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Vol. 17, Issue 5, 2158-2165, May 2006
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* Division of Shared Resources, Fred Hutchinson Cancer Research Center, Seattle, WA 98109;
Department of Zoology, University of Basel, CH-4056 Basel, Switzerland;
Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, NJ 08544; and
|| Department of Biophysics and Biochemistry, University of California, San Francisco, San Francisco, CA 94143-2240
Submitted January 17, 2006;
Accepted February 10, 2006
Monitoring Editor: Yixian Zheng
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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), referred to an unusual mechanism of genetic complementation at the bithorax complex, thought to require physical pairing between alleles. Since then, a variety of pairing-dependent phenomena have been documented in various species, including vertebrates. These include enhancer and silencer action in trans, the spreading of silenced states between homologous chromosomes, and pairing-dependent bypassing of insulator elements (reviewed in Pirrotta 1999; Duncan 2002; Kassis 2002). Pairing of alleles at specific Drosophila loci is also required to achieve wild-type levels of transcription (Goldsborough and Kornberg 1996), whereas the silencing effect of polycomb response elements (PREs) is often greatly enhanced by the pairing of two allelic copies of the PRE, the so-called pairing-sensitive silencing (reviewed in Pirrotta 1999). Although transvection phenomena are thought to rely on somatic pairing of homologous chromosomes, certain PRE regions, alone or aided by the presence of gypsy insulator elements, have been found to bypass this requirement (Hopmann et al., 1995; Sigrist and Pirrotta 1997; Müller et al., 1999). This finding suggested that Drosophila PREs might be able to pair autonomously, i.e., independently of the somatic pairing mechanism normally found in this organism. Cytological studies using conventional cytological methods have confirmed that copies of the Fab-7 PRE are able to pair in Drosophila tissues and embryos (Bantignies et al., 2003). Here, we used an in vivo assay based on green fluorescent protein (GFP)-Lac repressor/operator recognition to show that multiple remote copies of Mcp, another PRE from the Drosophila bithorax complex, are able to establish stable interactions in imaginal disk nuclei. Interactions of the type described here may provide the physical basis for the observed sensitivity of the bithorax complex to transvection effects. More importantly, the ability of Mcp to interact over large distances may be part of the mechanism through which this element performs its regulatory function within the bithorax complex. |
MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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) except for the presence of a 2.5-kb fragment with 64 lacO sites. Therefore, the P-element w#15 and the LacO-fragment clone pAFS153 (a gift from A. F. Straight, Stanford University, Stanford, CA; Robinett et al., 1996; Straight et al., 1996) served as a starting point for its construction. In a first step, w#15 was digested with KpnI and XhoI. The resulting vector fragment and the scs fragment were gel purified. Then, the 2.5-kb LacO fragment was isolated from pAFS153 after SalI and KpnI restriction digests. The w#15 vector fragment was subsequently ligated with the LacO fragment leading to the intermediate pOMws'. Compared with w#15, in pOMws', the scs fragment is replaced by the LacO fragment. In addition, the XhoI site separating the white enhancer from the LacO sites is abolished by the ligation of the SalI and XhoI compatible sticky ends. However, a new XhoI site located just next to the KpnI site in the LacO fragment was introduced. Hence, in the second cloning step, pOMws' was cut with KpnI and XhoI. Then, it was ligated with the scs KpnI/XhoI-fragment, which was isolated in the first cloning step. The resulting P-element was named psOMws'. The construction of the GFP-Lac repressor protein fusion in GAL UAS vector pUASP has been described previously (Vazquez et al., 2001).
Fly Stocks
Transgenic lines for psOMws' were established either according to standard procedures by injecting Df(1)w67c23,y– embryos (Rubin and Spradling 1982) or by mobilization (Robertson et al., 1998) of the X-linked transgene OM1 to chromosomes 2 or 3 (OM2, OM3, OM6, OM7, and OM10). Eight of 10 OM lines were pairing sensitive (80%). This is in good agreement with the value obtained for construct w#15, which lacks the lacO sites (71% pairing-sensitive lines; Müller et al., 1999). This indicates that the presence of the 2.5-kb DNA fragment containing 64 lacO sites does not significantly influence Mcp activity with respect to its pairing-dependent mini-white inactivation.
