INTRODUCTION

Top Abstract Introduction Materials and Methods Results and Discussion References

The homeotic genes of Drosophila melanogaster are found in two gene clusters, the Antennapedia complex and the bithorax complex (BX-C). The complexes are large and contain many enhancers, which act over long distances to drive segment-specific expression of the homeotic genes (reviewed in reference 12). The BX-C is divided into at least nine regulatory domains, each of which is responsible for activating homeotic gene transcription in a given parasegment (parasegments 5 to 13 [PS5 to -13]). The domains are aligned on the chromosome in the linear order of the parasegments they affect.

In early embryogenesis, the expression domains of the homeotic genes are established by a group of short-lived regulators, products of the gap and pair-rule genes. These factors disappear at approximately 5 h into embryogenesis, and control is transferred to another group of factors, the Polycomb group (PcG). The PcG acts to maintain the proper segmental patterns of the target genes, "remembering" the pattern over many cell division cycles. In PcG mutants, the homeotic genes are misexpressed and are transcribed in all segments of the embryo (reviewed in references 38 and 45).

Recent biochemical findings suggest that PcG factors are found in large multiprotein complexes (34, 44). It is not clear how these complexes are targeted to DNA sites or how they maintain repression. However, several pieces of evidence suggest that transcriptional repression by the PcG might mimic the formation of heterochromatin. The Polycomb protein, the first PcG factor identified, shares a protein motif, the chromodomain, with the heterochromatin-associated factor HP-1 (37). Like heterochromatic regions, the BX-C appears underreplicated in the polytene chromosomes of the Drosophila salivary gland (24), a tissue in which the BX-C is transcriptionally inactive, and is thought to be repressed by the PcG. Numerous transgene insertions have been recovered in the BX-C, almost all of which appear to respond to PcG regulation (3, 30). Moreover, transgenes outside of the BX-C bearing Polycomb response elements (PREs) show variegated repression of neighboring reporter genes (9).

While it is clear from these results that the PcG is able to repress many enhancer-promoter combinations and to act over long distances, there is little direct evidence for chromatin modification by the PcG. Indeed, it has been suggested that the PcG might exert its repressive effect specifically on promoter regions or by inhibiting promoter-enhancer interactions (5, 39). It has also been postulated, based on in vitro data, that the PcG might affect chromatin structure indirectly by blocking the activity of other chromatin remodeling complexes (44), such as the brahma complex (36). The brahma gene has been identified as a member of the trithorax group of factors, which act as genetic antagonists to the PcG (reviewed in reference 22).

PcG-mediated repression shares similarities not only to heterochromatic position effects, but also to silencing mediated by the SIR complex of proteins of Saccharomyces cerevisiae. The SIR proteins appear to mediate long-range transcriptional repression over the target loci, and transgene insertions into these loci are invariably silenced (reviewed in reference 14). The SIR proteins themselves are thought to coat the silenced regions, and several pieces of experimental evidence suggest that SIR silencing is associated with an altered chromatin structure. This evidence includes reduced accessibility of the transcriptionally repressed DNA (17, 25, 47). There have been several previous attempts to identify similar PcG-dependent changes in DNA accessibility. Boivin and Dura (6) tested changes in DNA accessibility by using Escherichia coli dam DNA methyltransferase as a probe. Using transgenes containing a presumptive PRE from the polyhomeotic locus as their target DNA, they demonstrated ~2-fold changes in the level of methylation in PcG mutant flies versus wild-type flies. However, Schloherr et al. (42) examined endogenous sequences of the BX-C for restriction enzyme accessibility and failed to find any difference. Similarly, in a previous study from this laboratory, bacteriophage T7 RNA polymerase (T7RNAP) was used to probe DNA accessibility in the BX-C, and no sensitivity to the PcG was seen (29). These studies suggested that if an accessibility block is imposed by the PcG, it must be incomplete or selective.

