A illustrates the mottled phenotype of wm4h, and Fig. 1B the enhancement of variegation due to the heterozygous mutation E(var)3-93E164. The mutation E(var)3-93E164 was found to be homozygous viable and fertile.

Fig. 1.

Effect on position-effect variegation of an insertional mutation at 93E. (A) Head of a male carrying the X-chromosome rearrangement white-mottled-4h. (wm4h/Y;+/Cy;+/Sb). (B) Head of a male carrying the enhancer mutation in the same background as in A (wm4h/Y;+/Cy;E(var)3-93E164/Sb). (C) Head of a male revertant for the P-induced enhancer mutation (wm4h/Y;+/Cy;excE(var)3-93E164/Sb). For details of crosses, see Materials and Methods.

Fig. 1.

Effect on position-effect variegation of an insertional mutation at 93E. (A) Head of a male carrying the X-chromosome rearrangement white-mottled-4h. (wm4h/Y;+/Cy;+/Sb). (B) Head of a male carrying the enhancer mutation in the same background as in A (wm4h/Y;+/Cy;E(var)3-93E164/Sb). (C) Head of a male revertant for the P-induced enhancer mutation (wm4h/Y;+/Cy;excE(var)3-93E164/Sb). For details of crosses, see Materials and Methods.

To ascertain that the phenotype is indeed caused by the insertion of the transposon, the element was mobilized to produce phenotypic revertants associated with an excision of the transposon. The P-induced mutant was crossed to a transposase-producing strain (P{ry+ Δ2-3}), and the progeny analyzed for the loss of the transposon as seen by the loss of the ry+ gene. Eleven independent ry lines were analyzed by Southern blot hybridizations. Eight were internal deletions in the transposon, and three apparent precise excisions. As illustrated in Fig. 1C, one of the three apparent precise excisions clearly reverts the enhanced variegation phenotype. Molecular analysis reported below confirmed the correspondence between the mutation and the phenotype.

Molecular cloning of the E(var)3-93E locus

The modified P element P{pUChsneo ry+} allows plasmid rescue of genomic DNA on one side of the transposon. Digestion with SalI or with EcoRI followed by ligation allowed us to recover plasmids of 9.1 and 7.1 kb respectively. The plasmids contain 4.4 kb of pUC plasmid sequence and the remaining DNA consists of flanking Drosophila genomic sequence. As a control, the rescued plasmids were found to hybridize to the same 93E locus on wild-type polytene chromosomes, thus demonstrating that the genomic sequence originates as expected from that region (not shown). With the genomic fragment as a probe, we screened a genomic library (Maniatis et al., 1978) to isolate sequences extending on both sides of the P-element. The region encompassing the 5′ end of the transcription unit was isolated in a second step by using the 5′ end of a cDNA clone (described below) as a probe. The resulting map of the genomic region and the position of the transposon is depicted in Fig. 2. The 5′-end exon is separated from the main coding region by a 12-kb-long intron.

Fig. 2.

Genome and transcript organization of the Drosophila E2F locus. (A) Genomic map and position of the P{pUChsneo ry+} transposon insertion. Probe 2.0 is the EcoRI restriction fragment used as probe on northern blots (see text). E, EcoRI; H, HindIII; X, XhoI; S, SalI. (B) Maps of the transcription unit and of the largest cDNA clone. Exons 4, 5, and protein coding part of exon 6 are from Duronio et al. (1995). (C) Features of the deduced protein sequence: the scheme is aligned on the cDNA restriction sites; * marks the position of the polymorphism QQLQQQ (see text); E2F DNA binding domain and Rb binding domain determined by Nevins (1992) and Ohtani and Nevins (1994). The intron/exon junctions have been sequenced showing the absence of additional miniexon within the large first intron by comparison with cDNA sequence.

Fig. 2.