Recombinants between two transgenes were established by crossing P1,+/+, P2 virgin flies to w1 males. The progeny of these crosses was screened for recombinant males of the genotype P1,P2/+,+ by looking for the characteristic eye pigmentation indicative of long-distance pairing interaction. Such males were crossed individually with appropriate balancer lines and stocks were established. At the same time, recombination frequencies between pairs of interacting inserts were obtained and the inserts could be positioned relative to each other. To determine the actual position of each insert more precisely, inserts on the second and third chromosomes were also mapped relative to known markers. The combination of all the recombination data allowed an approximate mapping of OM2, OM3, OM4, OM5, OM6, and OM7 (Table 1). Details are available upon request.
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) and OM4, OM6, or OM7 were established as follows. gpp1A +/+ OM virgins were crossed to homozygous gpp1A males. Among the male progeny, gpp1A OM/gpp1A + flies could be selected due to the eye pigmentation of the OM transgene and the strong loss of pigmentation in abdominal segments A5 and A6 characteristic of homozygous gpp1A males. Such males were crossed individually with appropriate balancer lines, and stocks were established. These lines served as a basis to establish OM4 gpp1A OM6, OM4 gpp1A OM7, gpp1A OM6 OM7 triple recombinants. Such recombinants have clearly darker eye color than flies heterozygous for a single OM transgene and homozygous for gpp1A. To drive expression of the GFP-Lac repressor protein, we used line 1.3hs. This recombinant line carries 2 P-element insertions on chromosome 2: a GFP-lac repressor fusion gene, which is expressed under the control of the UAS enhancer in vector pUASP (line 1.3; for details, see Vazquez et al., 2001), and a heat shock-inducible hsp70::GAL4 insertion (Brand and Perrimon 1993; transgenic line was a gift from Markus Affolter, University of Basel, Basel, Switzerland). Control P[lacO, white] constructs M2.1 and M6.1 that do not carry Mcp were described in Vazquez et al. (2001).
Scoring Eye Colors
The pigmentation of the fly eye as a consequence of mini-white gene expression depends strongly on the age and sex of the fly (Qian and Pirrotta 1995). Therefore, care was taken to only compare and score eye color of flies of very similar age and sex, and also in the absence of balancer chromosomes. Flies were collected within a 4-h window after eclosion and subsequently aged for 3 d before scoring their eye color. Pictures were taken with a Nikon Coolpix 4500 digital camera mounted on a Leica MZ75 stereomicroscope, and processed with Adobe Photoshop (Adobe Systems, Mountain View, CA).
Microscopy
To induce expression of the GFP-Lac repressor protein, young third instars were heat shocked for 45–90 min in a 36°C incubator and were left to recover at room temperature for at least 16 h before imaging. Typically, the imaging was done 24–48 h after the initial heat shock. The relatively mild heat shock conditions and long recovery time were used to minimize potential heat-induced artifacts. Larvae were rinsed and dissected in saline (Drosophila testis isolation buffer; Casal et al., 1990). For short-term imaging, a small chamber was made by applying a ring of several layers of nail polish to a microscopy slide. After the nail polish had dried out, tissues were placed inside the ring in a drop of buffer and covered with a coverslip. For longer term imaging, tissues were imaged in Drosophila SL3 medium (Invitrogen, Carlsbad, CA) supplemented with 7% fetal calf serum, inside a sealed microscope chamber, as described previously (Vazquez et al., 2001, 2002). Similar imaging conditions have been shown to preserve spermatocyte viability for up to 12 h, including their progression through the meiotic cell cycle. Dead or damaged cells typically show a much greater degree of chromatin Brownian motion, possibly due to the destruction of chromatin–nuclear cytoskeleton interaction. Therefore, nuclei with unusual Brownian motion patterns were not included in the present study. To ascertain that the animals under study had the desired number of P[Mcp, lacO] inserts, polytene tissues and/or young spermatocytes dissected from the same animals were also analyzed. In those tissues, Mcp elements do not associate, therefore yielding one GFP spot per insertion site. With our lines, expression of GFP-Lac repressor protein was achieved primarily in the posterior end of the eye-antenna imaginal disk, behind the morphogenetic furrow. Therefore, the cells analyzed are photoreceptor cells in their differentiating phase and are most likely to be in G2. Imaging was done on an inverted Olympus IX-70 microscope through a high numerical aperture 60x/1.4 PlanApo or 100x/1.35 UPlanApo objective. Time-lapse series and three-dimensional (3-D) stacks were deconvolved using Applied Precision (Issaquah, WA) SoftWoRx software. Pairing