In this study, we expand our analysis of DNA accessibility in the BX-C by using Gal4, T7RNAP, and FLP recombinase as probes. Each assay relies on in situ hybridization to fixed embryos, so that the products can be visualized cell by cell. The comparison of PcG-repressed and nonrepressed segments provides an internal control within each animal. By introducing Gal4, we tested for the ability of a foreign activator to elicit transcription from the fly's own polymerase II (Pol II) machinery under PcG-repressed conditions. Similarly, we compared the ability of a foreign polymerase, T7RNAP, to transcribe in PcG-repressed versus nonrepressed cells. T7RNAP has been used as a tool for recognizing altered chromatin states in yeast (10), trypanosome (33), and mammalian (19) systems. Although we had previously found no effect of PcG on T7RNAP, it seemed possible that the PcG might be more effective in blocking large protein complexes, such as the RNA Pol II transcription apparatus. Therefore, we created an enlarged version of T7RNAP, called "Goliath polymerase," and compared it to the wild-type T7RNAP in its sensitivity to PcG modification of the DNA. We also tested the ability of the site-specific recombinase, FLP, to find and synapse its target sites and to recombine a circular episome out of the chromosome. We performed these assays with multiple P element insertions, spread throughout the BX-C, each of which contains target sites for Gal4, T7RNAP, and FLP. We found consistent effects by the PcG on all three proteins.

Our observations with the Gal4, T7RNAP, and FLP probes suggest that the DNA within our P insertions is somehow altered when the control region surrounding it is actively PcG repressed. In addition to reduced DNA accessibility, SIR silencing has been shown to correlate in vivo with an altered topology over the repressed DNA (4, 11). Changes in nucleosome density, nucleosome conformation, or the association of other DNA binding factors can alter the packaging of the DNA, and all have been shown to have measurable effects on DNA topology (27, 31, 41). Similarly, PRC1, a purified complex containing a subset of the PcG factors, has been shown to inhibit SWI/SNF remodeling of chromatin in vitro (44). The assay used involved visualization of a change in the topology of a nucleosomal template, because SWI/SNF removed negative supercoils. Thus, we might expect PcG-repressed DNA of the BX-C to have a greater negative superhelix density than nonrepressed DNA. In this study, we examined the supercoiling of PcG target DNA in vivo by a method very similar to that used with yeast to study SIR silencing. Circular episomes were produced from our FLP-inducible cassettes in order to trap the structure of the PcG-modified DNA. In contrast to SIR silencing, we do not see a difference between the topologies of PcG-repressed DNA and nonrepressed DNA.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results and Discussion References

Plasmid constructions. Each FRT in the construct illustrated in Fig. 1A is a 48-bp sequence derived from pJFS36 (43). The two tandem 49-bp T7 promoters were cloned by PCR from pT7-7 (kindly provided by Stan Tabor). The UAS sequence was derived from pUAST (8), and the LacZ reporter was derived from pCaSpeR-AUG--gal (51). The complete T7 promoter/UAS-LacZ cassette was subcloned into the pCasper-4 transformation vector and transformed into Drosophila to produce the control lines. The miniwhite reporter was removed from this construct and replaced with the homing fragment/rosy transformation marker cassette from the "homing pigeon" (3) to produce the P element of Fig. 1A. This P element was used to produce the five BX-C insertions shown in Fig. 2.

View larger version (26K): [in this window] [in a new window]   FIG. 1.   The P element construct serves as a target for Gal4, T7RNAP, and FLP. (A) Diagram of the P element. The initial P insertions contain two cassettes, each flanked by directly oriented FRT sequences. The left cassette contains the rosy transformation marker, and a "homing fragment," which targets P elements to the BX-C chromosome region. The right cassette contains two tandem T7 promoters and a Gal4 activatable UAS-LacZ reporter. (B) Trimmed-down insertions, which lack the homing/rosy + cassette, were recovered as rosy LacZ+ individuals following FLP induction in the germ line. The T7 promoters are poised to transcribe the genomic DNA flanking the 3' P end. (C) Expression of FLP recombinase in a whole embryo results in excision of the T7 promoter/UAS-LacZ cassette from the chromosome, resulting in formation of a 4.9-kb circular episome in somatic cells. The T7 promoters are then poised to produce antisense copies of LacZ RNA.