Genome and transcript organization of the Drosophila E2F locus. (A) Genomic map and position of the P{pUChsneo ry+} transposon insertion. Probe 2.0 is the EcoRI restriction fragment used as probe on northern blots (see text). E, EcoRI; H, HindIII; X, XhoI; S, SalI. (B) Maps of the transcription unit and of the largest cDNA clone. Exons 4, 5, and protein coding part of exon 6 are from Duronio et al. (1995). (C) Features of the deduced protein sequence: the scheme is aligned on the cDNA restriction sites; * marks the position of the polymorphism QQLQQQ (see text); E2F DNA binding domain and Rb binding domain determined by Nevins (1992) and Ohtani and Nevins (1994). The intron/exon junctions have been sequenced showing the absence of additional miniexon within the large first intron by comparison with cDNA sequence.

Deduced protein sequence of transcripts from the E(var)3-93E locus identifies the Drosophila E2F gene

We mapped transcription units in the vicinity of the transposon. Restriction fragments on each side of the insertion site (Fig. 2) were used as probes on northern blots of embryonic RNA. A 2.0 kb EcoRI fragment (localized on the genomic region in Fig. 2A) detects three overlapping transcripts of 3.6, 4.2 and 4.7 kb (Fig. 3A). This fragment, located 3.0 kb 3′ of the P transposon and which gave a strong signal on northern blots, was used to select cDNA clones in a 4-8 hour cDNA library (Brown and Kafatos, 1988). About 50,000 colonies were screened and 26 clones were isolated. Eight clones were analysed in detail to establish the complete map of the transcripts depicted in Fig. 2. cDNA sequencing allowed us to detect an open reading frame, which was compared to known sequences in databases, and identified the cDNAs as encoding the Drosophila homolog of E2F, dE2F (Ohtani and Nevins, 1994; Dynlacht et al., 1994). The eight clones analyzed can be arranged in three classes, varying in the length of their 3′ untranslated region (not shown), but we could not unambiguously assign these classes to the three sizes of transcripts seen on northern blots.

Fig. 3.

The promoter region of the dE2F locus. (A) Sequence of the promoter region and transcription starts of dE2F. The bent arrows indicate the two transcription start sites, the major one being in bold. The two boxes are the potential E2F binding sites with differences from the consensus sequence (TTTCGCGC) noted with an asterisk. pNB14.3 and pNB1.4 are the longest cDNAs analyzed. The backwards arrows show the two oligonucleotides used to perform the primer extension experiment. O&N marks the beginning of the sequence published by Othani and Nevins (1994). The two vertical arrows indicate the start and the end of the first 12 kb intron. (B) Primer extension done with the most 5′ oligonucleotide. Sequencing reactions on the side (order G-A-T-C) were from the same oligonucleotide on a 6% sequencing gel. The other oligonucleotide gave the same result (not shown). The translation starts 86 nucleotides downstream of the sequence presented in the figure.

Fig. 3.

The promoter region of the dE2F locus. (A) Sequence of the promoter region and transcription starts of dE2F. The bent arrows indicate the two transcription start sites, the major one being in bold. The two boxes are the potential E2F binding sites with differences from the consensus sequence (TTTCGCGC) noted with an asterisk. pNB14.3 and pNB1.4 are the longest cDNAs analyzed. The backwards arrows show the two oligonucleotides used to perform the primer extension experiment. O&N marks the beginning of the sequence published by Othani and Nevins (1994). The two vertical arrows indicate the start and the end of the first 12 kb intron. (B) Primer extension done with the most 5′ oligonucleotide. Sequencing reactions on the side (order G-A-T-C) were from the same oligonucleotide on a 6% sequencing gel. The other oligonucleotide gave the same result (not shown). The translation starts 86 nucleotides downstream of the sequence presented in the figure.

Fig. 4.

