A Complex Array of DNA-Binding Proteins Required for Pairing-Sensitive Silencing by a Polycomb Group Response Element From the Drosophila engrailed Gene

Corresponding author: Judith A. Kassis, NICHD, NIH, 6 Center Dr., MSC 2785, Bethesda, MD 20892., jkassis{at}mail.nih.gov (E-mail)

Communicating editor: J. A. BIRCHLER

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUDING REMARKS LITERATURE CITED

Regulatory DNA from the Drosophila gene engrailed causes silencing of a linked reporter gene (mini-white) in transgenic Drosophila. This silencing is strengthened in flies homozygous for the transgene and has been called "pairing-sensitive silencing." The pairing-sensitive silencing activities of a large fragment (2.6 kb) and a small subfragment (181 bp) were explored. Since pairing-sensitive silencing is often associated with Polycomb group response elements (PREs), we tested the activities of each of these engrailed fragments in a construct designed to detect PRE activity in embryos. Both fragments were found to behave as PREs in a bxd-Ubx-lacZ reporter construct, while the larger fragment showed additional silencing capabilities. Using the mini-white reporter gene, a 139-bp minimal pairing-sensitive element (PSE) was defined. DNA mobility-shift assays using Drosophila nuclear extracts suggested that there are eight protein-binding sites within this 139-bp element. Mutational analysis showed that at least five of these sites are important for pairing-sensitive silencing. One of the required sites is for the Polycomb group protein Pleiohomeotic and another is GAGAG, a sequence bound by the proteins GAGA factor and Pipsqueak. The identity of the other proteins is unknown. These data suggest a surprising degree of complexity in the DNA-binding proteins required for PSE function.

IN Drosophila, it is well documented that expression of a gene on one chromosome can be influenced by an allele of that gene on the homolog (for reviews, see HENIKOFF and COMAI 1998 ; PIRROTTA 1999 ; WU and MORRIS 1999 ; KENNISON and SOUTHWORTH 2002 ). This phenomenon was first described at the bithorax complex by E. B. Lewis and was termed "transvection" (LEWIS 1954 ). In that case, two alleles of Ultrabithorax were unexpectedly found to complement one another, and this complementation was dependent on chromosome pairing. Subsequently, many additional examples of allelic complementation have been described. At the yellow (y) gene, molecular studies have shown that enhancers located on one chromosome can activate the y promoter on the homolog (GEYER et al. 1990 ; MORRIS et al. 1998 , MORRIS et al. 1999 ). This may be a common mechanism for many cases of allelic complementation. There are many other pairing-dependent effects on gene expression described in Drosophila, and these may occur by different mechanisms. For example, a particular allele of the brown locus, bw^D, is able to inactivate a paired wild-type allele of bw. This silencing is thought to be due to the ability of heterochromatic sequences present at bw^D to bring the wild-type copy into an inactive part of the nucleus (CSINK and HENIKOFF 1996 ; DERNBURG et al. 1996 ).

Pairing-dependent silencing has also been seen for transgenes in Drosophila. The commonly used P-element transformation vector CaSpeR includes a minigene for the selectable marker white; expression of the white minigene causes white mutant (white-eyed) flies to have colored eyes (PIRROTTA 1988 ). Eye color is sensitive to the dosage of white, with higher levels of white mRNA leading to darker eye colors. Thus, flies homozygous for a CaSpeR transposon, having two copies of mini-white, have a darker eye color than heterozygotes. However, when particular fragments of regulatory DNA are included in pCaSpeR, the eye color of homozygotes is lighter than that of heterozygotes (reviewed in KASSIS 2002 ). This phenomenon has been called "pairing-sensitive silencing" (KASSIS 1994 ). DNA fragments that cause pairing-sensitive silencing are called pairing-sensitive elements (PSEs). The focus of our laboratory is to understand the molecular mechanisms and biological significance of pairing-sensitive silencing.

Pairing-sensitive silencing was first described for DNA sequences from the Drosophila engrailed gene and subsequently found for many other DNA fragments (KASSIS et al. 1991 ; FAUVARQUE and DURA 1993 ; CHAN et al. 1994 ; GINDHARDT and KAUFMAN 1995 ; KAPOUN and KAUFMAN 1995 ; HAGSTROM et al. 1997 ; FUJIOKA et al. 1999 ; MULLER et al. 1999 ; SHIMELL et al. 2000 ). Many of these DNA fragments are known to be regulatory elements for the Polycomb group genes [Polycomb group response elements (PREs); FAUVARQUE and DURA 1993 ; SIMON et al. 1993 ; CHAN et al. 1994 ; GINDHARDT and KAUFMAN 1995 ; HAGSTROM et al. 1997 ; MULLER et al. 1999 ; reviewed in PIRROTTA 1997A , PIRROTTA 1997B ]. The Polycomb group (PcG) genes are a diverse group of at least 15 genes, important for heritable gene silencing in Drosophila (reviewed in BIENZ and MULLER 1995 ; KENNISON 1995 ; HAGSTROM and SCHEDL 1997 ; PIRROTTA 1997A,B ). Many PcG genes encode chromatin-associated proteins that are components of large protein complexes (SATIJN and OTTE 1999 ; SHAO et al. 1999 ; NG et al. 2000 ; CHANG et al. 2001 ; SAURIN et al. 2001 ; TIE et al. 2001 ). The PcG proteins act in three or more distinct protein complexes that silence transcription by unknown mechanisms. These mechanisms may involve alterations in chromatin structure and modification of histones (reviewed in SATIJN and OTTE 1999 ; FRANCIS and KINGSTON 2001 ; MAHMOUDI and VERRIJZER 2001 ). Pairing-sensitive silencing may be caused by the actions of PcG proteins; however, the relationship is not simple. PREs seem to be composite elements that can be divided into many PSEs, and single PSEs often cannot work as PREs (HORARD et al. 2000 ; SHIMELL et al. 2000 ). Thus, a PSE may be a component of a PRE, but in general is not sufficient for PRE activity. The molecular mechanisms that account for PSE activity are far from clear.