View larger version (74K): [in this window] [in a new window]   FIG. 2.   Five P element insertions were recovered within the BX-C. Transcription units are marked for the three homeotic genes, Ubx, abd-A, and Abd-B. Coordinates from the published sequence are shown (in kilobases). Above the map, control regions for each of the genes are denoted by brackets, and the large arrows point to the most anterior parasegments that they regulate in the fly embryo (displayed with the anterior portion to the left). The sites of insertion of the five P elements are indicated below the map as triangles. The location of the original 4× T7 promoters in the bx region (29) is indicated above the map as a black rectangle. Arrows denote the orientation of the T7 promoters.

D. melanogaster stocks and crosses. Germ line transformants were made as previously described (46). Locations of the new P insertions were identified by inverse PCR with primers in the P element ends (35). To produce trimmed-down insertions, flies with the initial P insertions were treated with FLP, and rosy progeny were collected. These were screened by PCR for retention of the T7 promoter and UAS sequences; less than 1% of the rosy candidates had the desired structure. All crosses were carried out at 25°C. Tests of LacZ patterns in PcG mutants utilized embryos homozygous for Pc³, Psc^II, and l(429b) and hemizygous for DfJA52, for testing zygotic loss of the Polycomb, Posterior Sex Combs, pleiohomeotic, and polyhomeotic genes, respectively. For extra sex combs, embryos lacking both maternal and zygotic product were generated by crossing esc¹⁰/esc² males and females. To perform the FLP accessibility assays, females containing a heat shock-inducible source of FLP located on the X chromosome and a heat shock-inducible source of T7RNAP on the 2nd chromosome were crossed to males containing the accessibility constructs. The same source of FLP was used to produce circles for topology experiments in embryos and adults (Table 1). To generate circles from Polycomb mutant embryos, 70FLP4A, Pc³ /TM2 flies were crossed to either Pc³, p[Mcp] or Pc³, p[Abd-B] recombinant flies. Table 1 lists the transformant lines used in this paper.

Whole-mount immunolocalization in Drosophila embryos. Whole-mount immunolocalization of Goliath polymerase expression patterns was performed as previously described (21), with mouse anti--galactosidase monoclonal antibody (Promega) as the primary antibody and horseradish peroxidase-conjugated goat anti-mouse antibody (Bio-Rad) as the secondary antibody. Embryos were dissected as previously described (21) and mounted in Immu-mount (Shandon) for photography.

RNA in situ analysis of Drosophila embryos. RNA in situ analysis was performed as previously described (15) with the following modifications. Hybridizations were carried out overnight at 45 to 50°C, and the alkaline phosphatase detection buffer was prepared with 2-amino-2-methyl-1-propanol (Sigma) instead of Tris-HCl. Digoxigenin-labeled RNA probes were prepared according to the protocol outlined for the Genius system (Boehringer Mannheim). The lacZ probe is complementary to 3 kb of LacZ coding sequence. The probe for transcription from p[bx], as well from the 4× T7 promoters, is from a 1.2-kb fragment, representing BX-C sequence coordinates 272,997 to 274,194 (sequence 89E of reference 28). A 1.4-kb fragment representing BX-C sequence 219,410 to 220,834 was used as a probe for transcription from p[bxd]. A 1.4-kb fragment representing BX-C sequence 125,812 to 127,224 was used as a probe for p[iab-4]. A 1.1-kb fragment representing BX-C sequence 113,733 to 114,900 was used as a probe for p[Mcp]. A 1.1-kb fragment representing BX-C sequence 48,720 to 49,853 was used as a probe for p[Abd-B]. The circle probe is complementary to 3 kb of the antisense strand of the LacZ coding sequence. To produce a probe for the promoters on the X chromosome, inverse PCR was performed as previously described (35) to capture ~370 bp of the genomic sequences immediately adjacent to the X chromosome control insertion.