efficiency was determined on deconvolved 3-D stacks with sections typically collected at 0.2- to 0.3-µm spacing. Spots were counted through examination of the 3-D data sets. Because the intensity of the fluorescent spots was very low, counting of the spots was done only on the best data sets, i.e., those where the number of spots could be determined unambiguously for >90% of the nuclei in a given field. Nuclei that were not scored usually fell into two categories: those where no clear spots could be clearly identified, and those that showed a single blurred spot or two closely spaced spots. The former probably represent nuclei with unpaired spots that are too weak to clearly identify from the background GFP levels. The latter may represent either nuclei with unpaired spots or nuclei with a single spot that got blurred because of motion during imaging. Among nuclei that were suitable for scoring, although there was a great deal of variability in the intensity of GFP-Lac repressor spots, paired spots gave a signal that was on average twice as intense as that of unpaired spots (Figure 3), and the two types could therefore be easily distinguished. Because ambiguous nuclei most likely represent nuclei with fainter, unpaired spots, the proportion of unpaired nuclei may be slightly underestimated. Time-lapse movies were collected either as 3-D stacks or as optical sections of a single focal plane. Because of the lower optical resolution along the vertical axis, and the small size of the eye disk nuclei, all spots could generally been seen in single sections focused roughly at the central plane of the nucleus.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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; Mihaly et al., 1998; Müller et al., 1999). Mcp is located between the iab-4 regulatory region that directs expression of abd-A in parasegment 9, and iab-5, that directs expression of Abd-B in parasegment 10 (Lewis 1978; Karch et al., 1985). Deletions of Mcp cause ectopic activation of Abd-B in PS9 (Crosby et al., 1993), which led to the hypothesis that this element could function either as a silencer (Busturia and Bienz 1993; Busturia et al., 1997) or as a boundary element located between and functionally separating iab-4 and iab-5 (Gyurkovics et al., 1990; Karch et al., 1994). When present on a transgene, a 2.8-kb fragment containing Mcp was shown to mediate pairing-dependent silencing of a linked mini-white gene (Müller et al., 1999). The term pairing-dependent silencing describes a phenomenon in which the eye color of homozygous flies (containing two copies of the transgene) is lighter than the eye color observed in heterozygous flies (containing 1 copy of the transgene; reviewed in Kassis 2002). In general, the activity of the mini-white reporter gene is dosage dependent. Hence, eye pigmentation normally increases with the number of mini-white transgenes in the genome (Figure 1C). Pairing-dependent silencing is position dependent and its strength is variable (compare Figure 1, D–F). Similar silencing effects could also be observed when the two P[Mcp, mini-white] copies were inserted at different locations in the genome or when they were present on rearranged chromosomes. These genetic interactions suggested that two copies of the Mcp element could physically interact, independently of homologous chromosome pairing (Müller et al., 1999).
To obtain direct evidence for the physical interaction of Mcp elements, we used the GFP-Lac repressor/operator chromosome tagging technique (Robinett et al., 1996; Vazquez et al., 2001). This live approach has the advantage of minimizing possible artifacts due to fixation and hybridization procedures. Furthermore, it offers high spatial resolution, because a cluster of GFP-lac repressor molecules bound to integrated lacO arrays occurs as a diffraction-limited spot in Drosophila nuclei (Vazquez et al., 2001). The P-element construct psOMws' carrying the Mcp element, the mini-white gene, the white enhancer, and a 
2.5-kb array of lac operator (lacO) sequences, was generated and used to transform a white mutant Drosophila strain (Figure 1A). Out of 10 lines recovered, eight showed pairing-sensitive expression of mini-white, as demonstrated by the reduced and often variegated eye color of flies homozygous for the insert (Figure 1, D–F, and Table 1). Construct psOMws', therefore, behaved in a manner similar to that observed for a previously tested construct lacking the lacO array (construct w#15 in Müller et al., 1999). This result indicates that the lacO sequences did not interfere with the ability of Mcp to induce pairing-sensitivity silencing of white. Lines carrying a similar construct lacking the Mcp sequences did not show pairing sensitivity (Müller et al., 1999; Müller, Hogga and Pirrotta, unpublished data; and Vazquez, data unpublished), which indicates that Mcp is required for the pairing dependent reduction of mini-white gene expression levels in transgenic flies.