We have been studying the sequences important for the pairing-sensitive silencing activity of a PSE from the Drosophila engrailed gene. We have previously shown that the protein encoded by the PcG gene pleiohomeotic (pho) binds to sequences within an engrailed PSE and that those sequences are required for PSE activity (BROWN et al. 1998 ). In this report we further examine the sequences important for PSE activity by mutational analysis of a minimal PSE and by gel mobility shift assays, using nuclear extracts from Drosophila embryos to detect protein-binding sites. Our data show that at least five and perhaps as many as eight distinct protein-binding sites are required for full PSE activity. We compared the pairing-sensitive silencing activity of a 181-bp PSE with a larger engrailed fragment (2.6 kb) that contains both this PSE and another strong PSE and found that they behave similarly in most assays, although silencing by the larger fragment was more stable in one assay. Finally, we compared the PRE activities of the 2.6-kb fragment and the 181-bp fragment in embryos in the context of a bxd-Ubx-lacZ reporter transgene. We found that each of these fragments could act as a PRE, while the larger fragment contained additional silencing capabilities.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUDING REMARKS LITERATURE CITED

Construction of transgenes:
The en fragment in 2.6up, 2.6upR, 2.6D, and 2.6DR extends from -2.407 kb to -10 bp, just upstream of the transcription start site. A natural EcoRI site is present at the 5' end, and a synthetic EcoRI site was added to the 3' primer. The 3' primer differed from the natural sequence at two positions: at -20 bp, a G was present instead of a C, and at -21 bp, a C was present instead of a G. These changes are not expected to change pairing-sensitive silencing (PSS) activity since this activity has been mapped to sequences upstream of -400 bp (KASSIS et al. 1991 ). The en fragment in 181up, 181upR, and 181D extends from -576 to -395 bp. This is the same fragment used in KASSIS 1994 (construct 8) and in BROWN et al. 1998 (Figure 1). We previously stated that this fragment ended at -400 bp, at the beginning of a SphI site; however, this was incorrect, since the entire SphI site was present. Therefore, the correct 3' end is -395 bp. Synthetic EcoRI sites were added to the 5' and 3' primers used to amplify the 181-bp fragment. For cloning the en fragments upstream of the mini-white gene, pCaSpeR was cut with EcoRI, and the en fragments were cloned as EcoRI fragments. The orientations of the en fragments were determined by PCR. For cloning the en fragments downstream, pCaSpeW15 was cut with SstI, removing a fragment of the mini-white gene including an upstream EcoRI site, creating pCaSpeW15-SstI. The en fragments were then cloned as EcoRI fragments into EcoRI-cut pCaSpeW15-SstI, and the orientation of insertion was determined by PCR. The SstI fragment was then cloned back into the constructs.

For 1, 2, 4, and Mutsite1, the 181-bp fragment was amplified by PCR and cloned into M13mp19. The conserved sites were deleted or mutated using the Bio-Rad (Richmond, CA) Mutagene M13 in vitro mutagenesis kit. Mutagenized clones were identified either by DNA sequencing (for the deletion clones) or by the identification of clones with an additional RsaI site (for the mutations), followed by DNA sequencing. The mutagenized fragments were amplified by PCR and subcloned into pCaSpeR (PIRROTTA 1988 ). All clones were sequenced prior to injection.

For constructs 5, 12, 13, 14, 16/4, 139 bp, and 98 bp, PCR primers were designed to amplify the following en fragments: 5, -576 to -412; 12, -454 to -395; 13, -516 to -437; 14, -576 to -499; 16/4, -516 to -395; 139, -576 to -437; and 98, -550 to -452. The 5' primer always had a synthetic EcoRI site added, and the 3' primer always had a synthetic BamHI site added. The en fragments were cloned into BamHI/EcoRI cut CaSpeR. All constructs were then sequenced. The results from construct 12 were previously reported (KASSIS 1994 ). The 139-bp fragment was also cloned into a vector with three binding sites for the eye enhancer binding protein Glass (FUJIOKA et al. 1999 ).

