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Visualization of chromatin domains created by the gypsy insulator of Drosophila

Visualization of chromatin domains created by the gypsy insulator of Drosophila JCB Article Visualization of chromatin domains created by the gypsy insulator of Drosophila Keith Byrd and Victor G. Corces Department of Biology, Johns Hopkins University, Baltimore, MD 21218 nsulators might regulate gene expression by establishing proteins, subjecting the cells to a brief heat shock, or de- and maintaining the organization of the chromatin fiber struction of the nuclear matrix lead to disruption of the within the nucleus. Biochemical fractionation and in loop. Insertion of an additional gypsy insulator in the situ high salt extraction of lysed cells show that two known center of the loop results in the formation of paired loops protein components of the gypsy insulator are present in through the attachment of the inserted sequences to the the nuclear matrix. Using FISH with DNA probes located nuclear matrix. These results suggest that the gypsy insulator between two endogenous Su(Hw) binding sites, we show might establish higher-order domains of chromatin structure that the intervening DNA is arranged in a loop, with the and regulate nuclear organization by tethering the DNA to two insulators located at the base. Mutations in insulator the nuclear matrix and creating chromatin loops. Introduction Multicellular organisms use complex mechanisms to ensure 2001; Gerasimova and Corces, 2001). The gypsy insulator is proper spatial and temporal expression of genes. Packaging a 350-bp sequence that requires at least two proteins for of the DNA with histones to form chromatin prevents function: Su(Hw), a zinc finger DNA binding protein, and improper gene activation by restricting accessibility of tran- Mod(mdg4), a BTB domain protein that binds Su(Hw) and scription factors to the regulatory sequences of the gene. can associate with itself (Gerasimova et al., 1995; Gause et Current research has focused on the effects of histone modi- al., 2001; Ghosh et al., 2001). Although the gypsy insulator fication in promoting or preventing transcription (Jenuwein was originally identified in the gypsy retrotransposon, the and Allis, 2001; Berger, 2002). However, much less is Drosophila genome contains 500 binding sites for the known about the three-dimensional arrangement of DNA in Su(Hw) and Mod(mdg4) proteins that are presumed to be the nucleus and the role this organization plays in gene regu- endogenous insulators; out of the multiple isoforms encoded lation. There is some evidence suggesting that chromatin is by the mod(mdg4) gene, only the Mod(mdg4)2.2 protein packaged into 50–200-kb loops attached to a nuclear matrix appears to be present at the gypsy insulator (Mongelard et al., by sequences called matrix attachment regions (MARs) or 2002). We will refer to the insulator present in the gypsy scaffold attachment regions (SARs) (Kaufmann et al., 1986; retrotransposon as the gypsy insulator, whereas we will use Gasser et al., 1989; Laemmli et al., 1992; Pederson, 1998). “endogenous Su(Hw) binding sites” to designate genomic Other results suggest that insulators might similarly be binding sites for the Su(Hw) protein that do not contain involved in nuclear organization by bringing the chroma- copies of the gypsy retrotransposon but might act as insula- tin fiber to a perinuclear compartment (Gerasimova and tors, although this property has not yet been demonstrated Corces, 1998; Gerasimova et al., 2000; Ishii et al., 2002). experimentally. We have previously shown that Su(Hw) and Insulators are characterized by two properties consistent Mod(mdg4)2.2 colocalize in large foci, named insulator with their potential ability to create these loop domains: (1) bodies, located mostly at the nuclear periphery of diploid they prevent an enhancer from activating a promoter when cells. These insulator bodies are presumed to be formed by located between the two, and (2) when flanking a transgene, multiple individual insulator sites coming together at re- they prevent the local chromatin environment around the stricted nuclear locations. This hypothesis is supported by integration site from affecting its expression (Bell et al., observations indicating that the presence of the gypsy insulator at two distant chromosomal sites causes the DNA containing Address correspondence to Victor G. Corces, Department of Biology, Johns Hopkins University, 3400 N. Charles St., Baltimore, MD 21218. Abbreviations used in this paper: ct, cut; MAR, matrix attachment region; Tel.: (410) 516-8749. Fax: (410) 516-5456. email: [email protected] NPC, nuclear pore complex; SAR, scaffold attachment region; SCS, Key words: insulator; chromatin; transcription; nucleus; retrotransposon specialized chromatin structures; Ubx, Ultrabithorax.  The Rockefeller University Press, 0021-9525/2003/08/565/10 $8.00 The Journal of Cell Biology, Volume 162, Number 4, August 18, 2003 565–574 http://www.jcb.org/cgi/doi/10.1083/jcb.200305013 565 The Journal of Cell Biology 566 The Journal of Cell Biology | Volume 162, Number 4, 2003 these gypsy sequences to come together at the nuclear periph- ery (Gerasimova et al., 2000). If insulator bodies bring together individual insulator sites, the intervening DNA should form a loop. These loops might then represent func- tionally separate chromatin domains that allow independent regulation of transcription within each domain. Indirect molecular evidence for the possibility that the for- mation of chromatin loops might serve as a basis for insula- tor function comes from results obtained during a screen for proteins with boundary function in yeast (Ishii et al., 2002). Proteins involved in nucleo-cytoplasmic transport or com- ponents of the nuclear pore complex (NPC) were found to be able to buffer a flanked reporter gene from heterochro- Figure 1. Protein components of the gypsy insulator are present matic silencing effects. These results suggest that boundary in the nuclear matrix. A collection of Drosophila 6–18-h-old activity can be accomplished by attachment of both sides of embryos was subjected to a karyoskeletal or nuclear matrix fraction- the insulated DNA to a solid substrate, in this case the NPC, ation procedure, and equal amounts of the fractions were run on 7.5 and 10% polyacrylamide gels and subjected to Western blot in a perinuclear compartment. Although not directly shown analysis using antibodies to Mod(mdg4), Su(Hw), lamin, histone H3, in this work, attachment is likely to result in the formation and Ubx. Antibodies against Mod(mdg4) were specific to the of a small loop with its base at the NPC, and this loop might Mod(mdg4) 2.2 isoform, which is the isoform present in the gypsy be functionally similar to the ones hypothesized to mediate insulator (Ghosh et al., 2001; Mongelard et al., 2002). gypsy insulator activity. In spite of the widespread discussion of the loop domain model in the context of insulator function, neither the exist- found using a different nuclear matrix isolation protocol ence of these loops nor the basis for their location at the nu- that involves precipitation with ammonium sulfate (unpub- clear periphery has been demonstrated directly. Using FISH lished data). These results suggest that the gypsy insulator as- analysis of high salt–extracted nuclei, we show here that sociates with the nuclear matrix and provides a biochemical DNA sequences located between two insulators form a loop foundation for the observed formation of insulator bodies in in the interphase nucleus. The formation of this loop is de- the nuclear periphery. pendent on functional insulator proteins and an intact nu- clear matrix. The results suggest that insulators might regu- An intact nuclear matrix is required for the formation late nuclear organization by controlling the formation of of gypsy insulator bodies higher-order domains of chromatin structure. To further explore the association of gypsy insulator proteins with the nuclear matrix under more physiological condi- tions, we used antibodies against Su(Hw) and Mod(mdg4) Results 2.2 for immunofluorescence studies on specially pre- Gypsy insulator proteins are present in the nuclear pared nuclear matrices. Using a modified form of a proce- matrix fraction dure described by Gerdes et al. (1994) to isolate nuclear The perinuclear localization of the gypsy insulator bodies matrices for cytological analysis, we spun detergent-treated suggests an association of the insulator proteins with a fixed imaginal disk cells from third instar Drosophila larvae onto substrate such as the nuclear matrix. The nuclear matrix is coverslips and then extracted the nuclei with 2 M NaCl. An defined biochemically as the fraction remaining after extrac- extracted nucleus appears as a darkly stained nuclear matrix– tion of nuclei with 2 M NaCl (Pederson, 1998; Nickerson, containing core surrounded by a lightly stained DNA halo 2001). To biochemically confirm the association of Su(Hw) (for schematic see Fig. 2 A). The black and white images of and Mod(mdg4)2.2 proteins with the nuclear matrix, we nuclei stained with DAPI more clearly show the halo forma- isolated a karyoskeletal or nuclear matrix fraction (7% of tion around the intensely stained residual nuclear matrix nuclear proteins) from Drosophila embryos ranging in age (Fig. 2, F and J). Based on images of extracted nuclei ob- from 6 to 18 h post–egg laying. Fig. 1 shows the results of tained with the electron microscope (McCready et al., Western analyses of different protein fractions. Lamin, as ex- 1979), the light blue halo is composed of histone-free DNA pected, is predominantly located in the nuclear matrix frac- loops associated at the base to the more intensely stained nu- tion. In contrast, histones are extracted in previous steps and clear matrix that contains DNA, RNA, ribonucleoproteins, are not present in this fraction (Oegema et al., 1997; Ma et and other proteins resistant to high salt extraction. Along al., 1999). Both gypsy insulator components, Su(Hw) and with histones, 95% of nuclear proteins are extracted with Mod(mdg4)2.2, copurify almost entirely with the nuclear this technique; however, lamin and other known matrix- matrix fraction (Fig. 1). As a control, we tested whether the associated proteins remain (Fey et al., 1986; Ma et al., 1999). Ultrabithorax (Ubx) transcription factor is also present in We then performed immunofluorescence light microscopy the nuclear matrix fraction; the Ubx protein is extracted on nuclei that were either untreated or extracted with 2 M from nuclei under the same conditions as histones and, NaCl using antibodies to Mod(mdg4)2.2 and lamin. As ex- therefore, is not associated with the nuclear matrix (Fig. 1). pected from previous results with paraformaldehyde-fixed The same association of Su(Hw) and Mod(mdg4)2.2 was nuclei (Gerasimova et al., 2000), Mod(mdg4)2.2 is present The Journal of Cell Biology Insulators and chromatin domains | Byrd and Corces 567 Figure 2. Distribution of Su(Hw) and Mod(mdg4) insulator proteins after a 2 M NaCl extraction of nuclei. Imaginal disk cells from wild-type larvae were spun onto coverslips and either extracted with 2 M NaCl or left untreated, and then they were stained with antibodies to Su(Hw), Mod(mdg4), or lamin; in addition, the DNA was stained with DAPI (blue). (A) Schematic representation of a nucleus before and after extraction with 2 M NaCl. The red depicts the nuclear lamin and the blue indicates DNA. Treatment of these cells with 2 M NaCl removes histones and extracts 95% of nuclear proteins, leaving the DNA in large 50–200-kb loops attached to the residual nuclear matrix (McCready et al., 1979; Ma et al., 1999). B, F, J, and N are black and white images of DNA, corresponding to the panels on their right, visualized by DAPI staining. This staining is shown in blue in the rest of the panels. (C–E) Distribution of lamin (red) and Mod(mdg4)2.2 (green) in untreated nuclei. (G–I) Distribution of lamin (red) and Mod(mdg4)2.2 (green) in 2 M NaCl- extracted nuclei. (K–M) Distribution of Su(Hw) (red) and Mod(mdg4)2.2 (green) in 2 M NaCl-extracted nuclei. (O–Q) Distri- bution of Su(Hw) (red) and Mod(mdg4)2.2 (green) in nuclei extracted with 2 M NaCl followed by treatment with RNase A. in insulator bodies formed by the aggregation of multiple in- treated 2 M NaCl-extracted