Analysis of supercoiled circles. To prepare PcG-repressed DNA from adult flies, approximately 500 heads were severed by vortexing and sieved through a 710-µm-pore mesh. In a separate experiment, frozen flies were individually dissected, and their heads, thoraxes, and abdomens were collected separately. Whole adult flies were used to compare males and females containing the X chromosome insertion. To prepare genomic DNA from embryos, dechorionated embryos were ground in embryo grinding buffer (100 mM Tris [pH 8], 50 mM NaCl, 50 mM EDTA, 1% sodium dodecyl sulfate [SDS], 0.15 mM spermine, 0.5 mM spermidine). Adults were ground in fly grinding buffer (100 mM Tris [pH 9.1], 100 mM NaCl, 200 mM sucrose, 0.5% SDS). An equal volume of preheated phenol (saturated in 10 mM Tris [pH 8.0]- 1 mM EDTA [TE]) was immediately added, and samples were vortexed and then incubated at 65°C for >10 min. Chloroform was added 1:1 with the phenol, and samples were extracted. Samples were then phenol-chloroform extracted a second time and chloroform extracted once. Twenty micrograms of DNase-free RNase A was then added, and samples were incubated briefly at room temperature. DNA was precipitated in a mixture of 0.5 M NaCl and 12% polyethylene glycol (PEG) 6000 for 1 h at 4°C and collected by centrifugation. (Less of the DNA was nicked by PEG precipitation than by ethanol precipitation, although the distribution of topoisomers was the same with either method.) Pellets were washed in 70% ethanol, dried, and resuspended in TE. DNA samples were loaded into 1.5% SeaKem LE agarose gels, supplemented with 2 µg of chloroquine diphosphate (Sigma) per ml. Electrophoresis was performed in 40 mM Tris-acetate (pH 7.5)-1 mM EDTA (TAE) buffer, also supplemented with 2 µg of chloroquine per ml, at 1.6 V/cm, for about 36 h. Gels were capillary blotted to Magnacharge nylon membranes (Osmonics) and hybridized at 65°C against a random primed labeled probe representing the lacZ and SV40 sequences of the episomes. Images were analyzed with a Fujix PhosphorImager. Fujifilm Image Gauge V3.0 software was used for densitometric measurement of the areas under supercoiled DNA peaks used for computation of the average linking numbers.

	RESULTS AND DISCUSSION

Top Abstract Introduction Materials and Methods Results and Discussion References

New P element insertions into the BX-C. We constructed a P element with target sites for our three accessibility probes (Fig. 1). First, it carries binding sites for the yeast GAL4 transcriptional activator (GAL4-UAS) next to a minimal TATA-containing promoter driving a LacZ reporter. We can use LacZ to monitor Pol II-mediated transcription, with or without activation. Second, the P element carries two tandem 49-bp T7 promoters, oriented toward the outside of the P element. We can assay initiation and elongation by T7RNAP by looking for RNA copies of the genomic sequences flanking the 3' end of the P element. Finally, the cassette with the T7 promoters and the lacZ gene is flanked by FRT recombination sites for the FLP enzyme. Excision of the cassette as a circular episome can be monitored indirectly by looking for T7RNAP transcription around the circle (Fig. 1C).

