In Vivo Chromatin Accessibility Correlates With Gene Silencing in Drosophila

Corresponding author: Jean-Maurice Dura, Institut de Génétique Humaine, CNRS / UPR 1142, 141, rue de la Cardonille, 34396 Montpellier Cedex 5, France., jmdura{at}igh.cnrs.fr (E-mail).

Communicating editor: J. A. BIRCHLER

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Gene silencing by heterochromatin is a well-known phenomenon that, in Drosophila, is called position effect variegation (PEV). The long-held hypothesis that this gene silencing is associated with an altered chromatin structure received direct support only recently. Another gene-silencing phenomenon in Drosophila, although similar in its phenotype of variegation, has been shown to be associated with euchromatic sequences and is dependent on developmental regulators of the Polycomb group (Pc-G) of gene products. One model proposes that the Pc-G products may cause a local heterochromatinization that maintains a repressed state of transcription of their target genes. Here, we test these models by measuring the accessibility of white or miniwhite sequences, in different contexts, to the Escherichia coli dam DNA methyltransferase in vivo. We present evidence that PEV and Pc-G-mediated repression mechanisms, although based on different protein factors, may indeed involve similar higher-order chromatin structure.

WHEN the euchromatic gene white, which is responsible for the red color of the fruit fly eye, is relocated near heterochromatin because of an X-chromosome rearrangement, white function is extinguished in some cells, leading to a mottled pigmentation. This phenomenon is called position-effect variegation (PEV). To explain the silencing of the white gene in some cells and not in others, the most popular model proposes that the condensed and inactive conformation of pericentric heterochromatin spreads over the breakpoint of the rearrangement and randomly inactivates neighboring genes (LOCKE et al. 1988 ; EISSENBERG 1989 ; TARTOF et al. 1989 ; HENIKOFF 1990 ). About 150 genes have been identified by genetic modifier mutations, both suppressors and enhancers, of this phenomenon (REUTER and SPIERER 1992 ; HENIKOFF 1996 ). It has been proposed that the products of many of these genes encode chromatin components or modifiers of those components. Some suppressor mutations were indeed molecularly identified as heterochromatin constituents (EISSENBERG et al. 1990 , EISSENBERG et al. 1992 ; GARZINO et al. 1992 ; CLEARD et al. 1997 ) or as potential heterochromatin constituents (REUTER et al. 1990 ; TSCHIERSCH et al. 1994 ) with dose-dependent opposite effects. One such gene, Su(var)205, encodes the heterochromatin-associated protein HP-1 (EISSENBERG et al. 1990 ). More recently, Su(var)3-7 was identified as a nuclear protein that associates with pericentric heterochromatin and coimmunoprecipitates with HP-1, suggesting that the two proteins cooperate in building a silencing complex that is responsible for the genomic silencing associated with heterochromatin (CLEARD et al. 1997 ). Moreover, histone modification, such as deacetylation, might affect how tightly the DNA is associated with the histones and/or might be necessary to recruit proteins to establish the silencing (DE RUBERTIS et al. 1996 ).

Direct evidence of altered chromatin structure associated with the PEV phenomenon was first described by WALLRATH and ELGIN 1995 . Using standard chromatin analysis on isolated nuclei, they showed that one particular hsp26 promoter site inserted into pericentric heterochromatin via P elements presents both a reduced accessibility to restriction enzymes and a correlated, strongly variegated phenotype for these lines. Furthermore, micrococcal nuclease digests showed that these inserts were packaged in a more regular nucleosome array than that observed for euchromatic inserts. However, no such difference has been observed for classical PEV, i.e., white locus inversions (HAYASHI et al. 1990 ; LOCKE 1993 ; SCHLOSSHERR et al. 1994 ).

Striking structural and functional similarities exist between PEV and the activities of Polycomb-group genes (Pc-G) in regulating the homeotic complexes. The expression pattern of the homeotic genes of the Antennapedia and Bithorax complexes are initiated early in embryonic development by the gap and pair-rule genes (BIENZ and MULLER 1995 ). These genes are expressed transiently, whereas continuous homeotic gene activity is essential for implementing stable determination of cell fates. The Pc-G maintains the repressed state in those cells where the homeotic gene was originally inactive (PARO 1993 ; PIRROTTA et al. 1995 ), whereas the trithorax group (trx-G) sustains the active state in cells where the homeotic gene was originally expressed (KENNISON 1995 ). Genetic and molecular data fit well with the fact that Pc-G gene products may act in large protein complexes. Combinations of different Pc-G mutations have a much more severe phenotype than the additive effect of two single mutations (JURGENS 1985 ), and haplo-insufficient phenotypes of some mutants can be partially rescued by increasing the copy number of another member (CHENG et al. 1994 ). The Polycomb protein (PC) was found to be associated in a multimeric complex with at least one other member of the Pc-G, the PH protein (FRANKE et al. 1992 ). Furthermore, the roughly 100 polytene-binding sites for PC, PH, and PCL completely coincide and largely overlap with those for PSC, E(Z), and ASX (DECAMILLIS et al. 1992 ; FRANKE et al. 1992 ; RASTELLI et al. 1993 ; LONIE et al. 1994 ; CARRINGTON and JONES 1996 ; SINCLAIR et al. 1998A ).