In a second step, the long-distance interaction potential between OM transgenes located on the X, the second or the third chromosome was assessed by 21 pairwise crosses (Table 2). Typically, transheterozygous combinations on the same chromosome showed a significant reduction in eye pigmentation levels and variable degrees of variegation, indicating pairing-dependent silencing of mini-white. The strength of the genetic interaction can be estimated by the difference in eye pigmentation between transheterozygous and heterozygous control flies. Silencing seems to decrease as the distance between inserts increases (compare Figure 1, G–K), with the lowest degree of silencing achieved between inserts located on different chromosomes. In the latter case, even though expression of white seemed to be largely dosage dependent, a weak degree of variegation in a number of lines still alluded to the possibility of a small level of long-distance interaction between the transgenes involved (Figure 1 and Table 2).
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To visualize the inserts in live Drosophila, we used a heat shock-inducible system to express GFP-Lac repressor protein in the eye imaginal disk and other Drosophila tissues (see Materials and Methods). lacO-bound GFP-Lac repressor protein was detected by fluorescence deconvolution microscopy. Flies with a single P [Mcp, mini-white] insert in heterozygous condition showed a single GFP spot in 
98% of the nuclei (Table 3). In rare cases, two spots could be observed. Such cases probably represented cells undergoing chromosome replication or segregation. When the same P-element insert was present in two copies (homozygous condition), the same frequency of nuclei with single spots was observed. This result indicates that allelic copies of the P [Mcp, mini-white] insert are associated. This association does not require the presence of Mcp, because constructs containing only the lacO array and white showed similar levels of association (Table 3), and it is due to the normal pairing of homologous chromosomes in somatic nuclei. When the two copies were not allelic, but rather were present at two different locations on the same chromosome, two types of nuclei were observed. Nuclei with a single GFP spot were most abundant (>90%), indicating widespread physical association of the two elements (Figure 2, D–G, and Table 3). A small fraction of nuclei (<10%) showed two distinct GFP spots of approximately half the fluorescence intensity (Figure 3) and represented nuclei in which the remote P-elements were not associated. The association of remote elements was dependent on the presence of Mcp, because virtually all nuclei from eye imaginal discs of control flies carrying similar P-element insertions that lacked Mcp sequences had two spots and hence no significant degree of association (Figure 2, A–C). Various combinations of same chromosome inserts were tested and showed similar levels of pairing (Table 3). These results provide direct evidence for an efficient physical interaction between remote chromosomal sequences, mediated by the Mcp element.
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Previous studies have demonstrated a strong preference for intrachromosomal interactions between P[Mcp, mini-white] constructs in genetic tests (Table 2; Müller et al., 1999). Such preference might reflect an inability of nonhomologous chromosomes to interact, perhaps due to their confinement to distinct chromosome territories. To test this idea, we examined one line carrying one insertion of the psOMws' element on the second chromosome (OM3) and a second insertion of the same element on the third chromosome (OM7; Figure 2, J and K). Surprisingly, we found a high degree of physical association comparable with that observed for inserts located on the same chromosome, even though this combination of inserts showed little silencing of mini-white in the adult eye (Figure 1K; Tables 2 and 3). These results are consistent with early studies that showed that translocated copies of the bithorax complex with little or no ability to genetically interact still paired with high frequency in Drosophila embryos (Gemkow et al., 1998).