nuclei from wild-type larvae dividual insulator sites in intact nuclei (Fig. 2, B–E). To with RNase A. Under these conditions, the nuclear matrix confirm that the insulator bodies are associated with the nu- appears fragmented and disorganized. In addition, the insu- clear matrix, we stained nuclei extracted with 2 M NaCl lator bodies are destroyed, as determined by the localization with antibodies to lamin and Mod(mdg4)2.2 (Fig. 2, F–I). of Su(Hw) and Mod(mdg4)2.2 proteins (Fig. 2, N–Q). This The Mod(mdg4)2.2 protein, which marks the localization result strongly suggests that the formation of insulator bod- of gypsy insulator bodies, remains associated with the lamin- ies requires the existence of an intact nuclear matrix. containing nuclear matrix core. We then extracted nuclei Chromatin loops form between two insulator sites with 2 M NaCl and stained them with antibodies to both insulator proteins, Su(Hw) and Mod(mdg4)2.2, as well as If insulator bodies form by attaching multiple individual in- DAPI to visualize the DNA (Fig. 2, J–M). Extraction with sulator sites to the nuclear matrix at specific nuclear loca- such high salt concentrations that remove 95% of nuclear tions, they might help organize the chromatin fiber into proteins did not disrupt the interaction between the two in- loops representing domains of higher-order organization. sulator proteins or their interaction with the nuclear matrix. The formation of these loops should be testable using the It has been previously shown that the nuclear matrix, al- nuclear halo technique and FISH with DNA probes span- though not composed of one repeating protein unit, is com- ning the region contained within the loop. As a loop should posed of ribonucleoprotein complexes and, thus, is suscepti- form by the DNA located between two individual insulator ble to destruction by RNase (He et al., 1990; Ma et al., sites, we used immunostaining with Su(Hw) antibodies to 1999). To test whether disruption of nuclear matrix integ- map the location of individual endogenous gypsy insulators rity would affect the formation of gypsy insulator bodies, we on polytene chromosomes of third instar larvae. We then The Journal of Cell Biology 568 The Journal of Cell Biology | Volume 162, Number 4, 2003 Figure 3. Localization of DNA probes A, B, and C and Su(Hw) protein on polytene chromosomes. BAC clones A, B, or C were used as DNA probes for FISH of polytene chromosomes from salivary glands of Drosophila third instar larvae from wild-type or ct mutant strains. The chromosomes were simul- taneously stained with antibodies against the Su(Hw) protein. For all images, the DNA is visualized by DAPI staining (blue), and the locations of endogenous Su(Hw) binding sites at 7B2 and 7B8, indicated by Su(Hw) staining (red), are labeled with arrows. (A) Schematic representation of probes A, B, and C at the ct locus. The ct mutant contains a copy of the gypsy retrotransposon located in the region recognized by probe B. The location of the gypsy retrotransposon and overlap of probes A, B, and C are drawn to scale. (B–D) Immunolocalization of Su(Hw) (red) and FISH signal of probe A in polytene chromosomes from wild- type larvae. (E–G) Immunolocalization of Su(Hw) (red) and FISH signal of probe B (green) in polytene chromosomes from wild-type larvae. (H–J) Immunolocaliza- tion of Su(Hw) (red) and FISH signal of probe C (green) in polytene chromo- somes from wild-type larvae. (K–M) Immunolocalization of Su(Hw) (red) and FISH signal of probe B in polytene chromosomes from ct larvae. concentrated our analysis on a region of the X chromosome clone B (Fig. 3 A). As the gypsy retrotransposon contains the containing the cut (ct) locus flanked by two endogenous gypsy insulator, one would expect to detect a new site of Su(Hw) binding sites located at 7B2 and 7B8. This region is Su(Hw) staining in polytene chromosomes prepared from spanned by three different BAC clones, designated as probes third instar larvae carrying the ct mutation. This is indeed A, B, and C. Clone A is 154 kb in length, whereas clones B the case, as seen in Fig. 3 (K–M). The gypsy insulator present and C are 175 kb and 183 kb, respectively. The DNA se- in the gypsy retrotransposon contains 12 binding sites for the quence for these three clones has been determined by the Su(Hw) protein, giving rise to a strong immunofluorescence Drosophila Genome Project, and their location with respect signal on polytene chromosomes at chromosomal subdivi- to each other and the ct gene is diagrammed in Fig. 3 A. sion 7B4. This signal partially obscures the endogenous Clones A and B overlap by 28 kb, whereas clones B and C Su(Hw) binding sites located at 7B2 and 7B8. In addition, overlap by 16 kb. Using FISH analysis with these three analysis of DAPI-stained chromosomes in this region sug- probes and simultaneous immunolocalization with Su(Hw) gests that the presence of gypsy induces a condensation of the antibodies, we were able to determine the relative location of local chromatin (unpublished data), further decreasing the each BAC clone with respect to the location of the endoge- resolution between the Su(Hw) signals at 7B2, 7B4, and nous Su(Hw) binding sites surrounding the ct locus. Fig. 3 7B8; as a consequence, all three signals overlap in one band. D shows that probe A is located adjacent and partially over- Probe B overlaps with the gypsy retrotransposon insulator in lapping the Su(Hw) binding site present at chromosomal polytene chromosomes from ct mutant larvae (Fig. 3 M), subdivision 7B2. Probe B is located between the 7B2 and but does not overlap with the two endogenous Su(Hw) 7B8 Su(Hw) binding sites (Fig. 3 G), whereas probe C par- binding sites in wild-type animals (Fig. 3 G). Based on the tially overlaps the 7B8 site (Fig. 3 J). The ct allele is caused location of the two endogenous Su(Hw) binding sites and by the insertion of a copy of the gypsy retrotransposon in the the three BAC clones on polytene chromosomes, we expect 5 region of the ct gene (Jack, 1985). This insertion site is lo- that, if these sequences form a loop in interphase nuclei of cated 65 kb to the right of the telomere-proximal end of diploid cells as a consequence of the two Su(Hw) binding The Journal of Cell Biology Insulators and chromatin domains | Byrd and Corces 569 Figure 4. Effect of the gypsy insulator on the distribution of DNA in 2 M NaCl- extracted nuclei from male larvae. Various combinations of DNA probes A, B, and C from chromosomal subdivision 7B (see Fig. 3) were hybridized to imaginal disk cells that were spun onto coverslips and either extracted with 2 M NaCl or left untreated. Probes A and C are in green and probe B is in red. A, F, K, P, and U are black and white images of DNA visualized by DAPI staining. This staining is shown in blue in the rest of the panels. The red depicts probe B, and the green depicts probes A and C in panels G–I and probe A in the rest. E, J, O, T and Y show schematic representations of the results found to their left. (A–D) Probes A (green) and B (red) in an un- treated wild-type nucleus. (F–I) Probes A (green), B (red), and C (green) in a 2 M NaCl-extracted wild-type nucleus. (K–N) Probes A (green) and B (red) in a 2 M NaCl-extracted nucleus from larvae carrying the ct mutation. (P–S) Probes A (green) and B (red) in a 2 M NaCl- extracted nucleus from larvae of the 6 V genotype ct ; su(Hw) . (U–X) Probes A (green) and B (red) in a 2 M NaCl- extracted nucleus also treated with RNase A. sites coming together and attaching to the nuclear matrix, the nuclear matrix (Fig. 4 H). The DNA complementary to the DNA present in clones A and C would form the two the other ends of probes A and C, overlapping probe B and stems of the loop. The loop would be attached at the base of lacking endogenous Su(Hw) binding sites, is present in the these probes, where the endogenous Su(Hw) binding sites nuclear halo region partially colocalizing with the small reside, to the nuclear matrix at one location. In contrast, the overlapping regions at the ends of probe B (Fig. 4 I). To de- DNA spanning clone B would be present in the central dis- termine the statistical significance of these results, we mea- tal region of the loop, not attached to the nuclear matrix, be- sured the number of nuclei in which probes A and C form a cause the DNA in this region does not contain an insulator V structure with the vertex located in the darkly stained core in wild-type larvae. This hypothesis rests on the assumption region. We scored the V structure based on the appearance that, as polytene salivary gland cells are in interphase, the lo- of two linear FISH signals meeting at one location in the nu- cation of Su(Hw) and Mod(mdg4)2.2 proteins at endoge- clear matrix. 86% of wild-type nuclei show the characteristic nous Su(Hw) binding sites would be maintained in diploid V structure formed by probes A and C. The other 14% had cells of larval imaginal disks. either nonlinear signal(s), signal(s) that did not have a clear To test this hypothesis, we performed FISH on untreated matrix association, and/or signals that did not meet to form and 2 M NaCl-extracted imaginal disk nuclei spun onto a vertex. Differences are significant (P  0.001, chi-squared coverslips. As the ct locus is located in the X chromosome, test) when the presence of the V structure in wild type is 6 V we initially restricted our analysis to nuclei of cells from compared with ct ; su(Hw) , a null for Su(Hw) protein (Ta- imaginal disks of male larvae. We first probed untreated ble I). In imaginal disk cells from su(Hw) mutant male lar- wild-type nuclei with probes A and B and found, as ex- vae, only 16% of nuclei show the presence of a V structure pected, that they colocalize specifically in one region of the formed by probes A and C. This lack of matrix association nucleus (Fig. 4, A–D). We then performed FISH with of probes A and C in nuclei that lack Su(Hw) indicates that probes A, B, and C on nuclei extracted with 2 M NaCl from a functional Su(Hw) protein is required for DNA attach- imaginal disk cells of wild-type male larvae. Probes A and C ment to the nuclear matrix at that location. frequently appear attached to the nuclear matrix at the same Insertion of a new gypsy insulator in the center of a location at one of their ends, presumably through the puta- tive endogenous Su(Hw) binding sites located at the outer loop results in the formation of two smaller loops boundaries of probes A and C (Fig. 4 G). In contrast, we To further test the idea that the observed chromatin loops found that DNA sequences complementary to probe B are are formed between gypsy insulator sites, we then per- usually located in the halo region and do not associate with formed similar FISH experiments on nuclei that were ex- The Journal of Cell Biology 570 The Journal of Cell Biology | Volume 162, Number 4, 2003 Table I. Statistical analysis of FISH data presented in Fig. 4 Genotype No. of nuclei Probes A and C in V structure No. of nuclei Probe B in V structure %% Wild-type males 96 86 127 16 ct males 94 81 189 78 6 V ct ; su(Hw) males 83 16 112 19 Wild-type males, RNase treated 28 0 28 3 ct heat-shocked males ND ND 80 25 Wild-type females ND ND 79 18 ct females ND ND 95 80 Nuclei were scored blindly by two researchers independently, based on the presence or absence of a V structure in the DNA complementary to probe B, indicating an attachment to the nuclear matrix. Nuclei were also scored based on the presence of a V structure complementary to probes A and C with the vertex located in the nuclear matrix core. tracted with 2 M NaCl from male larvae carrying the ct To ensure that the formation of an additional loop was mutation, known to contain an additional gypsy insulator due to the presence of a new functional gypsy insulator, we in the 7B4 region. In these nuclei, the DNA hybridizing examined 2 M NaCl-extracted nuclei from male larvae of 6 V to probe B acquired a V shape, with the vertex in the the genotype ct ; su(Hw) , which carry the gypsy retrotrans- darkly stained central core, suggesting that this region of poson in the ct locus but lack Su(Hw) protein. Nuclei from the chromosome is now attached to the nuclear matrix these larvae failed to show DNA structures with the charac- through the new insulator present at the site of insertion teristic V shape in the region complementary to probe B of the gypsy retrotransposon in the ct allele (Fig. 4 M). (Fig. 4 R). The number of nuclei showing a V structure, Furthermore, the shape of this V structure is asymmetric, scored as described above, is 19%, which is significantly dif- with one side of the V longer than the other, as would be ferent (P  0.001, chi-squared test) when compared with expected given the asymmetric location of the gypsy ele- nuclei carrying the ct mutation but wild type for su(Hw) ment in the ct locus within the region covered by probe B (Table I). The formation of a V structure by probes A and C 6 V (Fig. 3 A). We scored the formation of a V structure by is also disrupted in nuclei from ct ; su(Hw) mutant male the DNA complementary to probe B based on the appear- larvae. In these nuclei, the DNA complementary to probes A ance of a continuous linear FISH signal attached to the and C only occasionally appears as a straight line going from nuclear matrix at one location. Probe B acquires a V shape the central region toward the surrounding halo; instead, the in 78% of nuclei from ct male larvae. Differences are sig- DNA appears disorganized in the nuclear halo region sur- nificant (P  0.001, chi-squared test) when we compare rounding the residual nuclear matrix core (Fig. 4, Q–S). The ct nuclei with wild-type nuclei in which such a structure number of times the V structure was observed in nuclei from 6 V is only observed in 16% of nuclei (Table I). In many ct ; su(Hw) larvae is significantly lower (P  0.001, chi- cases, it was possible to observe the position of the proxi- squared test) than wild type (Table I). In the absence of mal region of probe A overlapping with the distal end of Su(Hw) protein, both the insulator present in the gypsy ret- probe B (Fig. 4 N). This allowed us to conclude that the rotransposon in the ct locus as well as the putative insulators location of the vertex of the V approximately maps to the present at endogenous Su(Hw) binding sites are not func- region where the gypsy retrotransposon is inserted in the ct tional. The disorganized appearance of the DNA in nuclei 6 6 V allele. The 22% of nuclei from ct larvae not scored as hav- from ct ; su(Hw) male larvae implies that the lack of ing a V structure had either a nonlinear signal and/or a Su(Hw) protein results in the absence of loop structures, signal that did not have a clear matrix association. The probably because of a failure to form functional insulators, state of the cell cycle might explain the lack of V forma- suggesting that the insulators serve as attachment points of tion in those cells, as late G2 and mitotic cells do not have the DNA to the nuclear matrix. Similar experiments were insulator bodies. In contrast, a possible explanation for the also performed with nuclei from male larvae of the genotype 6 u1 V formation of probe B observed in 16% of nuclei from ct ; mod(mdg4) . The results were comparable to those ob- 6 V wild-type larvae could be the presence of replicating or tained with the ct ; su(Hw) (unpublished data), suggesting transcribing DNA in the region complementary to probe that both proteins, Su(Hw) and Mod(mdg4)2.2, are re- B associated with the nuclear matrix at the time of the 2 M quired for the formation of a functional insulator capable of NaCl extraction. This interpretation is supported by attaching the DNA to the nuclear matrix. findings suggesting that actively transcribing and replicat- ing sequences associate with the nuclear matrix (Gerdes et Loop formation requires an intact nuclear matrix al., 1994). These results suggest that the presence of a new As these results suggest the requirement of the nuclear ma- gypsy insulator in the 7B4 region between the two endoge- trix for the formation of DNA loops in the nucleus, we then nous Su(Hw) binding sites located at 7B2 and 7B8 causes examined the effect of destroying the nuclear matrix, with the original loop formed between 7B2 and 7B8 to become RNase, on the organization of the DNA flanked by endoge- divided into two smaller loops, due to the attachment of nous Su(Hw) binding sites. Nuclei from wild-type male lar- the new insulator at 7B4 to the nuclear matrix (Fig. 4 N). vae were extracted with 2 M NaCl, treated with RNase A, The Journal of Cell Biology Insulators and chromatin domains | Byrd and Corces 571 Figure 5. Insulator-mediated loop organization after heat shock and in nuclei from female larvae. (A–D) Immuno- fluorescence analysis of 2 M NaCl- extracted nuclei from imaginal disk cells of ct male larvae subjected to a 30-min heat shock at 37C using antibodies against Su(Hw) and Mod(mdg4)2.2. The Su(Hw) protein is shown in red and the Mod(mdg4)2.2 protein in green. DNA was stained with DAPI and is shown in A; DAPI staining is shown in blue in B–D. The rest of the panels in the figure show FISH analysis of 2 M NaCl- extracted nuclei from imaginal disk cells using probes A (green) and B (red); in all panels, DAPI staining is shown in gray (E, I, and M) or in blue. (E–H) Nuclei from ct male cells subjected to a 30-min heat shock at 37C. (I–L) Nuclei from imaginal disk cells of wild-type female larvae. (M–P) Nuclei from imaginal disk cells of ct female larvae. and then labeled with probes A, B, and C. The DNA com- of insulator bodies observed in cells grown at normal tem- plementary to probes A and C fails to form the characteristic perature (Fig. 3) is dramatically affected by heat shock. Nu- V structure with the vertex attached to the nuclear matrix clei from heat-shocked cells do not show large insulator (Fig. 4, compare V with G). The frequency of the V struc- bodies; instead, the Su(Hw) and Mod(mdg4)2.2 proteins ture in the RNase-treated nuclei is significantly lower (P  are distributed throughout the nucleus and do not appear 0.001, chi-squared test) when compared with wild-type nu- to be located in the nuclear periphery (Fig. 5, A–D). This clei that were not RNase treated using probes A and C or to result suggests that heat shock interferes with the ability ct nuclei using probe B (Table I). This result suggests that of individual Su(Hw) binding sites to come together at the nuclear matrix is important for the attachment of the specific nuclear locations to form insulator bodies. If this DNA and the establishment of the loop structures. is the case, heat shock should also interfere with the for- The formation of DNA loops should require not only mation of DNA loops, which are presumed to be caused attachment to the nuclear matrix, but also interactions by interactions between Su(Hw) binding sites. To test between individual Su(Hw) binding sites to form insula- this prediction, we performed in situ hybridizations with tor bodies. We have previously shown that the heat shock probes A and B to 2 M NaCl-extracted nuclei from ct response causes gypsy insulator bodies to fall apart, as a male larvae. As we have described above, nuclei from brief increase in temperature to 37C results in the disap- these larvae should show the formation of an asymmetric pearance of the typical punctate pattern of Su(Hw) and V structure by DNA homologous to probe B (Fig. 4, L–N). Mod(mdg4) observed in imaginal disk cells (Gerasimova Instead, in most nuclei from cells subjected to heat shock, et al., 2000). Under these conditions, the insulator bodies the DNA homologous to probe B appears disorganized disappear, and, instead, Su(Hw) and Mod(mdg4) pro- and located in the halo region of the extracted nuclei, teins are diffusely spread throughout the nucleus; this is away from the darkly stained core (Fig. 5, E–H). The accompanied by changes in the subnuclear localization of number of times the V structure was observed in nuclei insulator-containing sequences, suggesting a dissociation from heat-shocked ct male larvae is significantly lower of the chromatin loops (Gerasimova et al., 2000). To (P  0.005, chi-squared test) than in non–heat-shocked confirm the effect of heat shock on insulator body struc- animals (Table I). These results suggest that interactions ture and distribution under the assay conditions used in between Su(Hw) binding sites, which are disrupted by the experiments described above, we performed immu- heat shock, are normally required for the formation of the nolocalization experiments using Su(Hw) and Mod(mdg4) V structure and, therefore, for the formation of a loop 2.2 antibodies on 2 M NaCl-extracted nuclei from imagi- mediated by the gypsy insulator present in the ct locus of 6 6 nal disk cells of ct male larvae. The typical distribution ct flies. The Journal of Cell Biology 572 The Journal of Cell Biology | Volume 162, Number 4, 2003 Su(Hw)-mediated loop organization in nuclei nuclear matrix. In our experiments, we have used two differ- ent biochemical nuclear matrix purification procedures. In from female larvae both cases, it is clear that the Su(Hw) and Mod(mdg4)2.2 The results described so far were obtained in experiments proteins associate with the nuclear matrix fraction, whereas performed with X-linked DNA probes and cells from male other proteins, such as histones and Ubx, are extracted by larvae, which have only one X chromosome. In Drosophila, high salt. We have also used an in situ cell extraction proce- both the homologous autosomes and the two X chromo- dure followed by visualization of the extracted nuclei using somes in females are paired during interphase (Hiraoka et light microscopy as a means of confirming the association of al., 1993; Fung et al., 1998). The ability of the two homolo- insulator proteins with the nuclear matrix. Using this ap- gous chromosomes to pair is responsible for the phenome- proach, gypsy insulator proteins also appear to be associated non of transvection, in which two different mutant alleles of with the nuclear residue that is resistant to salt extraction. the same gene can complement each other, resulting in a Whether this resistant fraction is a filamentous network of pairing-dependent wild-type phenotype (Lewis, 1954; Heni- defined composition or a matrix formed by interactions koff, 1997). The nature of the factors responsible for chro- among different proteins and nucleic acids is not known, mosome pairing during interphase is not known. It is possi- but it is clear that insulator proteins are not extractable by ble that, if insulator proteins contribute to the organization 2 M NaCl and are not present in the chromosomal regions of the interphase chromatin fiber into loop domains, the that extrude from the nucleus after high salt extraction. The same proteins might also contribute to homologous chro- interaction of Su(Hw) and Mod(mdg4)2.2 with nuclear ma- mosome pairing. To test this possibility, we determined trix components supports previous observations indicating whether DNA sequences surrounding the ct locus are that gypsy insulator proteins are preferentially present in the present in one or two loops in nuclei obtained from female nuclear periphery of the interphase nucleus (Gerasimova et larvae. Probes A and B were used for in situ hybridization on al., 2000). 