For Mutsite2, MutGAGA, MutNC1, and MutNC2, mutations were made using mutated primers in the PCR reactions. The 5' primer started at -576 bp and the 3' primer started at -437 bp. For the synthetic constructs, the oligonucleotides (oligos) used are listed in Fig 5. Oligos for both strands were made, annealed, and cloned into pCaSpeR. For Synsites3, the annealed oligos were ligated in the presence of BglII prior to cloning into the BamHI site of pCaSpeR. The sequences of these clones were determined.

View larger version (20K): In this window In a new window Download PPT slide   Figure 1. Pairing-sensitive silencing of mini-white by engrailed DNA. Construct names are on the left, followed by a diagram of each construct and the number of lines showing PSS, over the total number of homozygous viable lines. The corresponding percentage of lines showing PSS is in parentheses. Black boxes represent P-element ends, open boxes are the mini-white gene, and stippled boxes are engrailed DNA. Constructs are approximately to scale. 5' and 3' represent the orientation of the engrailed DNA with respect to its promoter. Arrows indicate the start site and direction of mini-white transcription. (A) Constructs with the 2.6-kb engrailed fragment cloned upstream and downstream in both orientations. (B) Constructs with the 181-bp engrailed fragment cloned upstream in both orientations and in one orientation downstream. The starting construct CaSpeR is also shown to illustrate that the small percentage of lines showing PSS with the engrailed DNA cloned downstream of mini-white is greater than that obtained with CaSpeR alone. (C) The E. coli lacZ gene (blue box) was cloned between the mini-white promoter and engrailed DNA. (D) Three binding sites for the eye disc transcriptional activator protein Glass (GBS, red box) were cloned between the mini-white promoter and the engrailed fragment.

View larger version (94K): In this window In a new window Download PPT slide   Figure 2. Pairing-sensitive silencing by the 181-bp PSE. (A) The eye color of flies from line 181up-1C indicates that the mini-white gene is homogenously silenced in homozygotes. (B) The mini-white gene in line 181lacZ-11B, where the engrailed PSE is 3.9-kb upstream of the promoter, is silenced in a variegated manner. Homozygotes contain patches of red on a yellow background, and the distribution and extent of eye coloration differ from eye to eye.

View larger version (9K): In this window In a new window Download PPT slide   Figure 3. Glass binding sites darken the eye color of heterozygous flies. The number of lines scored is shown on the y-axis and the eye colors observed (RED, red; DO, dark orange; O, orange; LO, light orange; Y, yellow) are shown on the x-axis. Solid bars represent 139GBS heterozygotes; open bars represent heterozygotes with construct 139bp.

View larger version (29K): In this window In a new window Download PPT slide   Figure 4. Defining a minimal PSE. (A) A sequence comparison of D. melanogaster (top sequence) and D. virilis (bottom sequence) is shown (from KASSIS et al. 1989 ). Identical bases are capitalized. Nonidentical sequences are shown in lowercase letters. Dots indicate a gap put in the sequence to optimize alignment. Long stretches of sequence identity are boxed and labeled 1–5. The two nonconserved regions mutated in Fig 6 are shown by lines above and denoted NC1 and NC2. (B) The starting construct 181up is shown on the top line along with the position of the conserved sequence blocks (stippled and numbered boxes). The DNA present in each construct is shown by a rectangle(s). 1, 2, and 4 are all precise internal deletions of the conserved bases shown boxed in A. The number of lines that showed PSS over the total number of homozygous viable lines is shown on the right. Those constructs that were positive for PSS are shown in boldface type. The data from 1 have previously been reported (BROWN et al. 1998 ).

View larger version (15K): In this window In a new window Download PPT slide   Figure 5. PSE activities of synthetic constructs. The name of each construct is shown on the left, followed by the sequence. On the right, the number of lines with PSS is shown over the total number of homozygous viable lines.

For the en-bxd-Ubx constructs, the vector Ußglz containing 3.1 kb of the Ubx promoter fused to the lacZ gene was cut with XbaI-KpnI, and a 1.6-kb XbaI-KpnI fragment containing the BXD enhancer was inserted (vectors obtained from J. Müller; MULLER and BIENZ 1991 ). The en fragments extending from -2.407 kb to -10 bp (2.6en) and from -537 to -395 bp (181en) were amplified using primers containing synthetic XbaI sites. The orientation of the en fragments in this vector was 3' to 5' with respect to the Ubx promoter.

Generation and analysis of transgenic lines:
Injections were done into homozygous Df(1)w67c2, y embryos using procedures described in FUJIOKA et al. 2000 . Most of the lines described in this study were obtained directly from injection, but a few were generated by transposon mobilization using a P[2,3], 99B line (ROBERTSON et al. 1988 ). The eye color of homozygotes and heterozygotes was compared in flies of the same age and sex. For this report, lines were scored as having PSS only if the eye color of homozygotes was lighter than that of heterozygotes. In previous studies (KASSIS et al. 1991 ; KASSIS 1994 ), lines were also scored as having PSS if the eye color of homozygotes was the same as that of heterozygotes. We previously reasoned that the eye color of homozygotes should be darker, and thus, when it was not, PSS was occurring. However, in recent experiments we have observed that lines generated with either pCaSpeR alone or with pCaSpeR containing non-PSS DNA fragments yield flies with the same eye color in homozygotes and heterozygotes at a frequency of 5–10%. Therefore, in this report, lines with the same eye color in homo- and heterozygotes were scored as negative for PSS activity.