Moreover, PC and the Drosophila heterochromatin protein HP-1 share a domain of homology (the chromodomain), and, therefore, it was suggested that Pc-G proteins may function by regulating higher-order chromatin structures (PARO and HOGNESS 1991 ). This regulation is mediated by Pc-G response elements (PREs), cis-acting DNA elements that are able to maintain the expression boundaries of homeotic genes in a Pc-G-dependent manner (SIMON et al. 1993 ). When present in reporter gene constructs in transgenic flies, such elements cause additional PC binding at the insertion site on polytene chromosomes (ZINK et al. 1991 ), and they are able to silence neighboring reporter genes in a Pc-G-dependent manner (FAUVARQUE and DURA 1993 ; CHAN et al. 1994 ; GINDHART and KAUFMAN 1995 ; GINDHART et al. 1995 ; ZINK and PARO 1995 ). The analogy between the variegated phenotype of such reporter genes and PEV led us to call this phenomenon developmental regulator effect variegation (DREV; FAUVARQUE and DURA 1993 ). PREs are thus thought to act as sequences that recruit members of the Pc-G and nucleate the formation of Pc-G-silencing complexes on target genes (PIRROTTA and RASTELLI 1994 ; ORLANDO and PARO 1995 ). It was proposed that Pc-G complexes function by spreading from PREs over extended chromosomal domains. Therefore, as envisaged for heterochromatic silencing, Pc-G complexes may package chromatin into a compact structure that prevents the binding of transcriptional activators to enhancer or promoter sequences (PARO 1990 ). This model is supported by the findings that PC is associated with the entire inactive region of the BX-C in tissue culture cells (ORLANDO and PARO 1993 ) and that GAL4-dependent transcription is inhibited by Pc in the bx regulatory region of the Ubx gene in embryos (MCCALL and BENDER 1996 ). However, no significant difference in the accessibility of restriction endonucleases has been detected between the active and inactive states of the Antennapedia gene in imaginal discs (SCHLOSSHERR et al. 1994 ).

The introduction into yeast of the gene encoding the Escherichia coli Dam methyltransferase has allowed the study of chromatin structure in vivo without the complications of in vitro artifacts introduced during extraction or purification procedures. Telomeric sequences, which, in yeast, induce a gene silencing that resembles that of Drosophila PEV, are undermethylated compared to euchromatic sequences (GOTTSCHLING 1992 ). Other more recent studies confirm the ability of exogenous methylases to probe yeast chromatin structure in vivo (SINGH and KLAR 1992 ; KLADDE and SIMPSON 1994 ; FISHER-ADAMS and GRUNSTEIN 1995 ; KLADDE et al. 1996 ). As in yeast, the genomic DNA of Drosophila is not methylated to a detectable level (BIRD 1992 ). WINES et al. 1996 reported introduction of the dam gene into Drosophila. They were unable to detect a significant difference in methylation levels between euchromatic and heterochromatic sites because they compared the accessibility of multiple sites at different loci simultaneously rather than the same site in the two different contexts.

Using a novel strategy, we report here that the E. coli Dam methylase is able to detect chromatin variations correlated with gene silencing in vivo. Probing the same sequences in different contexts, we show that the accessibility to Dam methyltransferase activity is significantly reduced at the white locus in cases of PEV for pericentric P elements and for white chromosomal rearrangements. Furthermore, using the same experimental design, we report evidence that the DREV phenomenon, i.e., Pc-G-mediated repression, is also correlated with a detectable, altered chromatin structure. Regarding the results, these two gene-silencing mechanisms, although based on different protein factors, may involve similar higher-order chromatin structure.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Drosophila stocks:
All Drosophila stocks were raised on standard culture medium. Crosses were performed at 25°. Flies of similar ages were used for all comparisons. All variants used are described in LINDSLEY and ZIMM 1992 , FAUVARQUE and DURA 1993 , and WALLRATH and ELGIN 1995 . The In(1)w^m4hrx342 is an X-ray-induced revertant of the In(1)w^m4h mutant strain (G. REUTER, personal communication). The ph^w viable mutant allele was obtained for this study after imprecise excision of the P element (ROBERTSON et al. 1988 ) contained in the ph^lac+3 w¹¹¹⁸ stock (FAUVARQUE et al. 1995 ). White-eyed male derivatives were mated with ph^lac+3 w¹¹¹⁸ females, and the female progeny was checked for the darker eye phenotype. This new ph mutant allele was confirmed by demonstrating lethality over a null allele of ph. The deficiency Df(1)w^vco, derived from the Tp(1;3)w^vco strain, includes the ph and white loci, and it was chosen as a ph mutant background to test the same BclI sites of the white gene as in the PEV studies. As this deficiency is male lethal, females bearing either the wild-type chromosome or one mutant for ph (or Psc), as well as the P[ph] insert (either T30 or T3) and the Dam methylase, were analyzed.