2 M NaCl-extracted nuclei from wild-type imaginal disk Although the insulator DNA and associated proteins re- cells. As in the case of male nuclei, DNA homologous to main in the nuclear matrix fraction, the intervening DNA is probe A appears arranged in a straight line, with one end lo- extruded from the nucleus by high salt extraction and is cated in the darkly stained core and the other end pointing found in the form of a large loop. The DNA contained toward the surrounding halo region (Fig. 5, I–L). Interest- within this loop appears only as a small dot after FISH anal- ingly, only one such hybridization signal is observed per nu- ysis of unextracted nuclei. These two observations suggest cleus, suggesting that the two homologous chromosomes re- that the chromatin fiber present in the loop formed by two main paired after 2 M NaCl treatment and that the proteins insulators is not completely decondensed during interphase involved in chromosome pairing are resistant to high salt ex- and only becomes extended after extraction of histones and traction. As in male cells, sequences homologous to probe B other associated proteins. This suggests that the loop formed are located in the halo region (Fig. 5, I–L). We then under- by two insulators might represent a domain of higher-order took the same type of analysis using nuclei from female lar- chromatin structure. This higher-order structure might be vae carrying the ct mutation. Again, only one signal homol- established and/or maintained by specific covalent modifica- ogous to probe A was observed; in these nuclei, probe B tions of histone tails. For example, it has been found that the gives rise to a single hybridization signal displaying an asym- chicken -globin locus, which is flanked by two CTC-bind- metric V structure (Fig. 5, M–P). Probe B acquires a V ing factor insulators, contains histones H3 and H4, acety- shape in 80% of nuclei from ct female larvae. Differences lated in various lysine residues (Litt et al., 2001; Mutskov et are significant (P  0.005, chi-squared test) when we com- al., 2002). Covalent modification of histone tails might pare ct nuclei with wild-type nuclei in which such a struc- modulate internucleosome interactions, which, in turn, de- ture is only observed in 18% of nuclei (Table I). The obser- termine the degree of higher-order chromatin structure (Tse vation of only one V structure suggests that the two new and Hansen, 1997). loops formed as a consequence of the presence of the gypsy The existence of chromatin insulator-induced domains retrotransposon in the ct locus from each chromosome ho- would explain their unusual gene regulatory property of pre- mologue are still paired after high salt extraction. These re- venting an enhancer from activating a promoter in a differ- sults again support a conclusion suggesting that insulator ent domain while not preventing the same enhancer from proteins and/or other proteins present in the nuclear matrix activating a promoter located in its own domain. The ability are involved in maintaining homologous chromosome pair- of the insulator, when flanking a transgene, to provide posi- ing during interphase. tion-independent expression of the transgene is also consis- tent with the formation of a loop domain. This domain ap- Discussion pears to be created by the interaction of flanking insulators with each other and the nuclear matrix. In fact, recent exper- We have presented evidence suggesting that the gypsy insula- iments by Ishii et al. (2002) have shown that boundary func- tor creates chromatin loop domains via association to the tion in yeast can be elicited by tethering boundary-associated nuclear matrix. The existence and exact composition of the proteins to the NPC. This tethering would presumably re- nuclear matrix has been a subject of intense debate (Nicker- sult in the formation of a loop, similar to the ones we ob- son, 2001). Lamin, which is the main component of the nu- serve here, by the DNA located between the two boundary clear lamina, is present in the nuclear matrix fraction. It is elements, which would attach the base of the loop to the possible that protein components of the gypsy insulator inter- NPC. In the case of Drosophila, the requirement for interac- act with the nuclear lamina or with other components of the The Journal of Cell Biology Insulators and chromatin domains | Byrd and Corces 573 tions between individual Su(Hw) binding sites for the for- been shown to depend on insulator function, the existence mation of the loops is underscored by the observation that a of these transcriptional domains could have a structural basis brief heat shock interferes both with the formation of insula- in the formation of chromatin loops in which gene expres- tor bodies and with the ability of the gypsy retrotransposon sion is globally regulated. to form a new loop when inserted in the ct locus. The organization of the chromatin fiber into loops has also been shown for the Drosophila specialized chromatin Materials and methods structures (scs) and scs’ boundary sequences. The proteins Drosophila strains that interact with these elements have been shown to inter- Fly stocks were maintained in standard medium and grown at 22.5C and act with each other both in vitro and in vivo. Consistent allele is caused by a small deletion 75% relative humidity. The su(Hw) with the idea that interaction between the two proteins that also affects the RpII15 gene, resulting in embryonic lethality. The strain used in these studies carries a transgene containing the RpII15 su(Hw) might facilitate pairing of boundary elements and formation u1 al- gene that rescues this lethality (Harrison et al., 1992). The mod(mdg4) of chromatin loops, sequences corresponding to the scs and lele has been described previously (Gerasimova et al., 1995); this mutation scs’ elements can be found in close proximity to each other affects only the Mod(mdg4)2.2 protein isoform (Mongelard et al., 2002). strain was obtained from the Drosophila Stock Center. in Drosophila nuclei (Blanton et al., 2003). The formation of The ct similar loops by the gypsy insulator could also explain results by Cai and Shen (2001) and Muravyova et al. (2001), who Nuclear matrix preparations Nuclear matrices were prepared from wild-type, Oregon R, D. melano- demonstrated that two gypsy insulators inserted between an gaster embryos 6–18 h old, following the protocol described by Fisher et enhancer and promoter have no enhancer-blocking effect. al. (1982), except that 2 M NaCl was used for the final fractionation step. These results could be explained in the context of the loop Western analysis was performed according to standard protocols. Nuclear organization observed here by assuming that two closely halos were prepared according to Gerdes et al. (1994), except that the cells were obtained by dissecting and manually disrupting imaginal disk linked insulators, due to their proximity, may preferentially cells from third instar larvae with dissecting needles and spun onto cover- interact with each other. This interaction would take place slips at 350 g for 15 min. Samples that were treated with RNase were ex- at the expense of interactions with other insulators, and it tracted with 2 M NaCl, as before, and then were incubated with 200 g/ml would result in the formation of a minidomain within a of RNase A for 1 h at 4C. larger domain. Enhancers located within the larger domain would then be free to activate transcription from promoters Immunocytochemistry and FISH analysis Antibodies against Su(Hw) and Mod(mdg4)2.2 were prepared as previ- in the same domain (Mongelard and Corces, 2001). The ously described (Gerasimova and Corces, 1998; Mongelard et al., 2002). ability of the gypsy insulator to establish these chromatin Antibodies against Mod(mdg4)2.2 recognize only this isoform, which is loops raises the question of whether insulators/boundary ele- the only one present in the gypsy insulator (Mongelard et al., 2002). ments are functionally equivalent to MARs/SARs. These se- Monoclonal antibodies against lamin were obtained from P. Fisher (The State University of New York at Stony Brook, Stony Brook, NY), H. Saum- quences have been defined biochemically, based on their weber (Humboldt Universitaet Berlin, Berlin, Germany), and Y. Gruen- ability to attach to the nuclear matrix protein fraction in baum (The Hebrew University of Jerusalem, Jerusalem, Isreal); Ubx anti- vitro. In some cases, MARs/SARs have also been shown to bodies were obtained from J. Botas (Bayer College of Medicine, Houston, possess boundary activity using in vivo assays (McKnight et TX). Immunolocalization of proteins on nuclear halos was performed as follows: coverslips with samples were incubated 3  20 min in 1% BSA, al., 1992; Namciu et al., 1998), although most MARs lack 0.1% TX-100, 1 PBS (BBST) and then incubated in 1:100 dilution of pri- this activity (Poljak et al., 1994; van der Geest and Hall, mary antibody overnight at 4C in a humidified chamber. Samples were 1997). It is possible that insulators and MARs have similar then washed 3  1 min and 3  15 min in 1 BBST and then incubated in properties but play very different roles in the cell. MARs 1:500 FITC- or Texas red–conjugated secondary antibody in 1 BBST for 1 h at 37C in a humidified chamber. The samples were then washed 3 might have a fixed structural function in establishing chro- 1 min and then 3  15 min in 1 BBST without BSA, coated with DAPI- matin organization within the nucleus. MARs might only be containing Vectashield (Vector Laboratories), and then visualized with a functional during mitosis, when the interphase to metaphase Carl Zeiss MicroImaging, Inc. fluorescence light microscope using Meta- Morph (Universal Imaging Corp.) imaging software. For FISH analysis, and back to interphase transition requires orderly changes in probes A, B, and C were made from DNA from BAC clones 20K1, 35A, chromosome condensation and organization. Alternatively, and 26L11 respectively representing the X chromosome at subdivisions MARs might create a scaffold of proteins and DNA that 7B2–7B8. BAC clones were obtained from BACPAC Resources, and DNA is more or less permanent during cell differentiation and was prepared following their protocol. Probes were synthesized using DIG-UTP or Biotin-UTP following a protocol from Boehringer. FISH on the among various cell types. Insulators, on the other hand, untreated and 2 M NaCl-extracted halos was performed following proto- might act at a different level by creating an organization su- cols established by Gerdes et al. (1994) with the following exceptions. The perimposed to that of MARs. Contrary to MARs, insulator probes were denatured for 5 min at 95C. Washes after 16 h incubation with probe were 3  5 min in 2 SSC at 42C, 3  5 min in 0.1 SSC at activity might be regulatable, allowing this organization to 60C, and 2  30 min in 4 SSC with 1.5% BSA at 37C. The samples change during development as cells differentiate and differ- were then incubated in 1:500 secondary antibody in 4 SSC with 1.5% ent patterns of gene expression are established. BSA at 37C in a humidified chamber for 1 h. The samples were washed Functional analyses of genome-wide expression patterns 3  1 min in 4 SSC, 1  10 min in 4 SSC, 1  10 min in 4 SSC with 0.1% TX-100, 1  10 min in 4 SSC, 3  5 min in 1 PBS with 0.1% TX- in yeast, Drosophila, and mammals also support the idea of 100, and then 3  1 min in 1 PBS. The samples were then coated with the compartmentalization of the chromatin fiber into do- Vectashield and visualized as described above. Chi-squared analyses were mains of gene expression (Cohen et al., 2000; Caron et al., done using Statistica 4.0 (Statsoft Inc.). Polytene chromosomes for immu- 2001; Lercher et al., 2002; Spellman and Rubin, 2002). nostaining and FISH were prepared as previously described (Gerasimova and Corces, 1998) with the following exceptions. Before immunostaining, Studies in this diverse group of organisms have shown that FISH was performed by placing the liquid nitrogen–frozen slides in 70C genes located together in the same chromosomal region are 95% EtOH at RT for 3 h. Then the samples were air dried, incubated in 2 transcriptionally coregulated. Although coregulation has not SSC at 65C for 1 h, incubated in 65C 70% EtOH at RT 3  10 min, incu- The Journal of Cell Biology 574 The Journal of Cell Biology | Volume 162, Number 4, 2003 bated in 95% EtOH at RT 2  10 min, denatured in 0.14 M NaOH for 3 15-kilodalton subunit is essential for viability in Drosophila melanogaster. min, rinsed in 2 SSC for 5 min, rinsed in 70% EtOH three times, rinsed in Mol. Cell. Biol. 12:928–935. 95% EtOH two times, and then air dried. Probes were prepared and dena- He, D.C., J.A. Nickerson, and S. Penman. 1990. Core filaments of the nuclear ma- tured as above and added to each slide, covered with coverslips, sealed trix. J. Cell Biol. 110:569–580. with rubber cement, and incubated in humidified chambers overnight at Henikoff, S. 1997. Nuclear organization and gene expression: homologous pairing 37C. After hybridization, the rubber cement was removed, and the slides and long-range interactions. Curr. Opin. Cell Biol. 9:388–395. were placed in 2 SSC at 37C for 2 min, washed 2  10 min in 2 SSC Hiraoka, Y., A.F. Dernburg, S.J. Parmelee, M.C. Rykowski, D.A. Agard, and J.W. at 37C, 1  5 min in 1 SSC at 30C, 1  10 min in 0.1 SSC, 2  5 min Sedat. 1993. The onset of homologous chromosome pairing during Dro- in 1 PBS, 0.1% TX-100, and then rinsed 1  5 min in 1 PBS at room sophila melanogaster embryogenesis. J. Cell Biol. 120:591–600. temperature. Immunostaining was performed as previously described Ishii, K., G. Arib, C. 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Visualization of chromatin domains created by the gypsy insulator of Drosophila

Byrd, Keith; Corces, Victor G.