Northern blot analysis of the transcripts of the dE2F gene. (A) 20 μg of total RNA (overnight collection) from mutant (164) or wild-type embryos hybridized with probe 2.0 (see Fig. 2). The size of the transcripts is 3.6, 4.2, 4.7 kb. The blot was also probed with actin (Fyrberg et al., 1980) as a control of the amounts of RNA transferred per lane. Size markers are indicated on the side. Transcripts are difficult to see in the mutant in this experiment and exposure, but were weakly detectable in others and in whole-mount mutant embryos. (B) Developmental northern blot. 20 μg of five embryonic stages were used except for lane 0-2hr and 12-24hr where only 10 μg of RNA were loaded. The blots were probed with an EcoRI fragment containing the full length cDNA of dE2F (pNB1.4). The absence of the large transcripts in 0-2 hours embryos has been confirmed on other blots (not shown).

Fig. 4.

Northern blot analysis of the transcripts of the dE2F gene. (A) 20 μg of total RNA (overnight collection) from mutant (164) or wild-type embryos hybridized with probe 2.0 (see Fig. 2). The size of the transcripts is 3.6, 4.2, 4.7 kb. The blot was also probed with actin (Fyrberg et al., 1980) as a control of the amounts of RNA transferred per lane. Size markers are indicated on the side. Transcripts are difficult to see in the mutant in this experiment and exposure, but were weakly detectable in others and in whole-mount mutant embryos. (B) Developmental northern blot. 20 μg of five embryonic stages were used except for lane 0-2hr and 12-24hr where only 10 μg of RNA were loaded. The blots were probed with an EcoRI fragment containing the full length cDNA of dE2F (pNB1.4). The absence of the large transcripts in 0-2 hours embryos has been confirmed on other blots (not shown).

When comparing the eight cDNAs analyzed, it appeared that four of them have a deletion of 18 nucleotides in the open reading frame. This leads to the deletion of six amino acids (QQLQQQ). Sequencing of our genomic clone from the Maniatis library included DNA coding for these amino acids, without interruption of the open reading frame before or after the stretch. There is hence no evidence that this deletion results from alternative splicing. The sequence resembles opa repeats (Wharton et al., 1985). We assume that it is a polymorphism in the population used for making the cDNA library.

E2F is known to be autoregulated in mammals and E2F binding sites have been described in the promoter region of the mammalian E2F gene (Johnson et al., 1994; Neuman et al., 1994). We have mapped the 5′ end of the transcription unit on the genomic DNA by primer extension. The result is shown in Fig. 3, which also presents genomic sequence upstream of the two starts of transcription. It should be noted that this promoter has no obvious TATA box. Two potential E2F binding sites were found within 50 base pairs upstream of the start sites.

The mutation affects transcript levels

Next we looked for effects of the insertional mutation on transcripts of the locus. Fig. 4 depicts northern blot hybridizations of RNA extracted at selected developmental stages using the cDNA as a probe. Early embryos (0-4 hours) contain three different sized transcripts, but only the two larger ones are detected in later stages. In a separate experiment, RNA from 0-2 hours was blotted. Only the shorter transcript is visible, thus establishing its maternal origin.

As the mutation is homozygous viable, RNA was extracted from homozygous mutant embryos to examine possible alterations of dE2F expression. Fig. 4 shows a severe reduction of all RNA species in the mutant, compared to wild type. The distribution of transcripts was examined in whole-mount homozygous mutant embryos (not shown). Transcripts were detectable, but at a much lower level. The pattern was similar to that seen in wild-type embryos (see below).

R. Duronio and P. O’Farrell have provided us with a null and homozygous lethal allele of the Drosophila dE2F that they have determined to be a nonsense mutation at amino acid 31 (dE2F91, Duronio et al., 1995). We have found this allele to be a strong dominant enhancer, actually stronger than our P-induced mutation, thus confirming allelism between the dE2F gene and the enhancer of variegation phenotype (Fig. 5).

Fig. 5.