The P[y⁺; dam⁺] construct:
The E. coli dam methyltransferase gene was obtained from pDOX1 (HULSMANN et al. 1991 ); the dam gene was subcloned into pBluescript as a XhoI-SalI 1027-bp fragment, and then the blunted NotI/SalI fragment was subcloned into the transformation vector phy701 (FAUVARQUE et al. 1995 ), cleaved by NotI/XbaI, and then blunted between the hsp70 promoter and the polyadenylation site. phy701 is derived from pCaSpeR-hs (THUMMEL and PIRROTTA 1992 ), whose white gene has been replaced by the yellow gene from Dint plasmid (GEYER and CORCES 1987 ). Detailed maps of the plasmids are available on request. The use of the yellow gene rather than the miniwhite gene as the transformation marker avoids interference when probing either sequences of the endogenous white for classical PEV studies or sequences of the miniwhite transgene contained in P-element constructs. This plasmid, named phy705, is designated P[y⁺; dam⁺].

Generation of transgenic flies:
phy705 (0.3 mg/ml) was coinjected with helper plasmid pUChsP 2-3 (0.15 mg/ml) into y w^67c23 embryos following standard procedures (RUBIN and SPRADLING 1982 ). One transformant was recovered with an intact single copy of the P element, as checked by genomic Southern blot analysis. Several additional lines were recovered after standard mobilization procedure of the initial P[y⁺; dam⁺] element (ROBERTSON et al. 1988 ).

Chromatin structure analysis:
Flies of similar ages grown at 25° without heat shock were used because the basal transcription level of the hsp70 promoter provides sufficient methylase activity. Before DNA preparation, flies were frozen at -80°. Samples of genomic DNA were extracted from 25 males or from 20 females: typically, flies were homogenized in 0.1 M Tris-HCl, 0.1 M EDTA, and 1% SDS, and incubated at 65° for 30 min. After addition of potassium acetate (1 M final), samples were cooled on ice for 30 min. Supernatants were recovered after two rounds of centrifugation (10 min, 13000g, 4°). DNA was precipitated with isopropanol (0.6 volume), washed in 70% ethanol, dried in a Speedvac, and resuspended in 50 µl 10 mM Tris, pH 8, and 1 mM EDTA. The DNA preparation (25 µl) was cleaved to completion with the first enzyme to flank the tested sites. The enzymes used were SalI for the 5' region and Csp6I for the 3' region of the pericentric P inserts, BamHI for the lacZ transgene and for the classical PEV studies, BglII for the PEV-unrelated sequence, and HindIII for the euchromatic P[ph] transgenes. The DNA was then cleaved with BclI at 50° for 5–6 hr, except for the 3' region of miniwhite or white studies, in which MboI was used at 37°. The DNA was size fractionated on a 1% agarose gel and transferred to a reinforced cellulose nitrate membrane. Blots were then hybridized with the appropriate ³²P probe, labeled by random priming (Random Prime-IT kit; Stratagene, La Jolla, CA) in 50% deionized formamide, 5x SSPE, 5x Denhardt's, and 0.1% SDS at 42° for 12 hr, then washed three times for 10 min with 3 mM Tris, pH 9, at room temperature. The probes used were the 650-bp BclI fragment from the 5' region of the white locus (see Figure 1A, Figure 3A, and Figure 5A), a 587-bp PCR-amplified fragment from the 3' region of the white locus (Figure 1B), the 977-bp BglII-BclI fragment from the lio locus (Figure 3D), and a 650-bp, PCR-amplified fragment from lacZ sequence (Figure 2B). Primers used for PCR-amplified probes were, for the 3' region of white, 5'-GCCGCTGGACTACGTGGG-3' near an MboI site and 5'-CGAAATGAACCACTCGGAACC-3' near a Csp6I site, and for the lacZ study, 5'-CTGGGTCGGTTACGGCCAGG-3' near a SalI site and 5'-GCACCATCGTCTGCTCATCC-3' near an EcoRV site. Autoradiograms were visualized on 3M Hyperfilm. Southern blots were scanned using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). The relative intensity of each band was calculated by measuring the intensity of the band compared with the total intensity of the bands in a given lane.

View larger version (33K): In this window In a new window Download PPT slide   Figure 1. Methylation levels at the miniwhite locus relative to the PEV phenomenon. Heterochromatic P transgenes were compared to euchromatic ones. (A) A schematic map of the P element tested (WALLRATH and ELGIN 1995 ) is given as the corresponding DNA fragments obtained after a SalI-BclI double digest. A typical Southern blot analysis shows the variation of the proportion of the methylated fragment between a uniformly expressed euchromatic line (39C-X) and variegated ones (39C-4 and 118E-12). (B) Relative intensity of a methylated fragment quantified with a PhosphorImager. Numbers over square brackets indicate the fold-factor difference between two lines (in bold type) and the probability that the difference observed, as determined by the Student's statistical test, results by chance (in parentheses). Values are 40.7% ± 2.7 (n = 6) for 39C-X hemizygous males, 21.1% ± 2.0 (n = 6) for 39C-4 heterozygous males, and 16.5% ± 1.5 (n = 6) for 118E-12 heterozygous males. (C) The sites in the 3' region assayed for accessibility are given on the schematic map of the P transgene. Cs is an abbreviation for Csp6I, and GATC is the tested site, which is cleaved by MboI. Values are 32.6% ± 0.5 (n = 6) for 118E-X hemizygous males, 26.5% ± 0.4 (n = 6) for 39C-4 heterozygous males, and 22.7% ± 1.7 (n = 7) for 118E-12 heterozygous males.