The Journal of Cell Biology , Volume 162 (4) – Aug 18, 2003
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Copyright © 2003, The Rockefeller University Press
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Abstract

JCB Article Visualization of chromatin domains created by the gypsy insulator of Drosophila Keith Byrd and Victor G. Corces Department of Biology, Johns Hopkins University, Baltimore, MD 21218 nsulators might regulate gene expression by establishing proteins, subjecting the cells to a brief heat shock, or de- and maintaining the organization of the chromatin fiber struction of the nuclear matrix lead to disruption of the within the nucleus. Biochemical fractionation and in loop. Insertion of an additional gypsy insulator in the situ high salt extraction of lysed cells show that two known center of the loop results in the formation of paired loops protein components of the gypsy insulator are present in through the attachment of the inserted sequences to the the nuclear matrix. Using FISH with DNA probes located nuclear matrix. These results suggest that the gypsy insulator between two endogenous Su(Hw) binding sites, we show might establish higher-order domains of chromatin structure that the intervening DNA is arranged in a loop, with the and regulate nuclear organization by tethering the DNA to two insulators located at the base. Mutations in insulator the nuclear matrix and creating chromatin loops. Introduction Multicellular organisms use complex mechanisms to ensure 2001; Gerasimova and Corces, 2001). The gypsy insulator is proper spatial and temporal expression of genes. Packaging a 350-bp sequence that requires at least two proteins for of the DNA with histones to form chromatin prevents function: Su(Hw), a zinc finger DNA binding protein, and improper gene activation by restricting accessibility of tran- Mod(mdg4), a BTB domain protein that binds Su(Hw) and scription factors to the regulatory sequences of the gene. can associate with itself (Gerasimova et al., 1995; Gause et Current research has focused on the effects of histone modi- al., 2001; Ghosh et al., 2001). Although the gypsy insulator fication in promoting or preventing transcription (Jenuwein was originally identified in the gypsy retrotransposon, the and Allis, 2001; Berger, 2002). However, much less is Drosophila genome contains 500 binding sites for the known about the three-dimensional arrangement of DNA in Su(Hw) and Mod(mdg4) proteins that are presumed to be the nucleus and the role this organization plays in gene regu- endogenous insulators; out of the multiple isoforms encoded lation. There is some evidence suggesting that chromatin is by the mod(mdg4) gene, only the Mod(mdg4)2.2 protein packaged into 50–200-kb loops attached to a nuclear matrix appears to be present at the gypsy insulator (Mongelard et al., by sequences called matrix attachment regions (MARs) or 2002). We will refer to the insulator present in the gypsy scaffold attachment regions (SARs) (Kaufmann et al., 1986; retrotransposon as the gypsy insulator, whereas we will use Gasser et al., 1989; Laemmli et al., 1992; Pederson, 1998). “endogenous Su(Hw) binding sites” to designate genomic Other results suggest that insulators might similarly be binding sites for the Su(Hw) protein that do not contain involved in nuclear organization by bringing the chroma- copies of the gypsy retrotransposon but might act as insula- tin fiber to a perinuclear compartment (Gerasimova and tors, although this property has not yet been demonstrated Corces, 1998; Gerasimova et al., 2000; Ishii et al., 2002). experimentally. We have previously shown that Su(Hw) and Insulators are characterized by two properties consistent Mod(mdg4)2.2 colocalize in large foci, named insulator with their potential ability to create these loop domains: (1) bodies, located mostly at the nuclear periphery of diploid they prevent an enhancer from activating a promoter when cells. These insulator bodies are presumed to be formed by located between the two, and (2) when flanking a transgene, multiple individual insulator sites coming together at re- they prevent the local chromatin environment around the stricted nuclear locations. This hypothesis is supported by integration site from affecting its expression (Bell et al., observations indicating that the presence of the gypsy insulator at two distant chromosomal sites causes the DNA containing Address correspondence to Victor G. Corces, Department of Biology, Johns Hopkins University, 3400 N. Charles St., Baltimore, MD 21218. Abbreviations used in this paper: ct, cut; MAR, matrix attachment region; Tel.: (410) 516-8749. Fax: (410) 516-5456. email: [email protected] NPC, nuclear pore complex; SAR, scaffold attachment region; SCS, Key words: insulator; chromatin; transcription; nucleus; retrotransposon specialized chromatin structures; Ubx, Ultrabithorax.  The Rockefeller University Press, 0021-9525/2003/08/565/10 $8.00 The Journal of Cell Biology, Volume 162, Number 4, August 18, 2003 565–574 http://www.jcb.org/cgi/doi/10.1083/jcb.200305013 565 The Journal of Cell Biology 566 The Journal of Cell Biology | Volume 162, Number 4, 2003 these gypsy sequences to come together at the nuclear periph- ery (Gerasimova et al., 2000). If insulator bodies bring together individual insulator sites, the intervening DNA should form a loop. These loops might then represent func- tionally separate chromatin domains that allow independent regulation of transcription within each domain. Indirect molecular evidence for the possibility that the for- mation of chromatin loops might serve as a basis for insula- tor function comes from results obtained during a screen for proteins with boundary function in yeast (Ishii et al., 2002). Proteins involved in nucleo-cytoplasmic transport or com- ponents of the nuclear pore complex (NPC) were found to be able to buffer a flanked reporter gene from heterochro- Figure 1. Protein components of the gypsy insulator are present matic silencing effects. These results suggest that boundary in the nuclear matrix. A collection of Drosophila 6–18-h-old activity can be accomplished by attachment of both sides of embryos was subjected to a karyoskeletal or nuclear matrix fraction- the insulated DNA to a solid substrate, in this case the NPC, ation procedure, and equal amounts of the fractions were run on 7.5 and 10% polyacrylamide gels and subjected to Western blot in a perinuclear compartment. Although not directly shown analysis using antibodies to Mod(mdg4), Su(Hw), lamin, histone H3, in this work, attachment is likely to result in the formation and Ubx. Antibodies against Mod(mdg4) were specific to the of a small loop with its base at the NPC, and this loop might Mod(mdg4) 2.2 isoform, which is the isoform present in the gypsy be functionally similar to the ones hypothesized to mediate insulator (Ghosh et al., 2001; Mongelard et al., 2002). gypsy insulator activity. In spite of the widespread discussion of the loop domain model in the context of insulator function, neither the exist- found using a different nuclear matrix isolation protocol ence of these loops nor the basis for their location at the nu- that involves precipitation with ammonium sulfate (unpub- clear periphery has been demonstrated directly. Using FISH lished data). These results suggest that the gypsy insulator as- analysis of high salt–extracted nuclei, we show here that sociates with the nuclear matrix and provides a biochemical DNA sequences located between two insulators form a loop foundation for the observed formation of insulator bodies in in the interphase nucleus. The formation of this loop is de- the nuclear periphery. pendent on functional insulator proteins and an intact nu- clear matrix. The results suggest that insulators might regu- An intact nuclear matrix is required for the formation late nuclear organization by controlling the formation of of gypsy insulator bodies higher-order domains of chromatin structure. To further explore the association of gypsy insulator proteins with the nuclear matrix under more physiological condi- tions, we used antibodies against Su(Hw) and Mod(mdg4) Results 2.2 for immunofluorescence studies on specially pre- Gypsy insulator proteins are present in the nuclear pared nuclear matrices. Using a modified form of a proce- matrix fraction dure described by Gerdes et al. (1994) to isolate nuclear The perinuclear localization of the gypsy insulator bodies matrices for cytological analysis, we spun detergent-treated suggests an association of the insulator proteins with a fixed imaginal disk cells from third instar Drosophila larvae onto substrate such as the nuclear matrix. The nuclear matrix is coverslips and then extracted the nuclei with 2 M NaCl. An defined biochemically as the fraction remaining after extrac- extracted nucleus appears as a darkly stained nuclear matrix– tion of nuclei with 2 M NaCl (Pederson, 1998; Nickerson, containing core surrounded by a lightly stained DNA halo 2001). To biochemically confirm the association of Su(Hw) (for schematic see Fig. 2 A). The black and white images of and Mod(mdg4)2.2 proteins with the nuclear matrix, we nuclei stained with DAPI more clearly show the halo forma- isolated a karyoskeletal or nuclear matrix fraction (7% of tion around the intensely stained residual nuclear matrix nuclear proteins) from Drosophila embryos ranging in age (Fig. 2, F and J). Based on images of extracted nuclei ob- from 6 to 18 h post–egg laying. Fig. 1 shows the results of tained with the electron microscope (McCready et al., Western analyses of different protein fractions. Lamin, as ex- 1979), the light blue halo is composed of histone-free DNA pected, is predominantly located in the nuclear matrix frac- loops associated at the base to the more intensely stained nu- tion. In contrast, histones are extracted in previous steps and clear matrix that contains DNA, RNA, ribonucleoproteins, are not present in this fraction (Oegema et al., 1997; Ma et and other proteins resistant to high salt extraction. Along al., 1999). Both gypsy insulator components, Su(Hw) and with histones, 95% of nuclear proteins are extracted with Mod(mdg4)2.2, copurify almost entirely with the nuclear this technique; however, lamin and other known matrix- matrix fraction (Fig. 1). As a control, we tested whether the associated proteins remain (Fey et al., 1986; Ma et al., 1999). Ultrabithorax (Ubx) transcription factor is also present in We then performed immunofluorescence light microscopy the nuclear matrix fraction; the Ubx protein is extracted on nuclei that were either untreated or extracted with 2 M from nuclei under the same conditions as histones and, NaCl using antibodies to Mod(mdg4)2.2 and lamin. As ex- therefore, is not associated with the nuclear matrix (Fig. 1). pected from previous results with paraformaldehyde-fixed The same association of Su(Hw) and Mod(mdg4)2.2 was nuclei (Gerasimova et al., 2000), Mod(mdg4)2.2 is present The Journal of Cell Biology Insulators and chromatin domains | Byrd and Corces 567 Figure 2. Distribution of Su(Hw) and Mod(mdg4) insulator proteins after a 2 M NaCl extraction of nuclei. Imaginal disk cells from wild-type larvae were spun onto coverslips and either extracted with 2 M NaCl or left untreated, and then they were stained with antibodies to Su(Hw), Mod(mdg4), or lamin; in addition, the DNA was stained with DAPI (blue). (A) Schematic representation of a nucleus before and after extraction with 2 M NaCl. The red depicts the nuclear lamin and the blue indicates DNA. Treatment of these cells with 2 M NaCl removes histones and extracts 95% of nuclear proteins, leaving the DNA in large 50–200-kb loops attached to the residual nuclear matrix (McCready et al., 1979; Ma et al., 1999). B, F, J, and N are black and white images of DNA, corresponding to the panels on their right, visualized by DAPI staining. This staining is shown in blue in the rest of the panels. (C–E) Distribution of lamin (red) and Mod(mdg4)2.2 (green) in untreated nuclei. (G–I) Distribution of lamin (red) and Mod(mdg4)2.2 (green) in 2 M NaCl- extracted nuclei. (K–M) Distribution of Su(Hw) (red) and Mod(mdg4)2.2 (green) in 2 M NaCl-extracted nuclei. (O–Q) Distri- bution of Su(Hw) (red) and Mod(mdg4)2.2 (green) in nuclei extracted with 2 M NaCl followed by treatment with RNase A. in insulator bodies formed by the aggregation of multiple in- treated 