PEV depends on the dose of dE2F. Two wild-type doses of the dE2F gene (Canton S control) in a wm4h background (wm4h/Y;+/Cy;+/Sb) (top). One wild-type dose of dE2F (heterozygous for the null mutation dE2F91; Duronio et al., 1995) in a wm4h background (wm4h/Y;+/Cy;dE2F91/Sb) (bottom left), and two wild-type doses of dE2F and one copy of a heat inducible transgene in the same background (wm4h/Y;+/Cy;P{pNHT4-E2F ry+}/Sb) (bottom right). Flies were all raised in mild heat shock condition (29°C).

Fig. 5.

PEV depends on the dose of dE2F. Two wild-type doses of the dE2F gene (Canton S control) in a wm4h background (wm4h/Y;+/Cy;+/Sb) (top). One wild-type dose of dE2F (heterozygous for the null mutation dE2F91; Duronio et al., 1995) in a wm4h background (wm4h/Y;+/Cy;dE2F91/Sb) (bottom left), and two wild-type doses of dE2F and one copy of a heat inducible transgene in the same background (wm4h/Y;+/Cy;P{pNHT4-E2F ry+}/Sb) (bottom right). Flies were all raised in mild heat shock condition (29°C).

These two experiments, namely reduction of transcript level in the insertional mutant and the enhancer effect of the null mutant, show that the dominant enhancer effect is due to reduced activity of dE2F.

The dE2F mutation enhances yellow variegation

To assess whether the dose of dE2F also affects other variegating rearrangements, the null mutation was placed in trans with two minichromosomes bearing a variegating yellow+ gene (Dp(1;f)1187 and Dp(1;f)8-23, Karpen and Spradling, 1990; Tower et al., 1993). The color phenotype was scored on the middle bristles of the triple row at the anterior wing margin. For both minichromosomes, the percentage of yellow bristle was significantly higher in heterozygous flies compared to the siblings bearing two wild-type copies of dE2F. With Dp (1;f)1187, 23% (mean) of the bristles were yellow in flies with a mutant copy of dE2F, in contrast to 12% in control flies (two copies of dE2F). A t-test confirms that these two values are significantly different at the 99% confidence level. Using the Dp(1;f)8-23 rearrangement, heterozygous mutant flies showed 33% of yellow bristles versus 20% in the control. A t-test confirms that these two values are significantly different at the 95% confidence level. We conclude that a reduced dose of dE2F acts as a dominant enhancer of the variegation of different genes in different variegating chromosomal rearrangements.

Overexpression of dE2F suppresses position-effect variegation

A clear-cut demonstration that the transcripts indeed encode the functional protein can be provided by introducing the cDNA into the genome by genetic transformation. It is expected that this overexpression will have the opposite effect of gene dose reduction in the mutant. cDNAs containing the complete ORF were cloned in a P-transposon-derived transformation vector (pNHT4; Schneuwly et al., 1986) that allowed us to place the cDNA under the control of the hsp70 heat shock promoter. We used cDNAs either with the short polymorphic insertion or without. They both gave the same effect on PEV. After injection into a ry background, transformant lines were selected by the ry+ marker of the transformation vector. The effect on variegation was determined by crossing transformants in the white-mottled background. Transformant flies and wild-type controls were grown at 29°C, a mild heat shock condition. Fig. 5 shows that the presence of one copy of the hs-dE2F transgene (in a background of two wild-type resident copies of dE2F) produces a clear suppressor effect. Flies grown at lower temperature (25°C) do not show this effect. Robin Wharton and Maki Asano provided us with their transgenic lines tagged with the miniwhite gene as a marker (unpublished data). Though the miniwhite background rendered the experiment more difficult, we have verified that these lines also suppress position effect variegation (not shown).

Though we have not embarked on a detailed comparison, it seems, at a qualitative level, that the suppressor phenotype is the same whether or not the transgene contains the six amino acid polymorphic stretch. We conclude that overexpression of dE2F has the opposite effect to the reduction of gene dosage. E(var)3-93E164 is homozygous viable, but null mutants are homozygous lethals (Duronio et al., 1995). Under mild heat shock conditions (29°C), both of these constructs save the lethality of the null allele dE2F91 (data not shown). Rescue of a null allele demonstrate clearly that the transgene is expressed and functional.