View larger version (75K): In this window In a new window Download PPT slide   Figure 2. Relation between Dam activity and transcriptional activity. (A) Northern blot analysis of lacZ transcription. (Lane a) mRNA extracted from heterozygous flies containing both P[UASlacZ] (BRAND and PERRIMON 1993 ) and P[daGAL4] (WODARZ et al. 1995 ). (Lane b) mRNA extracted from heterozygous flies containing only the P[UASlacZ]. (Upper panel) lacZ probe. (Lower panel) rp49 probe. Numbers indicate ratios of lacZ transcripts after normalization with the rp49 signal. (B) A schematic map of the P[UASlacZ] element and the probe used for this experiment. Diagram shows the relative intensity of the methylated fragment. Values are 32.9% ± 1.1 (n = 9) for P[UASlacZ];P[daGAL4] and 30.1% ± 1.2 (n = 9) for P[UASlacZ] heterozygous flies, and they are not significantly different (P > 0.2).

View larger version (32K): In this window In a new window Download PPT slide   Figure 3. Methylation levels at the white locus relative to the PEV phenomenon. (A) A schematic map of the white locus is given as the corresponding DNA fragments obtained after double digest BamHI-BclI. BH is for BamHI, Bp is for the proximal BclI site, and Bd is for the distal BclI site. A typical Southern blot analysis shows the variation of the proportion of methylated fragments between wild-type (Canton-S/Y), variegated [In(1)wm4/Y and In(1)wm4h/Y], and revertant males [In(1)wm4hrx342/Y]. (B) Relative intensity of methylated fragment for the proximal BclI site. Values are 32.8% ± 1.4 (n = 7) for Canton-S/Y males, 18.3% ± 2.5 (n = 5) for In(1)wm4/Y males, 21.2% ± 1.8 (n = 5) for In(1)wm4h/Y males, and 33.4% ± 1.1 (n = 4) for In(1)wm4hrx342/Y males. (C) Relative intensity of methylated fragment for the distal BclI site. Values are 39.0 ± 1.7 (n = 7) for Canton-S/Y males, 27.8% ± 1.4 (n = 5) for In(1)wm4/Y males, 32.5% ± 2.0 (n = 5) for In(1)wm4h/Y males, and 43.1 ± 2.0 (n = 4) for In(1)wm4hrx342/Y males. (D) A schematic map of the PEV-unrelated linotte locus and the probe used. Relative intensity of methylated fragment for the BclI site at this locus. Values are 20.0% ± 0.6 (n = 4) for Canton-S/Y males, 20.4% ± 0.3 (n = 4) for In(1)wm4/Y males, 20.6 ± 0.7 (n = 4) for In(1)wm4h/Y males, and 22.2% ± 1.0 (n = 4) for In(1)wm4hrx342/Y males, and they are not significantly different, as P is never <0.3. (E) Relative intensity of the methylated fragment for the distal BclI site of the white locus in XO males. Values are 43.3% ± 1.6 (n = 5) for Canton-S/Y males, 26.1% ± 0.6 (n = 5) for In(1)wm4/Y males, 25.9% ± 1.0 (n = 7) for In(1)wm4h/Y males, and 38.5% ± 1.4 (n = 11) for In(1)wm4hrx342/Y males.

View larger version (84K): In this window In a new window Download PPT slide   Figure 4. Eye phenotype of some of the strains used in this study. (A) In(1)wm4h/Y male. (B) 39C-4/+ male. (C) In(1)wm4h/0 male. (D) In(1)wm4hrx342/Y revertant male. (E) +/+;T30/T30 female. (F) Psc1/+;T30/T30 female.

View larger version (26K): In this window In a new window Download PPT slide   Figure 5. Methylation levels at the miniwhite locus relative to the DREV phenomenon. (A) A schematic map of the P[ph] (FAUVARQUE and DURA 1993 ) and the DNA fragments obtained after double-digest HindIII-BclI is given. A typical Southern blot analysis shows the variation of the proportion of methylated fragment in different genetic backgrounds. + corresponds to y w67c23/y w67c23, Psc1/+ corresponds to y w67c23/y w67c23;Psc1/+, Def ph/+ to Df(1)wvco/y w67c23, and phw corresponds to y phw/y phw. (B) Relative intensity of the methylated fragment for the proximal BclI site of the T30 insert. Values are 15.5% ± 0.9 (n = 5) for +, 25.1% ± 0.9 (n = 6) for Def ph/+, 25.5% ± 0.7 (n = 6) for Psc1/+, and 32.0% ± 0.7 (n = 4) in case of phw. (C) Relative intensity of methylated fragment for the distal BclI site of the T30 insert. Values are 20.6% ± 1.0 (n = 5) for +, 30.9% ± 1.3 (n = 6) for Def ph/+, 30.8% ± 0.4 (n = 6) for Psc1/+, and 38.2% ± 1.5 (n = 4) for phw. (D) Relative intensity of methylated fragment for the proximal BclI site of the T3 insert. Values are 22.1% ± 0.2 (n = 8) for the + and 33.9% ± 2.5 (n = 8) for phw. (E) Relative intensity of methylated fragment for the distal BclI site of the T3 insert. Values are 30.6% ± 0.4 (n = 8) for + and 39.6% ± 2.7 (n = 8) for phw.