2 M NaCl-extracted nuclei from wild-type larvae dividual insulator sites in intact nuclei (Fig. 2, B–E). To with RNase A. Under these conditions, the nuclear matrix confirm that the insulator bodies are associated with the nu- appears fragmented and disorganized. In addition, the insu- clear matrix, we stained nuclei extracted with 2 M NaCl lator bodies are destroyed, as determined by the localization with antibodies to lamin and Mod(mdg4)2.2 (Fig. 2, F–I). of Su(Hw) and Mod(mdg4)2.2 proteins (Fig. 2, N–Q). This The Mod(mdg4)2.2 protein, which marks the localization result strongly suggests that the formation of insulator bod- of gypsy insulator bodies, remains associated with the lamin- ies requires the existence of an intact nuclear matrix. containing nuclear matrix core. We then extracted nuclei Chromatin loops form between two insulator sites with 2 M NaCl and stained them with antibodies to both insulator proteins, Su(Hw) and Mod(mdg4)2.2, as well as If insulator bodies form by attaching multiple individual in- DAPI to visualize the DNA (Fig. 2, J–M). Extraction with sulator sites to the nuclear matrix at specific nuclear loca- such high salt concentrations that remove 95% of nuclear tions, they might help organize the chromatin fiber into proteins did not disrupt the interaction between the two in- loops representing domains of higher-order organization. sulator proteins or their interaction with the nuclear matrix. The formation of these loops should be testable using the It has been previously shown that the nuclear matrix, al- nuclear halo technique and FISH with DNA probes span- though not composed of one repeating protein unit, is com- ning the region contained within the loop. As a loop should posed of ribonucleoprotein complexes and, thus, is suscepti- form by the DNA located between two individual insulator ble to destruction by RNase (He et al., 1990; Ma et al., sites, we used immunostaining with Su(Hw) antibodies to 1999). To test whether disruption of nuclear matrix integ- map the location of individual endogenous gypsy insulators rity would affect the formation of gypsy insulator bodies, we on polytene chromosomes of third instar larvae. We then The Journal of Cell Biology 568 The Journal of Cell Biology | Volume 162, Number 4, 2003 Figure 3. Localization of DNA probes A, B, and C and Su(Hw) protein on polytene chromosomes. BAC clones A, B, or C were used as DNA probes for FISH of polytene chromosomes from salivary glands of Drosophila third instar larvae from wild-type or ct mutant strains. The chromosomes were simul- taneously stained with antibodies against the Su(Hw) protein. For all images, the DNA is visualized by DAPI staining (blue), and the locations of endogenous Su(Hw) binding sites at 7B2 and 7B8, indicated by Su(Hw) staining (red), are labeled with arrows. (A) Schematic representation of probes A, B, and C at the ct locus. The ct mutant contains a copy of the gypsy retrotransposon located in the region recognized by probe B. The location of the gypsy retrotransposon and overlap of probes A, B, and C are drawn to scale. (B–D) Immunolocalization of Su(Hw) (red) and FISH signal of probe A in polytene chromosomes from wild- type larvae. (E–G) Immunolocalization of Su(Hw) (red) and FISH signal of probe B (green) in polytene chromosomes from wild-type larvae. (H–J) Immunolocaliza- tion of Su(Hw) (red) and FISH signal of probe C (green) in polytene chromo- somes from wild-type larvae. (K–M) Immunolocalization of Su(Hw) (red) and FISH signal of probe B in polytene chromosomes from ct larvae. concentrated our analysis on a region of the X chromosome clone B (Fig. 3 A). As the gypsy retrotransposon contains the containing the cut (ct) locus flanked by two endogenous gypsy insulator, one would expect to detect a new site of Su(Hw) binding sites located at 7B2 and 7B8. This region is Su(Hw) staining in polytene chromosomes prepared from spanned by three different BAC clones, designated as probes third instar larvae carrying the ct mutation. This is indeed A, B, and C. Clone A is 154 kb in length, whereas clones B the case, as seen in Fig. 3 (K–M). The gypsy insulator present and C are 175 kb and 183 kb, respectively. The DNA se- in the gypsy retrotransposon contains 12 binding sites for the quence for these three clones has been determined by the Su(Hw) protein, giving rise to a strong immunofluorescence Drosophila Genome Project, and their location with respect signal on polytene chromosomes at chromosomal subdivi- to each other and the ct gene is diagrammed in Fig. 3 A. sion 7B4. This signal partially obscures the endogenous Clones A and B overlap by 28 kb, whereas clones B and C Su(Hw) binding sites located at 7B2 and 7B8. In addition, overlap by 16 kb. Using FISH analysis with these three analysis of DAPI-stained chromosomes in this region sug- probes and simultaneous immunolocalization with Su(Hw) gests that the presence of gypsy induces a condensation of the antibodies, we were able to determine the relative location of local chromatin (unpublished data), further decreasing the each BAC clone with respect to the location of the endoge- resolution between the Su(Hw) signals at 7B2, 7B4, and nous Su(Hw) binding sites surrounding the ct locus. Fig. 3 7B8; as a consequence, all three signals overlap in one band. D shows that probe A is located adjacent and partially over- Probe B overlaps with the gypsy retrotransposon insulator in lapping the Su(Hw) binding site present at chromosomal polytene chromosomes from ct mutant larvae (Fig. 3 M), subdivision 7B2. Probe B is located between the 7B2 and but does not overlap with the two endogenous Su(Hw) 7B8 Su(Hw) binding sites (Fig. 3 G), whereas probe C par- binding sites in wild-type animals (Fig. 3 G). Based on the tially overlaps the 7B8 site (Fig. 3 J). The ct allele is caused location of the two endogenous Su(Hw) binding sites and by the insertion of a copy of the gypsy retrotransposon in the the three BAC clones on polytene chromosomes, we expect 5 region of the ct gene (Jack, 1985). This insertion site is lo- that, if these sequences form a loop in interphase nuclei of cated 65 kb to the right of the telomere-proximal end of diploid cells as a consequence of the two Su(Hw) binding The Journal of Cell Biology Insulators and chromatin domains | Byrd and Corces 569 Figure 4. Effect of the gypsy insulator on the distribution of DNA in 2 M NaCl- extracted nuclei from male larvae. Various combinations of DNA probes A, B, and C from chromosomal subdivision 7B (see Fig. 3) were hybridized to imaginal disk cells that were spun onto coverslips and either extracted with 2 M NaCl or left untreated. Probes A and C are in green and probe B is in red. A, F, K, P, and U are black and white images of DNA visualized by DAPI staining. This staining is shown in blue in the rest of the panels. The red depicts probe B, and the green depicts probes A and C in panels G–I and probe A in the rest. E, J, O, T and Y show schematic representations of the results found to their left. (A–D) Probes A (green) and B (red) in an un- treated wild-type nucleus. (F–I) Probes A (green), B (red), and C (green) in a 2 M NaCl-extracted wild-type nucleus. (K–N) Probes A (green) and B (red) in a 2 M NaCl-extracted nucleus from larvae carrying the ct mutation. (P–S) Probes A (green) and B (red) in a 2 M NaCl- extracted nucleus from larvae of the 6 V genotype ct ; su(Hw) . (U–X) Probes A (green) and B (red) in a 2 M NaCl- extracted nucleus also treated with RNase A. sites coming together and attaching to the nuclear matrix, the nuclear matrix (Fig. 4 H). The DNA complementary to the DNA present in clones A and C would form the two the other ends of probes A and C, overlapping probe B and stems of the loop. The loop would be attached at the base of lacking endogenous Su(Hw) binding sites, is present in the these probes, where the endogenous Su(Hw) binding sites nuclear halo region partially colocalizing with the small reside, to the nuclear matrix at one location. In contrast, the overlapping regions at the ends of probe B (Fig. 4 I). To de- DNA spanning clone B would be present in the central dis- termine the statistical significance of these results, we mea- tal region of the loop, not attached to the nuclear matrix, be- sured the number of nuclei in which probes A and C form a cause the DNA in this region does not contain an insulator V structure with the vertex located in the darkly stained core in wild-type larvae. This hypothesis rests on the assumption region. We scored the V structure based on the appearance that, as polytene salivary gland cells are in interphase, the lo- of two linear FISH signals meeting at one location in the nu- cation of Su(Hw) and Mod(mdg4)2.2 proteins at endoge- clear matrix. 86% of wild-type nuclei show the characteristic nous Su(Hw) binding sites would be maintained in diploid V structure formed by probes A and C. The other 14% had cells of larval imaginal disks. either nonlinear signal(s), signal(s) that did not have a clear To test this hypothesis, we performed FISH on untreated matrix association, and/or signals that did not meet to form and 2 M NaCl-extracted imaginal disk nuclei spun onto a vertex. Differences are significant (P  0.001, chi-squared coverslips. As the ct locus is located in the X chromosome, test) when the presence of the V structure in wild type is 6 V we initially restricted our analysis to nuclei of cells from compared with ct ; su(Hw) , a null for Su(Hw) protein (Ta- imaginal disks of male larvae. We first probed untreated ble I). In imaginal disk cells from su(Hw) mutant male lar- wild-type nuclei with probes A and B and found, as ex- vae, only 16% of nuclei show the presence of a V structure pected, that they colocalize specifically in one region of the formed by probes A and C. This lack of matrix association nucleus (Fig. 4, A–D). We then performed FISH with of probes A and C in nuclei that lack Su(Hw) indicates that probes A, B, and C on nuclei extracted with 2 M NaCl from a functional Su(Hw) protein is required for DNA attach- imaginal disk cells of wild-type male larvae. Probes A and C ment to the nuclear matrix at that location. frequently appear attached to the nuclear matrix at the same Insertion of a new gypsy insulator in the center of a location at one of their ends, presumably through the puta- tive endogenous Su(Hw) binding sites located at the outer loop results in the formation of two smaller loops boundaries of probes A and C (Fig. 4 G). In contrast, we To further test the idea that the observed chromatin loops found that DNA sequences complementary to probe B are are formed between gypsy insulator sites, we then per- usually located in the halo region and do not associate with formed similar FISH experiments on nuclei that were ex- The Journal of Cell Biology 570 The Journal of Cell Biology | Volume 162, Number 4, 2003 Table I. Statistical analysis of FISH data presented in Fig. 4 Genotype No. of nuclei Probes A and C in V structure No. of nuclei Probe B in V structure %% Wild-type males 96 86 127 16 ct males 94 81 189 78 6 V ct ; su(Hw) males 83 16 112 19 Wild-type males, RNase treated 28 0 28 3 ct heat-shocked males ND ND 80 25 Wild-type females ND ND 79 18 ct females ND ND 95 80 Nuclei were scored blindly by two researchers independently, based on the presence or absence of a V structure in the DNA complementary to probe B, indicating an attachment to the nuclear matrix. Nuclei were also scored based on the presence of a V structure complementary to probes A and C with the vertex located in the nuclear matrix core. tracted with 2 M NaCl from male larvae carrying the ct To ensure that the formation of an additional loop was mutation, known to contain an additional gypsy insulator due to the presence of a new functional gypsy insulator, we in the 7B4 region. In these nuclei, the DNA hybridizing examined 2 M NaCl-extracted nuclei from male larvae of 6 V to probe B acquired a V shape, with the vertex in the the genotype ct ; su(Hw) , which carry the gypsy retrotrans- darkly stained central core, suggesting that this region of poson in the ct locus but lack Su(Hw) protein. Nuclei from the chromosome is now attached to the nuclear matrix these larvae failed to show DNA structures with the charac- through the new insulator present at the site of insertion teristic V shape in the region complementary to probe B of the gypsy retrotransposon