Gel mobility shift assays:
Nuclear extracts were made from 0- to 22-hr Drosophila embryos as described in TSUKIYAMA and WU 1995 . Three microliters of nuclear extract was incubated in a total volume of 10 µl with 20 fmol labeled oligo, 10 µg tRNA, 4 µg poly(dI-dC), 100 mM KCl, 35 mM HEPES pH 7.9, 1 mM dithiothreitol, 50 µM ZnCl₂, 12% glycerol, 2 mM spermidine, and 5 mg/ml BSA. Samples were incubated for 20 min at 25°, supplemented with 2 µl 5% BSA and 2 µl gel-loading buffer (2.5% Ficoll 400, 0.5x TBE and tracking dyes), and then electrophoresed on a 1% agarose, 0.5x TBE gel. The gel was dried onto DE81 paper (Whatman) and autoradiographed.

	RESULTS AND DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUDING REMARKS LITERATURE CITED

We previously showed that a 2.6-kb fragment containing the engrailed promoter and 2.4 kb of upstream sequences mediated pairing-sensitive silencing of the mini-white gene in CaSpeR, at 60% of insertion sites (KASSIS et al. 1991 ). Here we tested the orientation dependence of that activity. We found that this fragment works equally well in either orientation when positioned upstream of the mini-white transcription unit, but that the activity was significantly reduced when placed downstream, in either orientation (Fig 1A). We also tested the activity of a 181-bp subfragment (extending from -576 to -395 bp, construct 8 in KASSIS 1994 ) in both orientations upstream and in one orientation downstream of the mini-white gene (Fig 1B). Like the larger fragment, there was no difference in the percentage of lines showing pairing-sensitive silencing when the 181-bp fragment was tested in either orientation upstream. However, the pairing-sensitive silencing activity was greatly reduced when cloned downstream of mini-white. To test if this decreased activity was the result of an increased distance between the mini-white promoter and the PSE, we inserted the lacZ gene of Escherichia coli between the mini-white promoter and the engrailed DNA (Fig 1C). This separated the engrailed fragments from the mini-white promoter by 3.9 kb, similar to the 3.7-kb distance between them when the engrailed fragments were downstream of mini-white. The increased distance had no significant effect on the percentage of lines showing pairing-sensitive silencing (Fig 1C). Therefore we conclude that the engrailed PSEs can function at a considerable distance upstream of the mini-white promoter.

Interestingly, pairing-sensitive silencing from the upstream position by the 2.6-kb engrailed fragment was more stable than that by the 181-bp fragment, in that the 2.6lacZ construct resulted in eyes of a homogeneously lighter eye color, similar to that seen when either the 181-bp or the 2.6-kb fragment was located adjacent to the mini-white promoter (Fig 2A). In contrast, with 181lacZ in the upstream position, the repression was not homogenous. Instead, the eye color of homozygotes was often white or yellow with red spots (Fig 2B), suggesting that while the transgene was silenced in some portions of the eye, in others both copies were expressed, resulting in the darker eye color. The size and location of the spots varied from eye to eye, suggesting that loss of repression could occur either early or late in development and that the unrepressed state was thereafter heritably transmitted through multiple cell divisions. Of the 10 181lacZ lines that gave pairing-sensitive silencing, 9 had such variegated eyes in homozygotes. In contrast, of the three 2.6lacZ lines that gave pairing-sensitive silencing, none had variegated eyes. Thus, the stability of pairing-sensitive silencing through development and cell division is a distinct property of PSEs independent of the strength of repression that they produce within single cells.

We wondered why pairing-sensitive silencing occurs at only 50–60% of chromosomal insertion sites. Studies on pairing-sensitive silencing by the bxd PRE have shown that this PRE can completely silence mini-white at some chromosomal positions even in heterozygotes (SIGRIST and PIRROTTA 1997 ). If the engrailed fragment behaved in a similar manner, then there would exist a class of transgenic lines with white eyes, which would not be recovered, and this would result in the percentage of pairing-sensitive lines recovered being artificially low. We therefore tested the minimal 139-bp PSE (defined below) in a vector that contained three binding sites for the eye enhancer-activator protein Glass (ELLIS et al. 1993 ). We reasoned that the eye color of lines recovered should be darker using this glass-binding site (GBS) vector and that we might recover a greater percentage of lines with pairing-sensitive silencing. As expected, the eye color of heterozygous flies was on average darker with the GBS vector (Fig 3). However, the percentage of lines showing pairing-sensitive silencing was the same with both vectors (Fig 1D and Fig 4B, 139bp construct). Thus, the 139-bp PSE does not appear to be a strong silencer in the heterozygous state. These data also show that the 139-bp PSE is able to silence a strong eye enhancer.