The transcripts are ubiquitous in early embryonic development

The distribution of transcripts was examined in whole mount embryos. The results are illustrated in Fig. 6 for ten selected developmental stages. They extend previous work of Hao et al. (1995) and Duronio et al. (1995). The transcripts are ubiquitous in preblastoderm embryos. Incomplete cellular blastoderm embryos (stage 4) show a pattern reminiscent of the one reported by Hao et al. (1995) for dDP1, the factor that makes a heterodimer with dE2F in its active form (Dynlacht et al., 1994) and for string and twine (Alphey et al., 1992), two genes implicated in cell cycle progression. Staining is preponderant under the peripheral nuclei. At cellular blastoderm (stage 5) the zygotic transcripts appear in three broad bands. Ventral expression seems stronger. By the beginning of gastrulation (stage 6) a very transient banded pattern appears, showing at least 10 bands, having similar spacing to that of segment polarity genes. The ventral furrow is strongly stained. Slightly later (stage 7), the bands have already disappeared leaving most of the labeling in the ventral furrow (mesoderm). At stage 8, transcripts accumulate in mesoderm tissues, and at stages 9-10, the embryo shows staining principally in the neuroblasts and mesoderm. At stages 10-11, the neuroblast staining stands out. At stage 13, anterior and posterior midgut staining appears. By stage 16 the midgut and foregut are also stained.

Fig. 6.

Pattern of expression of the dE2F transcripts in embryos. RNA in situ hybridisation was performed on whole mounts as described by Cléard et al. (1995) with the longest cDNA as a probe. (A) Stage 1 embryo. The transcripts are ubiquitous. (B) Incompleted cellular blastoderm (stage 4). Staining is preponderant under the peripheral nuclei. (C) Cellular blastoderm (stage 5). The zygotic transcripts start in three broad bands. (D) Beginning of gastrulation (stage 6). A very transient banded pattern appears showing at least 10 bands having similar spacing to that of segment polarity genes. The ventral furrow is strongly stained. (E) Gastrulation (stage 7). The bands have already disappeared leaving most of the labeling in the ventral furrow (mesoderm). (F) Stage 8 embryo. Transcripts accumulate in mesoderm tissues. (G) Stage 9-10. Staining principally in the neuroblasts and mesoderm. (H) Stage 10-11. Neuroblasts staining stands out. (I) Stage 13. Anterior and posterior midgut staining appears. (J) Stage 16. The midgut and foregut are also stained.

Fig. 6.

Pattern of expression of the dE2F transcripts in embryos. RNA in situ hybridisation was performed on whole mounts as described by Cléard et al. (1995) with the longest cDNA as a probe. (A) Stage 1 embryo. The transcripts are ubiquitous. (B) Incompleted cellular blastoderm (stage 4). Staining is preponderant under the peripheral nuclei. (C) Cellular blastoderm (stage 5). The zygotic transcripts start in three broad bands. (D) Beginning of gastrulation (stage 6). A very transient banded pattern appears showing at least 10 bands having similar spacing to that of segment polarity genes. The ventral furrow is strongly stained. (E) Gastrulation (stage 7). The bands have already disappeared leaving most of the labeling in the ventral furrow (mesoderm). (F) Stage 8 embryo. Transcripts accumulate in mesoderm tissues. (G) Stage 9-10. Staining principally in the neuroblasts and mesoderm. (H) Stage 10-11. Neuroblasts staining stands out. (I) Stage 13. Anterior and posterior midgut staining appears. (J) Stage 16. The midgut and foregut are also stained.