Northern analysis:
Total RNA was isolated (SAMBROOK et al. 1989 ) from flies containing the P[UAS-lacZ] insertion (BRAND and PERRIMON 1993 ) with or without the P[daughterless-GAL4] (WODARZ et al. 1995 ). Total mRNA was purified using the PolyATtract IV System (Promega, Madison, WI) and fractionated on a 2% agarose gel with formaldehyde. After transfer to a nylon membrane and baking for 1 hr at 80° under vacuum, the membrane was probed with a PCR-amplified lacZ fragment and with a plasmid containing the rp49 gene (WONG et al. 1981 ). The intensity of the bands was quantified using a PhosphorImager (Molecular Dynamics). Differences in the amount of mRNA loaded in each lane were corrected using the signal intensity of rp49.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Expressing Dam methyltransferase in flies:
Expression of the E. coli Dam methylase in Drosophila did not cause detectable defects, neither developmental nor fertility related, in accordance with previous work (WINES et al. 1996 ). Eight P[y⁺; dam⁺] lines were tested for their ability to methylate DNA (Table 1). We had previously checked that DNA extracted from wild-type flies lacking methylase activity was completely cleaved by BclI (data not shown). All experiments were carried out at 25° without heat shock; the basal transcription level of the hsp70 promoter provided sufficient methylase activity. Wild-type Canton-S females were mated to males carrying different P[y⁺; dam⁺] insertions. In the case of autosomal P[y⁺; dam⁺] insertions, the male progeny were analyzed. For X insertions, the heterozygous females were tested. To assess the activity of the methylase, we have used the BclI restriction enzyme, which is sensitive to the state of methylation of its TGATCA target site: if this sequence is methylated on the first adenine residue by the Dam activity, BclI does not cut it. Genomic DNA was first cleaved with BamHI, which is insensitive to methylase, to flank the two BclI sites of the 5' region of the white sequence (see Figure 3A). DNA was then treated with BclI. The fraction of DNA uncut by BclI, probed by the 650-bp BclI fragment, and measured by a PhosphorImager directly reflects the level of the in vivo-methylated DNA. In this case, a combination of four bands was obtained, and the relative intensity of each band was calculated by measuring the intensity of one band compared with the total intensity of the bands in a given lane. Accessibility of the more proximal BclI site was given by the sum of the relative intensity of the 3-kb band, which results from methylation of both sites on the same DNA fragment, and the 1.2-kb band, where only the site proximal to the probe was methylated (see Figure 3A). Results are shown in Table 1. Dam activity ranged from 21.8 to 51.2% on this site. This seems to reflect a position-dependent activity regarding the insertion site of the P[y⁺; dam⁺]. Without heat shock, transcription of the dam gene could reflect neighboring enhancer activity, with the level of activity depending on the strength of the enhancer. However, it seems that 50% of methylated DNA is the upper limit that can be obtained in our experiments, as it was for WINES et al. 1996 . Nucleosomal conformation of DNA, nucleosome phasing, repair system modification of "damaged" methylated DNA, or some demethylase system may explain this upper limit.

The y w^67c23;P[y⁺; dam⁺]³⁴,CyO/+ strain (named dam34) was chosen for further tests as the P[y⁺; dam⁺] was easily monitored by the CyO marker.

Measure of accessibility of miniwhite sites in heterochromatic P elements:
Direct evidence of altered chromatin structure in the PEV phenomenon was first described by WALLRATH and ELGIN 1995 . Using standard chromatin analysis on isolated nuclei, they showed that one particular hsp26 restriction site presents a reduced accessibility to restriction enzyme in P elements inserted into pericentric heterochromatin (lines 39C-4 and 118E-12 have an insert at the base of chromosome arms 2L and 3R, respectively) compared to the accessibility of the same site localized in a euchromatic position (lines 39C-X and 118E-X have an insert at cytological regions 2D and 19A-B, respectively). The eye phenotypes of flies containing the euchromatic P element are wild type, while the eye phenotypes of flies containing heterochromatic P element are strongly variegated. An example is shown in Figure 4B (39C-4). Using Dam activity in vivo, two new sites of these transposons, one in the 5' region and the other in the 3' region of the miniwhite sequence, were tested (Figure 1A). To ensure that endogenous white sequences could not interfere with the studies, experiments for the 5' site were performed in a w^67c23 background, which is deficient for the 5' region of the white locus, including the sequence corresponding to the 650-bp BclI fragment used as probe. Similarly, experiments for the 3' site were performed in a w^IR8 background, which is deficient for the 3' region of white, including the 587-bp MboI-Csp6I fragment used as probe. In these cases, only one BclI site or MboI site was tested between flanking sites—SalI and Csp6I, respectively—and a simple combination of two bands was observed. Accessibility was measured as the intensity of the upper band compared to the total intensity of the two bands in a given lane. This relative quantification is independent of the quantity of DNA loaded in each lane. The results for the 5' and 3' regions (Figure 1B and Figure C), show a significant decrease in the level of methylation for the heterochromatic inserts compared to the euchromatic ones. For the 5' site, 2.5- and 2.0-fold factors were observed between the accessibility of the euchromatic line 39C-X and the variegating lines 118E-12 and 39C-4, respectively. A lower amount of material in lanes 39C-4 and 118E-12 may explain why the 1.1-kb product does not increase while a reduction of the corresponding 3.9-kb fragment is observed. In the 3' region, the differences were less pronounced, as 1.4- and 1.2-fold factors were observed between the euchromatic line 118E-X and the variegating lines 118E-12 and 39C-4, respectively. This is consistent with the lesser accessibility expected for those heterochromatic inserts. Dam activity in vivo correlates with previous results based on standard chromatin analysis using isolated nuclei. The fact that both sites in the 5' and 3' regions of miniwhite exhibit a reduction of the level of methylation when localized into heterochromatin, as with the previous study by WALLRATH and ELGIN 1995 on the XbaI site in the hsp26 promoter region, suggests strongly that the general accessibility of the pericentric P elements is reduced.