Gal4 activation of Pol II transcription is repressed by the PcG. In the absence of Gal4, the UAS-LacZ reporter in each BX-C insertion responds to the neighboring enhancer sequences, giving rise to a LacZ expression pattern that is spatially restricted to particular segments (Fig. 3B to E). The controls show no LacZ expression in the absence of Gal4 (3rd chromosome control, Fig. 3A [X chromosome control not shown]). For each of the insertions in the BX-C, LacZ expression reflects segments in which the segmental control region surrounding the P insertion is active. The segments lacking LacZ expression are those in which the region of the insertion is repressed by the PcG (30). When Gal4 is expressed in control embryos from a ubiquitous source (line 32B), the LacZ gene is strongly activated in all segments (3rd chromosome control, Fig. 3F). In contrast, activation by Gal4 is segmentally restricted in the BX-C insertions (Fig. 3G to J). Although some activation occurs in PcG-repressed segments, a far greater number of cells show a high level of LacZ transcription in nonrepressed segments. Gal4 does not activate equally in all cells, even within the nonrepressed segments. Moreover, the Gal4-activated pattern is not an amplification of the nonactivated pattern. Gal4 appears to be interacting with the neighboring enhancer elements in a complicated fashion. However, in all cases, Gal4 activation is substantially blocked in PcG-repressed segments.

View larger version (104K): [in this window] [in a new window]   FIG. 3.   Gal4 is partially blocked by the PcG. (A to E) Enhancer trap patterns in the P insertion lines. RNA in situ hybridizations showing LacZ expression (in the absence of Gal4) in germ band retracted embryos. (A) A control embryo containing the UAS-LacZ cassette on the 3rd chromosome outside of the BX-C shows no LacZ expression. (B to E) Trimmed-down insertions p[bx], p[iab-4], p[Mcp], and p[Abd-B], respectively. The most anterior parasegment in which transcription occurs is marked with a bracket. (F to J) Gal4 activation of LacZ expression. (F) Gal4 strongly activates LacZ expression throughout the control embryo. (G to J) Gal4 activation in p[bx], p[iab-4], p[Mcp], and p[Abd-B], respectively. LacZ expression is much weaker or absent in PcG-repressed segments. The anterior-most parasegment in which strong activation of LacZ transcription occurs is marked with a bracket. All embryos shown in this and subsequent figures are dissected along the dorsal midline and are displayed as "pelts," with the anterior oriented toward the top of the page.

Pattern changes in PcG mutants. In Polycomb (Pc) mutant embryos, the homeotic genes are turned on inappropriately in anterior segments. Likewise, in Pc embryos hosting our insertions, the LacZ pattern loses its segmental restriction. The Abd-B reporter shows strong expression throughout the thoracic and abdominal segments in a Polycomb mutant (Fig. 4D). Again, we suspect this reflects readthrough from the Abd-B promoter. However, in the bx, iab-4, and Mcp insertion lines, only a few cells in each segment turn on LacZ (Fig. 4A to C). In addition, the pattern is changed in posterior segments, with loss of expression in some cells. We also analyzed embryos with mutations specific for other PcG members, including extra sex combs, Posterior Sex Combs, polyhomeotic, and pleiohomeotic (see Materials and Methods for genotypes). All of the mutant embryos gave patterns similar to those of Pc embryos (data not shown). We were initially surprised that our insertions did not show widespread transcription in PcG mutant backgrounds. Previously discovered BX-C enhancer traps had shown strong and widespread misexpression in PcG mutant embryos (30). Most of these enhancer traps were driven by the Ubx gene promoter (including 1.7 kb of 5' flanking sequences), and all included the rosy gene as a reporter. The strong misexpression observed may have resulted from activation of enhancer sequences within the P elements. We believe the weak promoter in our new insertions is completely dependent upon the neighboring homeotic gene enhancers for activity and thus better reflects the effect of loss of PcG upon these homeotic gene control regions. We tested other P elements in the BX-C, which contain only LacZ driven from the P promoter (derived from "homing pigeons" of reference 3). These behave similarly to our new insertions, showing weak expression in Pc embryos (not shown). These results show that the loss of the PcG does not, by itself, lead to widespread activation of the long-distance enhancers in the BX-C. Moreover, it shows that the PcG affects the activity of these enhancers not only in repressed segments, but also in transcriptionally active segments. However, the changes may be indirect, resulting from pleiotropic effects in the PcG mutants. The loss of the PcG should cause missexpression of genes other than the homeotics, some of which may be transcription factors capable of binding to the homeotic control regions.