Identification of sequences important for pairing-sensitive silencing:
Our primary goal is to understand the molecular mechanisms responsible for pairing-sensitive silencing. To begin to accomplish this, we attempted to identify all the DNA-binding proteins important for the activity of a single PSE. Our first goal was to identify a minimal PSE. We started with the 181-bp fragment that we had previously identified as sufficient for pairing-sensitive silencing activity (KASSIS 1994 ). We used sequence conservation with a distantly related Drosophila species, Drosophila virilis, to guide our analysis (estimated divergence time 60 million years; BEVERLEY and WILSON 1984 ). We previously showed that the homologous DNA fragment from D. virilis acts as a PSE in D. melanogaster (KASSIS 1994 ). A sequence comparison of these two fragments is shown in Fig 4A (sequence data from KASSIS et al. 1989 ). Long blocks of high sequence similarity are shown in boxes and are labeled 1–5. Other sequence identities are shown in capital letters. Two regions with no long stretches of sequence identity are overlined and labeled NC1 and NC2 (for nonconserved regions 1 and 2). Conserved box 1 contains the binding site for the PcG protein Pho (BROWN et al. 1998 ). Conserved box 3 contains the sequence GAGAG, a binding site for GAGA factor and Pipsqueak, two proteins thought to be important in PRE function (SOELLER et al. 1993 ; LEHMANN et al. 1998 ; HORARD et al. 2000 ; BUSTURIA et al. 2001 ; HODGSON et al. 2001 ). In gel mobility band shift experiments to detect DNA-binding proteins, we found evidence for protein binding to sequences present in conserved box 2, but not to sequences present in conserved boxes 4 and 5 (below and data not shown). We began our analysis by subdividing the 181-bp fragment and by deleting separately the sequences present in boxes 1, 2, 4, and 5. In Fig 4B, rectangular boxes represent the sequences present in each construct. The positions of the conserved sequences are indicated by stippling in the 181up diagram. Those constructs that retain pairing-sensitive silencing activity are shown in boldface type. The smallest active construct is 139bp, which contains conserved sequences 1–3. Thus, conserved regions 4 and 5 are not required for pairing-sensitive silencing.

We next made synthetic constructs using sequences present in boxes 1–3 and asked whether they were sufficient to function as PSEs. Three copies of the Pho-binding site (3X Pho, Fig 5) had no pairing-sensitive silencing activity. We also tested a synthetic construct containing a Pho site, site 2, and site 3 (GAGAG) as either one or three copies (Fig 5). With one copy, 1/23 lines showed pairing-sensitive silencing, and with three copies, 1/11 lines had this activity. While these numbers are very low and not significantly different from 0/23 or 0/11, we believe that the synthetic construct has a very low level of PSE activity because pairing-sensitive silencing is observed very infrequently or not at all with CaSpeR alone (Fig 1 and our unpublished results). However, it is clear that some of the sequences required for pairing-sensitive silencing were not present in these synthetic constructs.

We tested whether base substitutions that disrupt the Pho and GAGAG binding sites compromised the activity of the PSE. Both of these changes decreased the percentage of lines showing pairing-sensitive silencing to a very low level (MutSite1 and MutGAGA, Fig 6). Likewise, mutations in conserved site 2 similarly reduced the PSE activity (MutSite2, Fig 6). As a control, we introduced two groups of mutations into the less conserved region between sites 1 and 2. To our surprise, the 6-nucleotide substitution in the region labeled NC1 in Fig 4A greatly reduced pairing-sensitive silencing activity (MutNC1, Fig 6). Likewise, the 10-nucleotide substitution in NC2 disrupted the pairing-sensitive silencing activity of the 139-bp PSE (MutNC2, Fig 6). With these data in hand, we investigated whether proteins in nuclear extracts from Drosophila embryos could recognize these sequences in vitro.

View larger version (25K): In this window In a new window Download PPT slide   Figure 6. Effects of base pair substitutions on pairing-sensitive silencing. The name of each construct is shown on the left, followed by the wild-type sequence. The mutations introduced are shown above each wild-type sequence. The substitutions in MutSite1 were made in the 181-bp PSE, and the results were previously reported (BROWN et al. 1998 ). All other mutations were made in the 139-bp PSE. The number of lines with PSS is shown over the total number of homozygous viable lines.