Effect of transcriptional activity on methylation:
To examine whether the level of methylation in vivo might reflect only transcriptional activity rather than a higher-order chromatin structure that is independent of the transcriptional apparatus, we have checked the accessibility of one site in a lacZ sequence driven by the UAS activation sequence. Without the GAL4 activator, no lacZ transcript was detectable on a Northern blot (Figure 2A). However, with the GAL4 activator under the control of the ubiquitous daughterless promoter (WODARZ et al. 1995 ), transcription of lacZ was enhanced by at least 20-fold (see MATERIALS AND METHODS). A 650-bp lacZ fragment was used to probe the accessibility to methylase activity of genomic DNA of flies containing P[UAS lacZ] (BRAND and PERRIMON 1993 ) and P[y⁺; dam⁺] in the presence or absence of the GAL4 activator. No significant difference in the level of methylation on the lacZ sequence was detected whether or not transcription of lacZ was activated (Figure 2B). This result suggests that the transcriptional activity does not modify accessibility measured by Dam activity in vivo. We, therefore, conclude that differences of accessibility observed for transposons in heterochromatin vs. euchromatic environments reflect some higher-order modification of the chromatin structure.

Rearrangement of the white locus near heterochromatin is associated with a reduced DNA accessibility:
The wild-type strain Canton-S, variegating lines In(1)w^m4 and In(1)w^m4h, and the revertant line In(1)w^m4hrx342 derived from In(1)w^m4h were assayed on the two BclI sites of the 5' region of white (Figure 3A and Figure 4A and Figure D, for eye phenotype). As two BclI sites were simultaneously tested, a combination of four fragments was observed. Similarly to the proximal site described previously, accessibility of the BclI-distal site was given by the sum of the relative intensities of the 3- and 2.5-kb bands where only the distal site was methylated. Variegating lines showed a lower methylation level for both sites than did the wild-type and revertant strains (Figure 3B and Figure C). For the more proximal site, Canton-S differs from In(1)w^m4 and In(1)w^m4h by 1.8- and 1.5-fold, respectively. For the distal site, subtle but significant differences remain: 1.4- and 1.2-fold factors were observed between the same strains. The In(1)w^m4hrx342 strain presents essentially the same accessibility level as the wild type, showing that the relocalization of the white locus outside the heterochromatin region is correlated with the return of a "wild-type" chromatin conformation.

Similar experiments were also carried out with the dam27 line (see Table 1). Although the activity of the Dam enzyme in this line was higher, the difference remains similar: for the more proximal site, Canton-S differs from In(1)w^m4 and In(1)w^m4h by 1.8- and 1.6-fold, respectively. For the distal site, 1.4- and 1.2-fold factors were observed between the same strains (data not shown). This result shows that the whole DNA of the fly is not saturated by methylation. Nonmethylated DNA resulting from a lack of Dam activity is considered an inaccessible conformation by our test. Therefore, higher Dam activity enhances both wild-type and mutant accessibility. As a consequence, the same ratio of methylated DNA in experimental vs. control samples was observed regardless of the activity of the expressing Dam strain used.

As for transposon studies, localization near or in heterochromatic regions seems to be correlated with a higher-order chromatin compaction. One can note that in the case of inversion near heterochromatin of the entire white locus and its surrounding euchromatin, the differences observed are less pronounced than in the case of smaller transposons, i.e., 10 kb of euchromatic DNA inside heterochromatin.

Figure 3E shows the effect of the lack of the Y chromosome, a strong enhancer of PEV (compare Figure 4A and Figure C). In XO Canton-S males, the proximal site exhibits 1.9- and 2.0-fold differences with In(1)w^m4/0 and In(1)w^m4h/0, respectively (data not shown). For the distal site, differences were slightly enhanced as a 1.7-fold factor was observed between Canton-S and each variegating line.

Line specificity cannot account for the accessibility difference:
Contrary to transposon line studies where all inserts are in the same genetic background, PEV lines could potentially have particularities because of their own specific genetic background. To test this possibility, a PEV-unrelated euchromatic site in 37D at the linotte locus (DURA et al. 1995 ) was assayed for methylation level for each PEV line (Figure 3D). If modifiers of Dam activity are present in the genetic background of tested lines, corresponding variations would be expected at this locus. In fact, no significant difference was detected, indicating that strain specificity cannot account for the accessibility differences observed above.