View larger version (131K): [in this window] [in a new window]   FIG. 4.   Polycomb mutant embryos show a weak spread of LacZ expression, but allow Gal4 to activate LacZ transcription throughout the embryo. All panels show in situ hybridizations to LacZ RNA in germ band retracted embryos. (A to D) Enhancer trap patterns in Polycomb zygotic null embryos containing the p[bx], p[iab-4], p[Mcp], and p[Abd-B] insertions, respectively. (E to H) Gal4 activation of LacZ transcription in Polycomb zygotic null embryos.

Pc

Transcription by T7RNAP is partially blocked by the PcG. To test whether or not the PcG affects transcription by T7RNAP, we crossed the fly lines containing the new T7 promoter insertions to a source of T7RNAP that is constitutively expressed in cells of the CNS (29). Transcription by T7RNAP of the genomic sequences abutting the T7 promoter insertions was visualized by RNA in situ analysis of embryos (Fig. 5). As reported previously (29), brief treatment of the embryos at 37 to 39°C is required to see T7RNAP transcripts, perhaps due to increased transcript stability.

View larger version (178K): [in this window] [in a new window]   FIG. 5.   Transcription by T7RNAP is partially blocked by the PcG at multiple sites within the BX-C. RNA in situ hybridizations in germ band retracted embryos. All embryos contained a source of T7RNAP, which is expressed in the CNS. (A) The control is an embryo containing a P element with the T7 promoter/UAS-LacZ cassette inserted on the X chromosome. T7RNAP produces a transcript in the cells of the CNS from PS3 to -14, mirroring the expression pattern of the polymerase. (B to F) Transcription by T7RNAP in the BX-C insertions. In panels C and D, the initial full-length P elements were used, and in panels B, E, and F, the trimmed-down forms were used. The anterior-most parasegment in which the control region is active is indicated for each embryo. In the anterior segments repressed by the PcG, T7RNAP produces a transcript in only a subset of the polymerase-producing cells.

Discordance with prior T7 assays. A previous publication from this laboratory reported that T7RNAP was not affected by the PcG, as assayed by transcription from an insertion in the bx region containing four T7 promoters (29). Our repeats of that experiment verify the prior results. There are several differences between the present and previous experiments, which may explain the different outcomes. Each of our five new insertions contains only two T7 promoters, whereas most of the work in the previous study was performed on a single, four-promoter insertion. The probe used for RNA in situ analysis in the previous study included sequences immediately downstream of the promoters. In our study, the probe sequences begin ~300 bp downstream of the transcription start site, since the polymerase must transcribe through FRT and 3' P sequences before reaching the neighboring genomic DNA. The most significant difference may be the context of the T7 promoters. Although our new bx insertion line is located close to the position of the T7 promoter insertion described by McCall and Bender (29), it is possible that the ~250 bp between the two insertions is enough to cause differences in the behaviors of the T7 promoters. It is also quite possible that the UAS-TATA Pol II promoter in our new insertions affects the neighboring T7 promoters. However, as is illustrated below, we do detect an accessibility effect on the simple T7 promoter target (29) by an enlarged version of T7RNAP, and so the PcG block cannot be solely an effect of a nearby Pol II promoter. We have also found that following prolonged expression of T7RNAP, no PcG block to T7RNAP transcription is apparent (see Fig. 6 and 7). Clearly the PcG block to T7 transcription is partial and is sensitive to assay conditions.