Eight protein-binding sites in the 139-bp PSE:
We previously showed that conserved site 1 binds the PcG protein Pho (BROWN et al. 1998 ). Further sequence analysis identified an additional Pho site on the opposite strand, from -569 to -576: GCCATCTC, which matches the Pho consensus (G/t)CCATN(T/a)(T/g/c) (HYDE-DERUYSCHER et al. 1995 ). As previously stated, conserved site 3 contains the sequence GAGAG, a binding site for both GAGA factor and Pipsqueak (SOELLER et al. 1993 ; LEHMANN et al. 1998 ). An additional GAGAG sequence is present on the opposite strand and extends from -557 to -562. Thus, two Pho-binding sites and two GAGAG sequences are present in the 139-bp PSE. Within the 98-bp construct that does not have PSE activity, one Pho and one GAGAG site have been deleted. In the homologous D. virilis DNA fragment, the only GAGAG sequences are those present in conserved site 3; however, two Pho sites are present, one within conserved site 1 and one located in the nonconserved region (within NC2, GCCATAGT). Thus it is possible that two Pho sites may be required for PSE function. This has not yet been tested.

We examined whether conserved site 2 could bind a factor present in nuclear extracts from Drosophila embryos. Oligos containing the site 2 sequence were radioactively labeled and combined with nuclear extracts from Drosophila embryos in gel mobility shift assays. Since an 11-bp oligo containing only site 2 did not give a reproducible band shift, we used a 19-bp oligo for our experiments. Results from a representative gel shift experiment are shown in Fig 7A. In this experiment, one band was detected that was effectively competed with an unlabeled site 2 19-mer, but not by an oligo containing the 5-bp mutation used to test the function of conserved site 2 in vivo (Fig 6). Our data suggest that at least one protein binds conserved site 2 and that mutation of this site leads to a loss of pairing-sensitive silencing activity.

View larger version (33K): In this window In a new window Download PPT slide   Figure 7. Gel mobility shift assays. (A) The site 2 oligo (sequence shown) was radioactively labeled and used in a gel shift experiment with Drosophila embryonic nuclear extracts. A single specific complex was detected that was competed by a 100-fold excess of the site 2 oligo, but not by a site 2 oligo with 5 nucleotide substitutions (MutSite2 oligo). Nucleotide substitutions are shown above the wild-type sequence. The band labeled nonspecific (Non Sp.) can be competed by any oligonucleotide (data not shown). (B) The sequence of the two nonconserved regions, NC1 and NC2, is shown, with the nucleotide substitutions in mutated oligos shown above the wild-type sequence. The oligos used in the band-shift experiments in C are shown by lines labeled A–C below the sequence. The C oligo extends beyond NC2 by 2 bases, both G's. (C) Gel mobility shift assays using the A, B, and C oligos. (Left) Mobility shift of the A oligo in the absence of competitor DNA or in the presence of a 100-fold excess of unlabeled A oligo, site 2 oligo, Pho oligo, or the A oligo with the NC1 or the NC2 mutations. (Middle) B oligo mobility shift in the absence of competitor DNA or in the presence of a 100-fold excess of either unlabeled B or Pho oligo. (Right) C oligo mobility shift in the absence of competitor DNA or in the presence of a 100-fold excess of unlabeled C oligo, B oligo, or the C oligo with the NC2 mutations. (D) Summary of the factors that bind to the 139-bp minimal PSE. The stippled Pho and GAGA icons represent sites that show homology to the consensus-binding sites for these factors but that we have not tested for binding.

We next examined whether we could detect proteins binding to NC1 and NC2. We found that at least three proteins can bind within this region. The sequences of the NC1 and NC2 regions and the oligos used in gel shift experiments are shown in Fig 7B. We did not find evidence of a protein binding to the NC1 sequence alone. However, using a 19-bp oligo spanning the overlap between NC1 and NC2 (oligo A), we detected a reproducible band shift, suggesting that a protein binds to this region. The sequence of this region bears some similarity to a GAGA-element binding sequence (the sequence is GAGGGAG) but was not competed by a GAGAGAG oligo (data not shown). The band shift with the A oligo was specifically competed by itself (A oligo), but not by the site 2 or Pho oligo or by the A oligo with the mutations present in NC1. Thus this factor should not bind to the PSE present in lines carrying the MutNC1 construct (which showed very low level activity; Fig 6). In contrast, an A oligo carrying the NC2 mutations could still compete with the A oligo band shift. Therefore, binding of the A factor should be unaffected in the lines carrying the NC2 mutations. When the NC2 oligo was used in a band shift experiment, multiple specific bands were observed (not shown). We therefore divided it into two different oligos, B and C (Fig 7B). The B oligo has substantial overlap with the A oligo, but they did not cross-compete for shifted complexes (data not shown). Three specific bands were detected in a mobility gel shift experiment using the B oligo (Fig 7C). The mutated B oligo, carrying the NC2 mutations, partially competed with the unmutated B oligo (not shown), suggesting that the binding of factor B may be decreased in the MutNC2 construct. For the C oligo, a specific complex was detected, and this complex was not competed by either a B oligo or a C oligo carrying the same mutations as in the MutNC2 construct. Thus, the binding of both factors B and C is disrupted in the PSE carrying the NC2 mutation (MutNC2, Fig 6). Therefore, while we can surmise that the binding of at least one factor to the NC2 region is important for PSE function, we cannot state whether it is factor B or C or both. Taken together, these data indicate that there are eight protein-binding sites in the 139-bp PSE (Fig 7D). Our functional data show that the conserved Pho site, site A, site B or C (or both), site 2, and the conserved GAGAG sites are all important for PSE activity (Fig 6). Mutations in the nonconserved Pho and GAGAG sites have not yet been tested.