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To address the dynamics and stability of Mcp interaction, live eye disk nuclei carrying P[Mcp, mini-white] insertions tagged with the lacO array were tracked by time-lapse fluorescence microscopy. The difficulty of imaging live imaginal discs, combined with the need for high-resolution observation of relatively faint signals, imposes severe constraints on the length of time during which the tissues can be reliably observed. When tracked over periods of up to 10 min at frame rates of one image per second, paired loci were never seen to dissociate, despite substantial Brownian motion (Figure 4, A–L, and supplemental movies of the corresponding data sets). Similarly, de novo associations between the rare unpaired loci were never observed during similar time-intervals (Figure 4, M–R). Attempts at tracking the behavior of paired loci over extended periods (1–2 h) also failed to reveal any separation of the loci (our unpublished data). These results suggest that the interactions, once established, are stable for periods of minutes, and possibly hours. Similarly, the rare unpaired loci do not seem to be able to associate in nuclei of third instar larval eye discs.
The isolation of mutants that suppress Mcp-dependent silencing of mini-white could potentially uncover chromosomal proteins that play a role in chromosome–chromosome interaction. One such mutation, grappa (gpp), has been described in detail previously (Shanower et al., 2005). gpp encodes the Drosophila homologue of the yeast Dot1p, a Histone H3 methyltransferase that modulates chromatin structure and gene silencing in yeast (Singer et al., 1998; San-Segundo and Roeder 2000; Lacoste et al., 2002; van Leeuwen et al., 2002; van Leeuwen and Gottschling 2002; Ng et al., 2003). In Drosophila, the dominant grappa allele gpp1A is homozygous viable. When tested on various double recombinant P[Mcp, mini-white] chromosomes, long-distance Mcp-mediated mini-white silencing is often (but not always) suppressed in heterozygous gpp1A flies. If occurring, suppression is always enhanced in a homozygous gpp1A background (Shanower et al., 2005; Figure 1, M and N). Therefore, we wanted to examine the colocalization of inserts OM4 and OM7 in gpp1A/gpp1A flies. As shown in Figure 2, N–P, and Table 3, Mcp elements were paired in >90% of the nuclei, a frequency similar to that observed in flies wild-type for grappa. Although limited, these results suggest that gpp1A does not prevent the establishment or maintenance of chromosome–chromosome interactions.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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, 1996). More recently, long-range associations, both intra- and interchromosomal, were demonstrated in human T-helper cells (Spilianakis and Flavell, 2004; Spilianakis et al., 2005).
This study identifies a short chromosomal region from the bithorax complex, Mcp, that is able to interact with other copies of the same element present at remote locations in the genome. After the direct demonstration of pairing of the Fab-7 PRE (Bantignies et al., 2003), this is the second example of a discrete chromosomal region able to mediate sequence-specific, long-range chromosomal interactions in the Drosophila nucleus.
The frequency of association of our Mpc construct in the eye disk was very high; it was observed in 90% of nuclei. The frequency of association was substantially higher than that observed by in situ hybridization for Fab-7. It could be argued that the conditions used for in situ hybridization might disrupt potentially fragile interactions. Limited experiments in our laboratory, however, showed that the fixation procedures generally used for in situ hybridization did not significantly affect the frequency of paired sites in the eye disk, compared with the in vivo method (our unpublished data). The differences, therefore, may reflect variable strengths of different pairing-sensitive elements, or stage or tissue-specific effects. Indeed, although we observed pairing of the psOMws' element in other larval tissues such as brain and wing discs, the frequency of pairing in such tissues was often much lower (20–60% of that observed in the eye disk; our unpublished data). Other tissues, such as polytene nuclei, showed virtually no pairing (Figure 2). One possible explanation is that tissue-specific factors present in the eye disk might contribute to the pairing. Because the white gene present on our constructs is expressed in the eye, it is possible that white sequences might act in conjunction with Mcp to increase the level of association of the constructs in the eye disk. It is also possible that the embryonic stages analyzed in the Fab-7 studies may represent the early stages in the establishment of this type of long-range interactions (Bantignies et al., 2003). In agreement with the work of these authors, however, we found no evidence of pairing of the Mcp element in the male or female germ line (Figure 2I; our unpublished data).