An enlarged version of T7RNAP is more sensitive to PcG repression. To test whether size matters to the PcG, we made T7RNAP larger by fusing it to -galactosidase. This fusion protein forms a tight tetramer that is ~860 kDa in size, over eight times the size of wild-type T7RNAP, but still substantially smaller than the Pol II holoenzyme. We call this fusion protein "Goliath polymerase" (Fig. 6A).

View larger version (71K): [in this window] [in a new window]   FIG. 6.   An enlarged polymerase is more sensitive to PcG repression. (A) Drawing comparing wild-type T7RNAP and Goliath polymerase. (B) Pattern of Goliath polymerase protein in the CNS Goliath line in a germ band retracted embryo. The location of Goliath polymerase was detected with a monoclonal antibody to -galactosidase. PS5, the anterior limit of bx enhancer activity, is indicated with a bracket. (C) Transcription by Goliath polymerase on the 4× T7 promoters in the bx control region in a germ band retracted embryo. Transcription by Goliath is blocked anterior to PS5. (D) Transcription by Goliath polymerase in an extra sex combs mutant embryo (esc2/esc10, maternal and zygotic null). The repression in anterior segments is lost. (E to H) T7RNAP versus Goliath polymerase as shown by expression of the polymerases with the Gal4-UAS system. A ubiquitous Gal4 source (line 32B) was used to drive polymerase expression. All embryos are in the germ band extended stage. (E) Goliath polymerase protein pattern marked with an antibody to -galactosidase. (F) Transcription by T7RNAP on the 4× T7 promoters in the bx control region. Transcription appears unaffected by the PcG. (G) Transcription by Goliath polymerase. Transcription is repressed by the PcG anterior to PS5. (H) Transcription by Goliath polymerase in a Polycomb mutant (zygotic null). Repression in the anterior segments is lost.

FLP-mediated recombination is blocked by Polycomb. The cassette containing the UAS-LacZ reporter and T7 promoters is flanked by FRTs oriented as direct repeats. Introduction of FLP recombinase into the somatic tissues of the embryo allows the excision of this cassette from the chromosome into a 4.9-kb circular episome (Fig. 1C). The T7 promoters on this episome are now poised to transcribe around the circle, producing antisense copies of LacZ. If FLP recombination is blocked, no episome is formed, and the T7 promoters remain in the chromosome, poised to transcribe the flanking genomic DNA. We therefore introduced both a source of FLP and a source of T7RNAP into our trimmed-down insertion lines, to assay for FLP activity cell by cell (Fig. 7). We performed cRNA in situ hybridizations with the flanking chromosomal DNA as probe to highlight cells in which FLP failed to excise the cassette (Fig. 7F to J) and a probe to antisense LacZ to highlight cells in which circles were present (Fig. 7K to O).

View larger version (178K): [in this window] [in a new window]   FIG. 7.   FLP recombinase is partially blocked by the PcG. RNA in situ hybridizations were used to distinguish T7 promoters in the chromosome or in FLP-induced circles in germ band retracted embryos. (A to E) Control embryos lacking FLP. RNA probes detect transcription of flanking genomic sequences from the T7 promoters in the P insertions. In the absence of FLP, all T7 promoter cassettes remain in the chromosome and are poised to transcribe the flanking genomic DNA. Under these assay conditions, no segmental bias in transcription by T7RNAP is apparent in any of the fly lines. (F to J) Transcription of chromosome sequences by T7RNAP marks cells in which FLP has failed to access its FRT target sites, which flank the T7 promoter/UAS-LacZ cassette. (F) FLP efficiently excises the cassette from the majority of the cells in a control line, in which the cassette is located on the X chromosome. (G to J) In the BX-C insertion lines, FLP fails to excise the cassette in many cells in PcG-repressed segments. The block to circle formation appears to occur one parasegment anterior to the block to Pol II transcription. The posterior-most parasegment in which chromosomal transcription is strong is marked with a bracket. (K to O) Transcription of antisense LacZ RNA by T7RNAP marks cells in which FLP has succeeded in producing a circular episome. (K) Circles are visible in most cells in the control embryo. (L to O) A greater number of circles are visible in non-PcG-repressed segments in the BX-C insertion lines. The anterior-most parasegment in which robust circle formation is visible is marked with a bracket.