PRE activity of the engrailed fragments:
PREs often act as PSEs in CaSpeR vectors, and there are several reasons to suggest that the engrailed DNA we are studying is a PRE. First, the fact that it acts as a PSE suggests that it might be a component of a PRE. Second, Pho, a PcG protein, is required for pairing-sensitive silencing activity (BROWN et al. 1998 ). Third, STRUTT and PARO 1997 found that the PcG proteins Polycomb, Posterior sex combs (Psc), and Polyhomeotic could be cross-linked to sequences within the 2.6-kb engrailed fragment in cultured cells, while, of the proteins tested, only the Psc protein could be cross-linked to the 181-bp PSE. We tested whether engrailed PSEs could act as PREs in a vector designed to test for PRE activity in embryos. This vector includes a 3.1-kb Ubx promoter fused to lacZ and the 1.6-kb bxd enhancer (MULLER and BIENZ 1991 ). Without a PRE, lacZ is expressed throughout both the ectoderm and the nervous system in late-stage embryos, while addition of a PRE restricts lacZ expression to PS6 and posterior segments (MULLER and BIENZ 1991 ). This vector was used to study the iab-7 PRE (HAGSTROM et al. 1997 ). Interestingly, when the 2.6-kb engrailed fragment was tested in this vector, it largely silenced the activity of the bxd enhancer in a wild-type background. In the embryo shown in Fig 8A, lacZ was expressed in a few cells in the central nervous system, in very light stripes in the ectoderm (due to the "basal activity" of lacZ vectors), and in the head (due to regulatory sequences flanking the position of insertion). Twelve lines of en2.6-bxd-lacZ were recovered. Six had expression patterns that were due to the position of insertion and could not be interpreted. Three of the lines had very limited lacZ expression in the nervous system and no expression in the ectoderm, similar to the embryo shown in Fig 8A. Individual embryos varied as to the number and position of cells expressing lacZ, although expressing cells were always posterior to PS5. Thus, the decision of whether or not to silence was made on a cell-by-cell basis. In a heterozygous Pc background, some of this silencing was relieved, and lacZ was expressed more extensively in the nervous system. However, this expression was still restricted to PS6 and posteriorly (Fig 8B). Three en2.6-bxd-lacZ lines gave embryos with expression patterns similar to that seen in Fig 8B in a wild-type background. In Pc homozygotes, lacZ expression was derepressed anterior to PS6 in all six lines, while remaining restricted primarily to the nervous system. Although the bxd enhancer is also active in the embryonic ectoderm, lacZ was expressed only lightly in the ectoderm in all six en2.6-bxd-lacZ lines, even in Pc homozygotes. Thus, sequences within the 2.6-kb fragment were able to partially silence the bxd enhancer in the ectoderm in a Pc-independent manner. We suggest that these may be engrailed regulatory sequences that normally act to keep engrailed off in the anterior compartment of each segment. Since the 2.6-kb engrailed fragment does silence the Ubx promoter in a Polycomb-responsive manner in the nervous system, it is behaving as a PRE in that tissue. We also examined whether lacZ expression in these lines differed in embryos heterozygous or homozygous for the reporter construct. We saw no evidence for increased silencing in homozygotes, suggesting that the silencing activity is not pairing sensitive in embryos (data not shown).

View larger version (60K): In this window In a new window Download PPT slide   Figure 8. PRE activities of engrailed DNA. The DNA construct used to test for PRE activity is diagrammed at the bottom. One construct contained 2.6 kb of engrailed DNA in the position shown (2.6en-bxd-lacZ), while the other contained the 181-bp engrailed PSE in the same position (181en-bxd-lacZ, not diagrammed). ß-Galactosidase expression was visualized by immunoperoxidase staining. Embryos are anterior left, ventral (A), ventral-lateral (B and C), and lateral views (D and E), at 10 hr after egg laying. (A–C) 2.6en-bxd-lacZ in wild type (A), a Pc heterozygote (B), or a Pc homozygote (C). (D and E) 181en-bxd-lacZ in wild type (D) or a Pc homozygote (E).

In contrast to the 2.6-kb fragment, the 181-bp PSE behaves as a typical PRE in the bxd-Ubx-lacZ reporter. In wild-type embryos, lacZ was expressed in both the ectoderm and the nervous system in PS6 and posteriorly (Fig 8D). In Pc homozygotes, lacZ expression extended anteriorly (Fig 8E). There was no derepression in Pc heterozygotes (data not shown). For 181en-bxd-Ubx-lacZ we obtained 12 lines. Two had enhancer trap patterns, and of the remaining 10, 8 showed expression restricted to PS6 and posteriorly. The degree of restricted expression varied with the insertion site, with some lines showing very good anterior silencing and others having many individual lacZ-expressing cells anterior to PS6.