E(var)3-93E164 is a dominant mutation that enhances position-effect variegation. When one dose is lost or when expression is reduced, heterochromatic position-effect silencing is enhanced. This dominant enhancer effect makes E(var)3-93E164 a member of the group of haplo-insufficient enhancers of PEV. Overexpression with a transgene under the control of a heat shock promoter has an opposite suppressor effect on variegation. Hence, within the enhancers of PEV, this locus is a haplo-enhancer gene with a triplo-suppressor effect. Triplo-effect is not used here in a strict sense as there might be differences between the overexpression of a cDNA transgene and a duplication of the whole locus. E(var)3-93E164 is the first molecularly characterized gene with this opposite effect on PEV. Though the classical reporter for PEV is white-mottled, we have also determined a parallel enhancer effect for two other rearrangements variegating for the gene yellow, making it unlikely that there is a specific effect of the enhancer mutation on the transcription of the white gene.

E(var)3-93E164 encodes the Drosophila E2F transcriptional activator and cell cycle regulator. This is a surprising finding as the dose dependence argues in favor of an architectural effect on chromatin, although dE2F could also be the rate-limiting regulator of an architectural factor. However, previously characterized mutations enhancing PEV have identified other transcriptional activators such as the GAGA-factor (also known as trithorax-like, a regulator of homeotic genes (Farkas et al., 1994)), the E(var)3-93D gene, which is also a regulator of homeotic genes as well as a modifier of the activity of su(Hw), and encodes a protein involved in the insulation of chromosomal domains (Dorn et al., 1993a; Gerasimova et al., 1995), and possibly modulo (Garzino et al., 1992). Recently, zeste was reported as a recessive enhancer of PEV (Judd, 1995). zeste encodes a DNA binding protein with two previously known roles: transcriptional activator affecting chromatin configuration at a distance, and an effector of chromosome pairing-dependent effects (reviewed by Pirrotta, 1991). In contrast, suppressor mutations have identified the heterochromatin-associated proteins HP1 (Eissenberg et al., 1992) and Su(var)3-7 (Reuter et al., 1990; Cléard, 1993; Cléard et al., 1995).

In mammals, members of the E2F family of proteins form heterodimers with the DP family of proteins in different combinations, and activate genes involved in DNA metabolism, hence promoting the transition from G1 to S phase. These dimers can be sequestered by the retinoblastoma tumor suppressor protein (pRb) or other pRb-related proteins (p107, p130). Phosphorylation of the pRb proteins by cyclin-dependent kinases in the course of the G1 phase releases the E2F-DP dimers. Absence of E2F blocks the cell cycle at the G1/S transition (reviewed by La Thangue, 1994). In contrast, overexpression of E2F can lead to oncogenic transformation (Beijersbergen et al., 1994; Ginsberg et al., 1994; Singh et al., 1994), or apoptosis (Qin et al., 1994; Shan and Lee, 1994; Wu and Levine, 1994). As in mammalian cells, active dE2F is complexed with dDP in Drosophila (Dynlacht et al., 1994). It binds to the promoter of a dE2F-dependent gene, DNA polymerase alpha (Ohtani and Nevins, 1994), and is essential for the G1 to S progression (Duronio et al., 1995). Interaction of dE2F with pRb arrests cells in G1 phase, and dE2F binds to cyclin A/cdk2 and can be modulated by viral oncoproteins (Hao et al., 1995). These data, and our findings, show that the Drosophila dE2F protein parallels the function of mammalian E2F.