Dam activity reveals chromatin alteration in Pc-G-mediated silencing:
Using the same experimental design, namely assaying the accessibility of the BclI sites contained in the white sequence, we tested the role of the Polycomb group gene products on the euchromatic PRE-induced variegation (DREV). Chromatin accessibility assays were performed on the miniwhite gene of two euchromatic P[ph] inserts, T30 and T3 (FAUVARQUE and DURA 1993 ), respectively located in 86CD, which is a binding site of the PC/PH complex on the polytene chromosome, and 65F, which is not a site of binding of the complex (ZINK and PARO 1989 ). In these constructs, a ph regulatory region that induces variegation lies upstream of miniwhite driven by its own promoter. This variegation is not sensitive to classical PEV modifiers, but it is strongly suppressed in ph (see Figure 5 in FAUVARQUE and DURA 1993 ) and Psc¹ (Figure 4E and Figure F) mutant backgrounds. The results (Figure 5A and Figure 5B) show a significant rise in accessibility in derepressed mutants (ph and Psc) compared to the wild-type control for both tested inserts. For the more proximal 5' miniwhite BclI site of the T30 insert, a 2.1-fold difference was obtained between a wild-type background and the homozygous ph^w context. In a Psc¹ or heterozygous ph deficiency background, a 1.6-fold factor was observed. A similar derepression is observed for the distal BclI site, as a 1.9-fold factor was obtained between +/+ and ph^w/ph^w flies, and a 1.5-fold factor was obtained between wild type and Psc¹ or ph deficiency. One can note that variations are stronger in the case of the T30 insertion than for the T3 P element, where the difference never exceeded 1.5-fold. The PEV-unrelated lio sequence was also assayed for accessibility to know if a ph mutant background can influence Dam activity. No differences were observed: compare 15.4% ± 0.5 (n = 4) in a wild-type background to 15.1% ± 0.5 (n = 4) in the ph deficiency context. This indicates that methylase activity does not change in a ph mutant background.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Probing Drosophila chromatin structure in vivo with the E. coli Dam DNA methylase activity:
Using the E. coli Dam DNA methylase activity, we were able to show that a significantly reduced accessibility is detected at one specific BclI site at the miniwhite sequence of the pericentric hsp26 transgenes compared to euchromatic control transgenes. This result shows conclusively that the adult Drosophila chromatin structure can be assayed directly in vivo. Furthermore, Dam activity revealed that both the 5' and 3' regions of the gene were less accessible in the case of pericentric location, compared to euchromatic controls. This result indicates that the use of Dam is able to corroborate in vivo the previous results obtained by standard chromatin analysis (WALLRATH and ELGIN 1995 ), and that the whole P-element DNA seems to be in a "closed" chromatin conformation, entirely wrapped with heterochromatin proteins and consistent with the model of chromatin compaction (ZUCKERKANDL 1974 ; LOCKE et al. 1988 ).

Heterochromatin has been defined as those regions of the chromosomes that remain condensed throughout the cell cycle and that are associated with the absence of gene expression. The long-held hypothesis that this gene silencing is associated with an altered chromatin structure received direct evidence only recently (WALLRATH and ELGIN 1995 ). These authors reported a standard chromatin analysis using isolated nuclei in which heterochromatic insertions of P elements (hsp26 transgenes) were correlated with a lower accessibility to a specific restriction site than the control euchromatic P element. The overall nucleosomal organization showed a more packaged array than the controls did. There have been a few earlier studies of the chromatin structure in classical rearrangements giving rise to PEV (w^m4 inversion). A DNAse I digestion study using nuclei isolated from third instar larvae was unable to detect any difference in the packaging of DNA at the white locus, even though the relocation of the gene near the heterochromatin was correlated with dramatic differences in chromosomal morphology (HAYASHI et al. 1990 ). Monitoring the resistance of the heterochromatin-euchromatin junction fragment to degradation by endogenous nucleases showed that the w^m4 inversion did not induce significant effects on chromatin structure (LOCKE 1993 ). Using the ligation-mediated PCR procedure on nuclear extracts, no significant differences on chromatin accessibility were detected on the white locus, in a w^m4 chromosome, in the presence of either a strong suppressor or a strong enhancer of PEV (SCHLOSSHERR et al. 1994 ).

Following the same experimental scheme as for the transgenes study, we used the E. coli Dam DNA methylase for studying classical PEV. Significant variations can be detected on the two tested sites of the 5' region of the white locus. Moreover, the difference between control and variegated lines is greater for the proximal BclI site than for the distal one. This may reflect each site having its own accessibility level to the Dam methylase, perhaps because of a particular nucleosomal conformation. Those differences of accessibility were the first described at the white locus itself, as previous reports related no detectable difference between heterochromatic sites vs. euchromatic ones. The variations we detected at the white locus were subtle, and it is possible that the classical techniques requiring chromatin purification before an accessibility test sustain damage to an unstable chromatin architecture. Our method allows the reliable detection of differences of the magnitude twofold or less, simply and reproducibly.

On the basis of our own data, we believe that the contrasting published results of the hsp26 and the w^m4 studies reflect differences in the sensitivity of the techniques rather than in the mechanisms inducing PEV. The effects seen in our experiments are stronger with the hsp26 transgenes than with w^m4. This difference might result from the fact that in the variegated rearrangement, a large block of euchromatin is brought into juxtaposition with a block of heterochromatin, whereas in the case of the hsp26 transgene, a ±10-kb fragment of euchromatic DNA has been inserted within heterochromatin. One can note that in these two cases of PEV, the chromatin compaction, revealed by the in vivo Dam methylation, seems correlated with the strength of the variegated phenotype (compare Figure 4A and Figure B). However, only a slight increase of compaction was observed when w^m4h males lacking the Y chromosome were tested, although a clear increase of white repression was phenotypically observed (compare Figure 4A and Figure C).