	CONCLUDING REMARKS

TOP ABSTRACT MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUDING REMARKS LITERATURE CITED

Here we present a functional analysis of the sequences required for pairing-sensitive silencing and of the protein-binding capabilities of a 139-bp minimal PSE. This analysis suggests that binding sites for at least five different DNA-binding proteins are required for full activity of this PSE, with some sites still untested. One of the essential sites binds Pho, the product of the Polycomb group gene pleiohomeotic (BROWN et al. 1998 ). Another is the sequence GAGAG, a known binding site for two Drosophila proteins, GAGA factor and Pipsqueak (SOELLER et al. 1993 ; LEHMANN et al. 1998 ). The sequence GAGAG has been shown to be important for the pairing-sensitive silencing activity of the iab-7 PRE (MISHRA et al. 2001 ). In that case, the authors were able to show that mutations in Trl, the gene that encodes GAGA factor, reduce the silencing activity of the iab-7 PRE (HAGSTROM et al. 1997 ; MISHRA et al. 2001 ). In contrast, Trl mutations do not affect the eye color of flies with the 139-bp engrailed PSE (data not shown). The GAGAG sequence has also been found to be important for the silencing activity of the Mcp and bxd PREs (BUSTURIA et al. 2001 ; HODGSON et al. 2001 ). Biochemical and genetic evidence suggest a role for both GAGA factor and Pipsqueak in PcG silencing (HORARD et al. 2000 ; BUSTURIA et al. 2001 ; HODGSON et al. 2001 ).

It is unclear why pairing-sensitive silencing occurs at only 60% of insertion sites, although it presumably involves the nearby flanking sequences, which might either contribute to or prevent the effect. Here we have shown that addition of binding sites for the enhancer protein Glass, while it darkened the eye color of heterozygous flies, did not change the percentage of lines with pairing-sensitive silencing (Fig 1 and Fig 3). This suggests two things. First, it is unlikely that there is a class of lines with strong mini-white repression in heterozygotes that we failed to recover using the CaSpeR vector alone, and second, pairing-sensitive silencing is not reduced significantly by the presence of this activator. In fact, curiously, pairing-sensitive silencing was more efficient with the GBS vector, in that more lines had complete silencing (data not shown). These results differ somewhat from those obtained by MISHRA et al. 2001 , where a greater percentage of lines with pairing-sensitive silencing were obtained using a vector without an eye enhancer than with the enhancer. However, those authors used a different eye enhancer (from the white gene), so the results are not directly comparable.

Our data suggest that mutations in any one of five protein-binding sites reduce the level of pairing-sensitive silencing, although they do not reduce it to zero. Transgenic lines with CaSpeR alone rarely if ever show pairing-sensitive silencing. In contrast, in lines with the 139-bp PSE carrying mutations in the Pho, GAGAG, site 2, or NC1 sites, the percentage of lines with pairing-sensitive silencing was reduced from 60 to 10%. Even the synthetic constructs containing only the Pho, GAGAG, and site 2 sequences gave a low number of lines with pairing-sensitive silencing. These data suggest that flanking genomic sequences can contribute to the activity of an impaired PSE. The interplay between PSEs and flanking genomic DNA has not been thoroughly explored. We previously showed that a PSE-containing transgenic line with the insertion at a chromosomal site that did not allow pairing-sensitive silencing could be converted into a pairing-sensitive line by duplication of the transgene (KASSIS et al. 1991 ), effectively putting two PSEs near each other in the genome. These data suggested that a flanking genomic PSE may be an important component of pairing-sensitive silencing. Aside from one example (WHITELEY et al. 1992 ), this idea has not been further investigated.

Here we have shown that engrailed PSEs can work as PREs in a bxd-Ubx-lacZ reporter. We believe that this activity is significant, but offer the caveat that this reporter may contain a weak PRE (MULLER and BIENZ 1991 ). Thus, the engrailed PSE may not be able to stably recruit or maintain Polycomb-group protein complexes by itself, but may nonetheless be an essential component of PRE function (for a complete discussion see KASSIS 2002 ). Interestingly, in tissue culture cells, the 181-bp PSE appears to be bound only by Psc and not by Pc or Ph (STRUTT and PARO 1997 ). This Psc binding might potentiate the action of a weak Pc- and Ph-binding PRE present in the bxd-Ubx-lacZ vector. A functional distinction between pairing sites and PREs has also been suggested by HODGSON et al. 2001 . Clearly, further studies using different PRE vectors, as well as functional studies of the role of these PSEs at the engrailed locus itself, are required to fully understand the biological role of engrailed PSEs.

	ACKNOWLEDGMENTS

We thank Jim Kennison and Jürg Müller for many stimulating discussions, Jürg Müller and Pam Geyer for plasmids, and Carl Wu for the use of his fly facility. We also thank the reviewers for their helpful comments. This work was supported by National Institutes of Health (GM50231) and National Science Foundation (0110856) grants to J.B.J.

Manuscript received October 25, 2001; Accepted for publication January 31, 2002.

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