In the widely accepted ‘structural’ model of heterochromatic PEV, enhancers of variegation are constituents, or modifiers of constituents, of euchromatin that resist epigenetic silencing or promote derepression. They can also be negative regulators of heterochromatin constituents. We propose two hypotheses to explain how the dose of dE2F affects position-effect variegation. First, dE2F might be a positive transcriptional activator of another enhancer gene, or a negative regulator of a suppressor gene. Decreased amounts of E2F would decrease the expression of the other enhancer or increase the expression of the suppressor, producing the haplo-enhancer effect. In contrast, increased amounts of dE2F would increase the levels of the other enhancer or decrease the level of a suppressor, leading to the triplo-suppressor effect. It should be noted here, that though E2F was characterized as an activator of transcription during the S phase, a negative effect is also plausible via the complex between E2F and the hypophosphorylated form of pRb (Weintraub et al., 1995). The association of E2F and pRb could also prevent DNA condensation. In mammalian cells, the pRb protein indeed seems to localise not only over euchromatin but also as large nuclear granules located at the border between euchromatin and hete-rochromatin, suggesting that the protein may influence DNA condensation (Szekely et al., 1991). dE2F could also prevent the spreading of the heterochromatic conformation by its association with its many target sites on euchromatin. In such a model, there must be E2F binding sites between the variegation-inducing heterochromatin and the variegating gene. Among other known enhancers of PEV, the trithorax-like locus does indeed encode a protein, the GAGA factor, with binding sites both at its euchromatic targets sites and in heterochromatin (Raff et al., 1994).The product of E(var)3-93D, probably also a transcription factor, is associated with many sites in euchromatic arms of polytene chromosomes (Dorn et al. 1993a).

Our second hypothesis proposes that the dose of dE2F modulates PEV through its function as a cell cycle regulator. In this respect, it is interesting to note that involvement of mitosis control in variegation was suggested by the finding that mutation of the PP1 phosphatase gene suppresses variegation and leads to abnormal mitosis (Baksa et al., 1993) and that mutations in PCNA, an auxiliary factor in DNA replication, are recessive sup-pressors of PEV (Henderson et al., 1994). In humans, PP1 binds to the retinoblastoma protein, the same protein that releases E2F upon phosphorylation (Durfee et al., 1993). A lower dose of PP1 in flies could prevent some dephosphorylation of pRB (a protein yet to be isolated in Drosophila), resulting in an increased level of free dE2F, and hence suppressing PEV. In this second hypothesis, it remains to be determined how a perturbation of the G1/S transition (or in the length of the S phase) can affect PEV. During DNA replication is likely to be the critical time for establishing, erasing, or maintaining epigenetic programming. If this is true for the effect we see, then a delay in the G1/S transition or a modification of the S phase could have an enhancer effect on the establishment of silencing as detected by heterochromatic position effects. This could be explained, for example, if a longer S phase provides more time for building up the silenced state of large chromosomal domains. An old, and possibly related, observation is that heterochromatic regions are late replicating (Lima de Faria and Jarworska, 1968).

In conclusion, we emphasize that this possible link between epigenetic silencing and cell cycle was recently suggested in the analogous phenomena of mating type locus silencing and telomeric position effect in yeast. Indeed, telomeric silencing is variegated and spreads from the telomere (Renauld et al., 1993). Miller and Nasmith (1984) have shown that progression through the S phase of the cell cycle is required for establishing a repressed state of the silent mating type locus. Recently, Laman and collaborators have looked for mutations suppressing the silencer-defective mutation rap1 (Laman et al., 1995), an approach analogous to our search for enhancers of white-mottled variegation. Three suppressors were cloned and all encode components necessary for normal cell cycle progression. This analogy reinforces the idea that telomeric position effect in yeast and PEV are related. That mutations perturbing cell-cycle progression enhance silencing in both systems supports the hypothesis that establishment of epigenetic silencing occurs in a window of the cell cycle, which is critical either by its duration and/or its timing.

We thank Robin Wharton and Maki Asano for providing us with dE2F transformants, Robert Duronio and Pat O’Farrell for providing us with the dE2F null mutant, and Gary Karpen for yellow variegating rearrangements. We are also grateful to all the above-mentioned for sharing unpublished data. This work was supported by grants from the Swiss National Science Foundation (to P. S. and D. P.) and by grants from the Deutsche Forschung Gemeinschaft (DFG,SFB 363 to G. R. and J. S.).

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