Like PEV, the DREV phenomenon is correlated with a lesser DNA accessibility in vivo:
The repression of gene expression by the Polycomb-group gene products seems, in many aspects, similar to PEV. Again, using the same experimental scheme, we tested the role of the Polycomb group gene products on the euchromatic PRE-induced variegation. FAUVARQUE and DURA 1993 reported that euchromatic transgenes containing the miniwhite sequences and a ph PRE (P[ph]) display variegation that is sensitive to some Pc-G mutations. Testing the accessibility of the Dam activity to the same sites of miniwhite as those tested in the PEV studies of two of these transgenes, we have shown that in a ph or in a Psc mutant background, an enhancement of accessibility of these sites is correlated with the derepression of miniwhite expression observed in the eye. The strongest differences observed in the case of DREV, in a homozygous ph mutant background, are similar to those obtained in hsp26 transposons studies. This led us to think that the chromatin fiber that occurred in DREV might be as "closed" by compaction as for strong PEV. Although there may exist some proteins playing a role in both of these gene silencing mechanisms, such as E(z) (LAIBLE et al. 1997 ), E(Pc) (SINCLAIR et al. 1998B ), and ASX (SINCLAIR et al. 1998B ), little overlap has been noted between the proteins involved (FAUVARQUE and DURA 1993 ; FAUVARQUE et al. 1995 ; GINDHART and KAUFMAN 1995 ; KENNISON 1995 ).

One model has been proposed to explain gene silencing where the chromatin fiber is left unaltered, but the region to be silenced is assigned to a compartment within the nucleus to which not all transcription factors have access (PARO 1993 ; SCHLOSSHERR et al. 1994 ). If the Dam methylase is also excluded from this compartment, then it is clear that the use of this tool cannot discriminate between the "compartmentalization" model and the packaging model.

Moreover, in a review, SIMON 1995 schematizes the two extreme packaging models that can be elaborated, based on the available data, for stable repression by Pc-G proteins. In the first, the enhancer interference model, the Pc-G proteins coat only the PREs preventing, in one way or another, transcription [see also PIRROTTA and RASTELLI 1994 for another form of a similar model]. In the second, also called the chromatin accessibility model (adapted from PARO 1990 ), the Pc-G proteins coat the entire region of DNA to be inactivated. Our results can exclude the first model because differences of chromatin accessibility are detected outside the PREs. On the other hand, they fit well with the finding that, after formaldehyde cross-linking in fly tissue culture cells, the PC protein is associated continuously across tens of kilobases in regions that are transcriptionally silenced in the bithorax complex (ORLANDO and PARO 1993 ). Moreover, a similar, improved method has shown that the PC protein is not distributed homogeneously along the bithorax complex, but that it spreads locally over a few kilobases of DNA surrounding PREs (STRUTT et al. 1997 ). This is in good correlation with our results because the two tested sites in the miniwhite gene are <1 kb away from the PRE.

It would be interesting to assay the question of compaction during development. Preliminary tests on embryos and third instar larvae were negative, as the Dam was expressed at too low a rate to detect variation in methylation ratio.

MCCALL and BENDER 1996 examined the accessibility of DNA-binding proteins for DNA of the BX-C that was under Pc-G repression. They found that GAL4-dependent transcription is inhibited by Pc, whereas T7 polymerase-dependent transcription is not. This apparent contradiction is likely a result of the small size of the T7 polymerase because an enlarged T7 polymerase (T7-ß-galactosidase fusion) is blocked by Pc in early embryos (D. FITZGERALD and W. BENDER, personal communication). Thus, one means for reducing the background level of DNA methylation may be to engineer a bigger probe, i.e., a fusion of ß-galactosidase protein with the methylase. This might be a way to limit the access of the Dam to the chromatin and, therefore, enhance the site difference accessibility between an active and inactive gene in PEV and DREV phenomena.

	FOOTNOTES

¹ Present address: Institut de Génétique Humaine, CNRS/UPR 1142, 34396 Montpellier Cedex 5, France.

	ACKNOWLEDGMENTS

We thank Renato Paro and Ulrich Hahn for providing the pDOX plasmid, Gunter Reuter for providing In(1)w^m4h and revertant stocks, and Kathy Matthews at the Indiana Drosophila Stock Center. We also thank Jean Marie Rouillard and Francois Lacroute for the use of the PhosphorImager and Sébastien Bloyer for sharing unpublished results. We are particularly grateful to Roger Karess, Pradip Sinha, and David Cribbs for useful comments on the manuscript. This work was supported by grants from the Indo-French Centre for the Promotion of Advanced Research (Project 1603-1), from the Centre National de la Recherche Scientifique (ATIPE No. 7), from the Association pour la Recherche contre le Cancer (No. 6786), from the Actions Concertées Coordonées-Sciences du Vivant I (ACC-SV1) "Analyse fonctionnelle des génomes," and from the University Paris XI-Orsay (URA 2227). A.B. is supported by a fellowship from the Ministère de l'Education Nationale and by a grant from the Ligue Nationale Contre le Cancer.

Manuscript received May 18, 1998; Accepted for publication September 9, 1998.

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