in the ct allele (Fig. 4 M). (Fig. 4 R). The number of nuclei showing a V structure, Furthermore, the shape of this V structure is asymmetric, scored as described above, is 19%, which is significantly dif- with one side of the V longer than the other, as would be ferent (P  0.001, chi-squared test) when compared with expected given the asymmetric location of the gypsy ele- nuclei carrying the ct mutation but wild type for su(Hw) ment in the ct locus within the region covered by probe B (Table I). The formation of a V structure by probes A and C 6 V (Fig. 3 A). We scored the formation of a V structure by is also disrupted in nuclei from ct ; su(Hw) mutant male the DNA complementary to probe B based on the appear- larvae. In these nuclei, the DNA complementary to probes A ance of a continuous linear FISH signal attached to the and C only occasionally appears as a straight line going from nuclear matrix at one location. Probe B acquires a V shape the central region toward the surrounding halo; instead, the in 78% of nuclei from ct male larvae. Differences are sig- DNA appears disorganized in the nuclear halo region sur- nificant (P  0.001, chi-squared test) when we compare rounding the residual nuclear matrix core (Fig. 4, Q–S). The ct nuclei with wild-type nuclei in which such a structure number of times the V structure was observed in nuclei from 6 V is only observed in 16% of nuclei (Table I). In many ct ; su(Hw) larvae is significantly lower (P  0.001, chi- cases, it was possible to observe the position of the proxi- squared test) than wild type (Table I). In the absence of mal region of probe A overlapping with the distal end of Su(Hw) protein, both the insulator present in the gypsy ret- probe B (Fig. 4 N). This allowed us to conclude that the rotransposon in the ct locus as well as the putative insulators location of the vertex of the V approximately maps to the present at endogenous Su(Hw) binding sites are not func- region where the gypsy retrotransposon is inserted in the ct tional. The disorganized appearance of the DNA in nuclei 6 6 V allele. The 22% of nuclei from ct larvae not scored as hav- from ct ; su(Hw) male larvae implies that the lack of ing a V structure had either a nonlinear signal and/or a Su(Hw) protein results in the absence of loop structures, signal that did not have a clear matrix association. The probably because of a failure to form functional insulators, state of the cell cycle might explain the lack of V forma- suggesting that the insulators serve as attachment points of tion in those cells, as late G2 and mitotic cells do not have the DNA to the nuclear matrix. Similar experiments were insulator bodies. In contrast, a possible explanation for the also performed with nuclei from male larvae of the genotype 6 u1 V formation of probe B observed in 16% of nuclei from ct ; mod(mdg4) . The results were comparable to those ob- 6 V wild-type larvae could be the presence of replicating or tained with the ct ; su(Hw) (unpublished data), suggesting transcribing DNA in the region complementary to probe that both proteins, Su(Hw) and Mod(mdg4)2.2, are re- B associated with the nuclear matrix at the time of the 2 M quired for the formation of a functional insulator capable of NaCl extraction. This interpretation is supported by attaching the DNA to the nuclear matrix. findings suggesting that actively transcribing and replicat- ing sequences associate with the nuclear matrix (Gerdes et Loop formation requires an intact nuclear matrix al., 1994). These results suggest that the presence of a new As these results suggest the requirement of the nuclear ma- gypsy insulator in the 7B4 region between the two endoge- trix for the formation of DNA loops in the nucleus, we then nous Su(Hw) binding sites located at 7B2 and 7B8 causes examined the effect of destroying the nuclear matrix, with the original loop formed between 7B2 and 7B8 to become RNase, on the organization of the DNA flanked by endoge- divided into two smaller loops, due to the attachment of nous Su(Hw) binding sites. Nuclei from wild-type male lar- the new insulator at 7B4 to the nuclear matrix (Fig. 4 N). vae were extracted with 2 M NaCl, treated with RNase A, The Journal of Cell Biology Insulators and chromatin domains | Byrd and Corces 571 Figure 5. Insulator-mediated loop organization after heat shock and in nuclei from female larvae. (A–D) Immuno- fluorescence analysis of 2 M NaCl- extracted nuclei from imaginal disk cells of ct male larvae subjected to a 30-min heat shock at 37C using antibodies against Su(Hw) and Mod(mdg4)2.2. The Su(Hw) protein is shown in red and the Mod(mdg4)2.2 protein in green. DNA was stained with DAPI and is shown in A; DAPI staining is shown in blue in B–D. The rest of the panels in the figure show FISH analysis of 2 M NaCl- extracted nuclei from imaginal disk cells using probes A (green) and B (red); in all panels, DAPI staining is shown in gray (E, I, and M) or in blue. (E–H) Nuclei from ct male cells subjected to a 30-min heat shock at 37C. (I–L) Nuclei from imaginal disk cells of wild-type female larvae. (M–P) Nuclei from imaginal disk cells of ct female larvae. and then labeled with probes A, B, and C. The DNA com- of insulator bodies observed in cells grown at normal tem- plementary to probes A and C fails to form the characteristic perature (Fig. 3) is dramatically affected by heat shock. Nu- V structure with the vertex attached to the nuclear matrix clei from heat-shocked cells do not show large insulator (Fig. 4, compare V with G). The frequency of the V struc- bodies; instead, the Su(Hw) and Mod(mdg4)2.2 proteins ture in the RNase-treated nuclei is significantly lower (P  are distributed throughout the nucleus and do not appear 0.001, chi-squared test) when compared with wild-type nu- to be located in the nuclear periphery (Fig. 5, A–D). This clei that were not RNase treated using probes A and C or to result suggests that heat shock interferes with the ability ct nuclei using probe B (Table I). This result suggests that of individual Su(Hw) binding sites to come together at the nuclear matrix is important for the attachment of the specific nuclear locations to form insulator bodies. If this DNA and the establishment of the loop structures. is the case, heat shock should also interfere with the for- The formation of DNA loops should require not only mation of DNA loops, which are presumed to be caused attachment to the nuclear matrix, but also interactions by interactions between Su(Hw) binding sites. To test between individual Su(Hw) binding sites to form insula- this prediction, we performed in situ hybridizations with tor bodies. We have previously shown that the heat shock probes A and B to 2 M NaCl-extracted nuclei from ct response causes gypsy insulator bodies to fall apart, as a male larvae. As we have described above, nuclei from brief increase in temperature to 37C results in the disap- these larvae should show the formation of an asymmetric pearance of the typical punctate pattern of Su(Hw) and V structure by DNA homologous to probe B (Fig. 4, L–N). Mod(mdg4) observed in imaginal disk cells (Gerasimova Instead, in most nuclei from cells subjected to heat shock, et al., 2000). Under these conditions, the insulator bodies the DNA homologous to probe B appears disorganized disappear, and, instead, Su(Hw) and Mod(mdg4) pro- and located in the halo region of the extracted nuclei, teins are diffusely spread throughout the nucleus; this is away from the darkly stained core (Fig. 5, E–H). The accompanied by changes in the subnuclear localization of number of times the V structure was observed in nuclei insulator-containing sequences, suggesting a dissociation from heat-shocked ct male larvae is significantly lower of the chromatin loops (Gerasimova et al., 2000). To (P  0.005, chi-squared test) than in non–heat-shocked confirm the effect of heat shock on insulator body struc- animals (Table I). These results suggest that interactions ture and distribution under the assay conditions used in between Su(Hw) binding sites, which are disrupted by the experiments described above, we performed immu- heat shock, are normally required for the formation of the nolocalization experiments using Su(Hw) and Mod(mdg4) V structure and, therefore, for the formation of a loop 2.2 antibodies on 2 M NaCl-extracted nuclei from imagi- mediated by the gypsy insulator present in the ct locus of 6 6 nal disk cells of ct male larvae. The typical distribution ct flies. The Journal of Cell Biology 572 The Journal of Cell Biology | Volume 162, Number 4, 2003 Su(Hw)-mediated loop organization in nuclei nuclear matrix. In our experiments, we have used two differ- ent biochemical nuclear matrix purification procedures. In from female larvae both cases, it is clear that the Su(Hw) and Mod(mdg4)2.2 The results described so far were obtained in experiments proteins associate with the nuclear matrix fraction, whereas performed with X-linked DNA probes and cells from male other proteins, such as histones and Ubx, are extracted by larvae, which have only one X chromosome. In Drosophila, high salt. We have also used an in situ cell extraction proce- both the homologous autosomes and the two X chromo- dure followed by visualization of the extracted nuclei using somes in females are paired during interphase (Hiraoka et light microscopy as a means of confirming the association of al., 1993; Fung et al., 1998). The ability of the two homolo- insulator proteins with the nuclear matrix. Using this ap- gous chromosomes to pair is responsible for the phenome- proach, gypsy insulator proteins also appear to be associated non of transvection, in which two different mutant alleles of with the nuclear residue that is resistant to salt extraction. the same gene can complement each other, resulting in a Whether this resistant fraction is a filamentous network of pairing-dependent wild-type phenotype (Lewis, 1954; Heni- defined composition or a matrix formed by interactions koff, 1997). The nature of the factors responsible for chro- among different proteins and nucleic acids is not known, mosome pairing during interphase is not known. It is possi- but it is clear that insulator proteins are not extractable by ble that, if insulator proteins contribute to the organization 2 M NaCl and are not present in the chromosomal regions of the interphase chromatin fiber into loop domains, the that extrude from the nucleus after high salt extraction. The same proteins might also contribute to homologous chro- interaction of Su(Hw) and Mod(mdg4)2.2 with nuclear ma- mosome pairing. To test this possibility, we determined trix components supports previous observations indicating whether DNA sequences surrounding the ct locus are that gypsy insulator proteins are preferentially present in the present in one or two loops in nuclei obtained from female nuclear periphery of the interphase nucleus (Gerasimova et larvae. Probes A and B were used for in situ hybridization on al., 2000). 