The reduced accessibility of PcG-repressed DNA is not associated with a change in DNA supercoiling. We compared the superhelix density of PcG-repressed and nonrepressed DNAs by using our FLP cassettes to produce extrachromosomal episomes. We crossed our accessibility test lines to a heat shock-inducible source of FLP recombinase, induced FLP activity, and then collected total genomic DNA, which should include newly excised circles (Fig. 1C). This DNA was subjected to gel electrophoresis in the presence of chloroquine, which resolves supercoiled circles into a ladder of distict bands. Adjacent rungs of the ladder differ by a single superhelical turn, and the topoisomers of the circles describe a Gaussian distribution. Chloroquine gels were Southern blotted and probed with the lacZ sequences contained within our FLP-inducible cassette (Fig. 8). By this technique, a reproducible change in linking number as small as 1 can be seen.

View larger version (99K): [in this window] [in a new window]   FIG. 8.   PcG repression is not associated with altered DNA supercoiling. Circular episomes produced from the FLP cassettes were subject to electrophoresis through agarose gels supplemented with 2 µg of chloroquine per ml. Under these conditions, more negatively supercoiled DNA has a faster mobility. The gels were Southern blotted and probed with lacZ sequence. (A) Total genomic DNA was prepared from adult fly heads, either immediately following a 1-h heat shock treatment to induce FLP activity (60') or following 1 h of heat shock plus 2 h of recovery at room temperature (180'). All of the circles produced in the p[bx], p[iab-4], p[Mcp], and p[Abd-B] lines come from PcG-repressed DNA. The control is the 3rd chromosome insertion, which is not PcG repressed. The average linking number is approximately the same for all lanes. The locations of the unexcised DNA remaining in the chromosome (ch) and of nicked circles (nc) are marked with arrows. The average linking number for the sample in the first lane is indicated with an arrowhead (see Materials and Methods). (B) Total genomic DNA was prepared from whole embryos following 1 h of heat shock plus a half-hour of recovery at room temperature to induce FLP activity. This DNA was subjected to chloroquine gel and Southern blot analyses as described above. Circles produced from the p[Mcp] line with wild-type PcG activity are compared to circles produced from p[Mcp] and p[Abd-B] lines, which are null for the Polycomb protein. The loss of PcG repression does not alter the topology of the DNA in these regions.

Concluding remarks. Our accessibility assays show that PcG repression blocks the activities of the Gal4 activator, the FLP recombinase, and two forms of T7RNAP. At least for FLP and T7RNAP, there should be no specific interactions between our probe proteins and any Drosophila proteins involved in transcription, and thus, the segment-specific differences in their activities should reflect differences in their ability to access the DNA. Our assays do not address the precise cause of the block. The inhibition of Gal4 could reflect a block to binding or to activation. The inhibition of the T7 polymerases could reflect a block to binding or to progression of the polymerase along the DNA template. The inhibition of FLP could reflect a block to binding or to recombination by the enzyme. Since all three enzymes are affected by the PcG, a block to binding for all three proteins seems the most simple explanation. Regardless of the exact nature of the block, it seems clear that there is a structural difference to PcG-repressed DNA. We note that the two control lines used in this paper were both transcriptionally silent in the absence of Gal4, but both allowed access to Gal4, T7RNAP, Goliath polymerase, and FLP throughout the embryo. If the controls represent a "typical" chromatin configuration, our results suggest that PcG-repressed DNA is less accessible than average. This suggests that the PcG does not simply prevent positive chromatin remodeling complexes from targeting the DNA.