The eye color assay for long-distance interactions showed that insertions located on the same chromosome are much more likely to show genetic interaction (as evidenced by the stronger silencing of white). Our assay, however, revealed similar (and high) levels of association between sequences located on different chromosomes. These results are consistent with previous studies, where a substantial amount of residual pairing between alleles of the bithorax complex was still observed for translocations that abolished transvection (Gemkow et al., 1998). Our assay also revealed that the long-range association may involve at least up to four elements located at three different chromosomal loci. Although not tested in this study, such interactions are also likely to involve the endogenous Mcp elements (Bantignies et al., 2003). This raises the possibility that Mcp and similar elements may be involved in the formation of higher order chromatin complexes comprising multiple genes or regulatory regions.
Previous studies have identified a mutation in grappa, gpp1A, that substantially suppresses the pairing-dependent silencing of white mediated by Mcp (Müller et al., 1999). grappa encodes the Drosophila homologue of the yeast Histone H3 methyltransferase Dot1p. Our results clearly show that although gpp1A drastically reduces the level of pairing-dependent silencing mediated by Mcp, it has little or no effect on the observed pairing of Mcp elements in the eye imaginal disk. This suggests that pairing may be an initial necessary step in the regulatory process mediated by Mcp and that grappa acts subsequently to induce chromatin changes required for silencing. In the absence of additional data, however, other possibilities cannot be excluded. For example, the timing of pairing could be critical to allow developmentally regulated factors to associate to, and repress transcription around the Mcp element. In such a model, gpp1A could be delaying the onset of pairing, resulting in reduced levels of silencing. Additional studies will be necessary to establish the series of events that lead to pairing-dependent silencing of Mcp-associated genes.
The use of a live system has also allowed us, for the first time, to also address the dynamics of long-distance chromosome–chromosome interaction. Once established, the interactions seem to be stable, because we saw no evidence of separation of initially paired loci. Due to the finite resolution of the light microscope, this does not exclude local transient separation of short DNA regions. However, given that chromatin is naturally subject to diffusive motion, a complete separation of the paired regions, even for a brief moment, would be expected to lead to a drifting away of the tagged regions and the appearance of two separate GFP spots (Vazquez et al., 2001). Rare, unpaired loci were also never seen to associate. The presence of a small fraction of nuclei with unpaired loci at any given time therefore does not seem to be the result of an equilibrium state between a population of rapidly associating and dissociating loci. Therefore, Mcp elements, possibly by the action of specific chromosome-associated proteins, are able to lock remote chromosomal regions in the paired state for extended periods, even in the presence of substantial chromatin movement. The stable contacts we describe are in contrast with the short-lived dynamic interactions that have been postulated to occur between remote regulatory elements, such as between the human betaglobin LCR and promoter regions (Wijgerde et al., 1995). Our studies suggest that the rate-limiting step in the pairing process could be the establishment of the initial contact between remote Mcp elements early during development and possibly renewed early at the beginning of each new cell cycle. This situation is reminiscent of the rapid and stable pairing of homologous chromosomes observed in somatic cells (Fung et al., 1998) and of meiotic pairing in Drosophila spermatocytes (Vazquez et al., 2002). This interpretation is consistent with the hypothesis that interactions between Polycomb-group response elements might be involved in the transmission of chromatin states during Drosophila development (Bantignies et al., 2003).
We have presented a live system for the direct analysis of long-distance chromosome interactions in Drosophila. This system allowed us to identify a discrete DNA sequence from the bithorax complex, Mcp, that is able to promote stable physical interactions between distant chromosomal regions. The presence of pairing elements at the bithorax complex had long been suspected, due to the susceptibility of this locus to transvection effects. Although the pairing properties of Mcp (and Fab-7) were originally inferred from the ability of this element to silence a linked white gene in a pairing-dependent manner, it is not clear at the moment what function pairing serves in the context of the bithorax complex. It has been proposed that association between these elements might play a role in the transmission of regulatory chromatin states (Bantignies et al., 2003). It is also possible that pairing elements might play a role in bringing together remote regulatory regions or stabilize regulatory interactions within the complex. The ability to track such associations both in live and fixed tissues should help clarify the relationship between chromosome organization and gene regulation.
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Footnotes |
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Abbreviations used: PRE, polycomb response element.
The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org). 
These authors contributed equally to this work.
Address correspondence to: Julio Vazquez (jvazquez{at}fhcrc.org).
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