2 M NaCl-extracted nuclei from wild-type imaginal disk Although the insulator DNA and associated proteins re- cells. As in the case of male nuclei, DNA homologous to main in the nuclear matrix fraction, the intervening DNA is probe A appears arranged in a straight line, with one end lo- extruded from the nucleus by high salt extraction and is cated in the darkly stained core and the other end pointing found in the form of a large loop. The DNA contained toward the surrounding halo region (Fig. 5, I–L). Interest- within this loop appears only as a small dot after FISH anal- ingly, only one such hybridization signal is observed per nu- ysis of unextracted nuclei. These two observations suggest cleus, suggesting that the two homologous chromosomes re- that the chromatin fiber present in the loop formed by two main paired after 2 M NaCl treatment and that the proteins insulators is not completely decondensed during interphase involved in chromosome pairing are resistant to high salt ex- and only becomes extended after extraction of histones and traction. As in male cells, sequences homologous to probe B other associated proteins. This suggests that the loop formed are located in the halo region (Fig. 5, I–L). We then under- by two insulators might represent a domain of higher-order took the same type of analysis using nuclei from female lar- chromatin structure. This higher-order structure might be vae carrying the ct mutation. Again, only one signal homol- established and/or maintained by specific covalent modifica- ogous to probe A was observed; in these nuclei, probe B tions of histone tails. For example, it has been found that the gives rise to a single hybridization signal displaying an asym- chicken -globin locus, which is flanked by two CTC-bind- metric V structure (Fig. 5, M–P). Probe B acquires a V ing factor insulators, contains histones H3 and H4, acety- shape in 80% of nuclei from ct female larvae. Differences lated in various lysine residues (Litt et al., 2001; Mutskov et are significant (P  0.005, chi-squared test) when we com- al., 2002). Covalent modification of histone tails might pare ct nuclei with wild-type nuclei in which such a struc- modulate internucleosome interactions, which, in turn, de- ture is only observed in 18% of nuclei (Table I). The obser- termine the degree of higher-order chromatin structure (Tse vation of only one V structure suggests that the two new and Hansen, 1997). loops formed as a consequence of the presence of the gypsy The existence of chromatin insulator-induced domains retrotransposon in the ct locus from each chromosome ho- would explain their unusual gene regulatory property of pre- mologue are still paired after high salt extraction. These re- venting an enhancer from activating a promoter in a differ- sults again support a conclusion suggesting that insulator ent domain while not preventing the same enhancer from proteins and/or other proteins present in the nuclear matrix activating a promoter located in its own domain. The ability are involved in maintaining homologous chromosome pair- of the insulator, when flanking a transgene, to provide posi- ing during interphase. tion-independent expression of the transgene is also consis- tent with the formation of a loop domain. This domain ap- Discussion pears to be created by the interaction of flanking insulators with each other and the nuclear matrix. In fact, recent exper- We have presented evidence suggesting that the gypsy insula- iments by Ishii et al. (2002) have shown that boundary func- tor creates chromatin loop domains via association to the tion in yeast can be elicited by tethering boundary-associated nuclear matrix. The existence and exact composition of the proteins to the NPC. This tethering would presumably re- nuclear matrix has been a subject of intense debate (Nicker- sult in the formation of a loop, similar to the ones we ob- son, 2001). Lamin, which is the main component of the nu- serve here, by the DNA located between the two boundary clear lamina, is present in the nuclear matrix fraction. It is elements, which would attach the base of the loop to the possible that protein components of the gypsy insulator inter- NPC. In the case of Drosophila, the requirement for interac- act with the nuclear lamina or with other components of the The Journal of Cell Biology Insulators and chromatin domains | Byrd and Corces 573 tions between individual Su(Hw) binding sites for the for- been shown to depend on insulator function, the existence mation of the loops is underscored by the observation that a of these transcriptional domains could have a structural basis brief heat shock interferes both with the formation of insula- in the formation of chromatin loops in which gene expres- tor bodies and with the ability of the gypsy retrotransposon sion is globally regulated. to form a new loop when inserted in the ct locus. The organization of the chromatin fiber into loops has also been shown for the Drosophila specialized chromatin Materials and methods structures (scs) and scs’ boundary sequences. The proteins Drosophila strains that interact with these elements have been shown to inter- Fly stocks were maintained in standard medium and grown at 22.5C and act with each other both in vitro and in vivo. Consistent allele is caused by a small deletion 75% relative humidity. The su(Hw) with the idea that interaction between the two proteins that also affects the RpII15 gene, resulting in embryonic lethality. The strain used in these studies carries a transgene containing the RpII15 su(Hw) might facilitate pairing of boundary elements and formation u1 al- gene that rescues this lethality (Harrison et al., 1992). The mod(mdg4) of chromatin loops, sequences corresponding to the scs and lele has been described previously (Gerasimova et al., 1995); this mutation scs’ elements can be found in close proximity to each other affects only the Mod(mdg4)2.2 protein isoform (Mongelard et al., 2002). strain was obtained from the Drosophila Stock Center. in Drosophila nuclei (Blanton et al., 2003). The formation of The ct similar loops by the gypsy insulator could also explain results by Cai and Shen (2001) and Muravyova et al. (2001), who Nuclear matrix preparations Nuclear matrices were prepared from wild-type, Oregon R, D. melano- demonstrated that two gypsy insulators inserted between an gaster embryos 6–18 h old, following the protocol described by Fisher et enhancer and promoter have no enhancer-blocking effect. al. (1982), except that 2 M NaCl was used for the final fractionation step. These results could be explained in the context of the loop Western analysis was performed according to standard protocols. Nuclear organization observed here by assuming that two closely halos were prepared according to Gerdes et al. (1994), except that the cells were obtained by dissecting and manually disrupting imaginal disk linked insulators, due to their proximity, may preferentially cells from third instar larvae with dissecting needles and spun onto cover- interact with each other. This interaction would take place slips at 350 g for 15 min. Samples that were treated with RNase were ex- at the expense of interactions with other insulators, and it tracted with 2 M NaCl, as before, and then were incubated with 200 g/ml would result in the formation of a minidomain within a of RNase A for 1 h at 4C. larger domain. Enhancers located within the larger domain would then be free to activate transcription from promoters Immunocytochemistry and FISH analysis Antibodies against Su(Hw) and Mod(mdg4)2.2 were prepared as previ- in the same domain (Mongelard and Corces, 2001). The ously described (Gerasimova and Corces, 1998; Mongelard et al., 2002). ability of the gypsy insulator to establish these chromatin Antibodies against Mod(mdg4)2.2 recognize only this isoform, which is loops raises the question of whether insulators/boundary ele- the only one present in the gypsy insulator (Mongelard et al., 2002). ments are functionally equivalent to MARs/SARs. These se- Monoclonal antibodies against lamin were obtained from P. Fisher (The State University of New York at Stony Brook, Stony Brook, NY), H. Saum- quences have been defined biochemically, based on their weber (Humboldt Universitaet Berlin, Berlin, Germany), and Y. Gruen- ability to attach to the nuclear matrix protein fraction in baum (The Hebrew University of Jerusalem, Jerusalem, Isreal); Ubx anti- vitro. In some cases, MARs/SARs have also been shown to bodies were obtained from J. Botas (Bayer College of Medicine, Houston, possess boundary activity using in vivo assays (McKnight et TX). Immunolocalization of proteins on nuclear halos was performed as follows: coverslips with samples were incubated 3  20 min in 1% BSA, al., 1992; Namciu et al., 1998), although most MARs lack 0.1% TX-100, 1 PBS (BBST) and then incubated in 1:100 dilution of pri- this activity (Poljak et al., 1994; van der Geest and Hall, mary antibody overnight at 4C in a humidified chamber. Samples were 1997). It is possible that insulators and MARs have similar then washed 3  1 min and 3  15 min in 1 BBST and then incubated in properties but play very different roles in the cell. MARs 1:500 FITC- or Texas red–conjugated secondary antibody in 1 BBST for 1 h at 37C in a humidified chamber. The samples were then washed 3 might have a fixed structural function in establishing chro- 1 min and then 3  15 min in 1 BBST without BSA, coated with DAPI- matin organization within the nucleus. MARs might only be containing Vectashield (Vector Laboratories), and then visualized with a functional during mitosis, when the interphase to metaphase Carl Zeiss MicroImaging, Inc. fluorescence light microscope using Meta- Morph (Universal Imaging Corp.) imaging software. For FISH analysis, and back to interphase transition requires orderly changes in probes A, B, and C were made from DNA from BAC clones 20K1, 35A, chromosome condensation and organization. Alternatively, and 26L11 respectively representing the X chromosome at subdivisions MARs might create a scaffold of proteins and DNA that 7B2–7B8. BAC clones were obtained from BACPAC Resources, and DNA is more or less permanent during cell differentiation and was prepared following their protocol. Probes were synthesized using DIG-UTP or Biotin-UTP following a protocol from Boehringer. FISH on the among various cell types. Insulators, on the other hand, untreated and 2 M NaCl-extracted halos was performed following proto- might act at a different level by creating an organization su- cols established by Gerdes et al. (1994) with the following exceptions. The perimposed to that of MARs. Contrary to MARs, insulator probes were denatured for 5 min at 95C. Washes after 16 h incubation with probe were 3  5 min in 2 SSC at 42C, 3  5 min in 0.1 SSC at activity might be regulatable, allowing this organization to 60C, and 2  30 min in 4 SSC with 1.5% BSA at 37C. The samples change during development as cells differentiate and differ- were then incubated in 1:500 secondary antibody in 4 SSC with 1.5% ent patterns of gene expression are established. BSA at 37C in a humidified chamber for 1 h. The samples were washed Functional analyses of genome-wide expression patterns 3  1 min in 4 SSC, 1  10 min in 4 SSC, 1  10 min in 4 SSC with 0.1% TX-100, 1  10 min in 4 SSC, 3  5 min in 1 PBS with 0.1% TX- in yeast, Drosophila, and mammals also support the idea of 100, and then 3  1 min in 1 PBS. The samples were then coated with the compartmentalization of the chromatin fiber into do- Vectashield and visualized as described above. Chi-squared analyses were mains of gene expression (Cohen et al., 2000; Caron et al., done using Statistica 4.0 (Statsoft Inc.). Polytene chromosomes for immu- 2001; Lercher et al., 2002; Spellman and Rubin, 2002). nostaining and FISH were prepared as previously described (Gerasimova and Corces, 1998) with the following exceptions. Before immunostaining, Studies in this diverse group of organisms have shown that FISH was performed by placing the liquid nitrogen–frozen slides in 70C genes located together in the same chromosomal region are 95% EtOH at RT for 3 h. Then the samples were air dried, incubated in 2 transcriptionally coregulated. Although coregulation has not SSC at 65C for 1 h, incubated in 65C 70% EtOH at RT 3  10 min, incu- The Journal of Cell Biology 574 The Journal of Cell Biology | Volume 162, Number 4, 2003 bated in 95% EtOH at RT 2  10 min, denatured in 0.14 M NaOH for 3 15-kilodalton subunit is essential for viability in Drosophila melanogaster. min, rinsed in 2 SSC for 5 min, rinsed in 70% EtOH three times, rinsed in Mol. Cell. Biol. 12:928–935. 95% EtOH two times, and then air dried. Probes were prepared and dena- He, D.C., J.A. Nickerson, and S. Penman. 1990. Core filaments of the nuclear ma- tured as above and added to each slide, covered with coverslips, sealed trix. J. Cell Biol. 110:569–580. with rubber cement, and incubated in humidified chambers overnight at Henikoff, S. 1997. Nuclear organization and gene expression: homologous pairing 37C. After hybridization, the rubber cement was removed, and the slides and long-range interactions. Curr. Opin. Cell Biol. 9:388–395. were placed in 2 SSC at 37C for 2 min, washed 2  10 min in 2 SSC Hiraoka, Y., A.F. Dernburg, S.J. Parmelee, M.C. Rykowski, D.A. Agard, and J.W. at 37C, 1  5 min in 1 SSC at 30C, 1  10 min in 0.1 SSC, 2  5 min Sedat. 1993. The onset of homologous chromosome pairing during Dro- in 1 PBS, 0.1% TX-100, and then rinsed 1  5 min in 1 PBS at room sophila melanogaster embryogenesis. J. Cell Biol. 120:591–600. temperature. Immunostaining was performed as previously described Ishii, K., G. Arib, C. 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Published: Aug 18, 2003

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