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Mol Cell Biol. Apr 2004; 24(8): 3100–3111.
PMCID: PMC381657

Drosophila Nipped-B Protein Supports Sister Chromatid Cohesion and Opposes the Stromalin/Scc3 Cohesion Factor To Facilitate Long-Range Activation of the cut Gene

Abstract

The Drosophila melanogaster Nipped-B protein facilitates transcriptional activation of the cut and Ultrabithorax genes by remote enhancers. Sequence homologues of Nipped-B, Scc2 of Saccharomyces cerevisiae, and Mis4 of Schizosaccharomyces pombe are required for sister chromatid cohesion during mitosis. The evolutionarily conserved Cohesin protein complex mediates sister chromatid cohesion, and Scc2 and Mis4 are needed for Cohesin to associate with chromosomes. Here, we show that Nipped-B is also required for sister chromatid cohesion but that, opposite to the effect of Nipped-B, the stromalin/Scc3 component of Cohesin inhibits long-range activation of cut. To explain these findings, we propose a model based on the chromatin domain boundary activities of Cohesin in which Nipped-B facilitates cut activation by alleviating Cohesin-mediated blocking of enhancer-promoter communication.

The available evidence, derived largely from studies with Saccharomyces cerevisiae, supports the idea that transcriptional activator proteins recruit basal transcription machinery and chromatin-modifying enzymes to the promoter (43). In essence, activator proteins increase the local concentration of the transcriptional machinery near the promoter. Most yeast activators bind less than a kilobase upstream of the promoters they activate, and looping of the chromatin between the activator and the promoter accommodates interactions. Consistent with this model, the yeast activators tested work poorly when positioned more than a few hundred base pairs upstream of the promoter (53).

Although it explains much of yeast gene activation, the local-concentration model does not adequately explain long-range activation by metazoan enhancers, which are often tens of kilobases from the promoter. Based on theoretical calculations, two sequences separated by several kilobases in DNA, or in various forms of chromatin, do not have a local concentration relative to each other greater than that of the estimated total concentration of all the promoters in a nucleus (46). In addition to these theoretical considerations, it was found experimentally that site-specific recombination between two sites separated by several kilobases in mammalian cells occurs slowly compared to recombination between two sites separated by a kilobase or so (45). Thus, an enhancer located several kilobases from a promoter is not expected to contact the promoter specifically or efficiently if it is dependent on random diffusion alone. Studies with the human β-globin locus using two different techniques, however, indicate that an enhancer in the locus control region located some 40 to 60 kb from the globin genes is in close proximity to a globin gene promoter when that promoter is activated (8, 56). This suggests that mechanisms exist to facilitate enhancer-promoter contact over large distances.

Studies of Drosophila melanogaster have identified sequences that facilitate particular long-range enhancer-promoter interactions. The large interval separating the iab-7 enhancer and the Abd-B gene helps tether iab-7 to the Abd-B promoter (51). This interval contains a transvection-mediating region that allows iab-7 to activate Abd-B even when they are separated by chromosomal translocations (25, 26). The transvection-mediating region contains a promoter-targeting sequence (PTS) that enables an enhancer to selectively activate a single promoter (34). A similar promoter-proximal tethering element facilitates specific long-range enhancer-promoter interactions in the antennapedia gene complex (6). Other promoter-proximal sequences involved in long-range enhancer-promoter communication are in the white and engrailed genes (42, 44). The engrailed sequence binds the GAGA protein, which can link two DNA molecules in vitro to support enhancer-promoter communication (35). In all these cases, it is probable that specialized, gene-specific proteins facilitate particular enhancer-promoter interactions.

In addition to gene-specific facilitators, it is likely that there are also general facilitators that act widely to support enhancer-promoter communication in a large number of genes (15). Such proteins could, for example, act between enhancers and promoters to stabilize folded or looped structures. The existence of general facilitators could explain how certain insulator sequences, such as the insulator in the Drosophila gypsy transposon, block enhancer-promoter interactions in diverse genes (15). Insulators block activation only when positioned between an enhancer and a promoter (5, 15, 19, 30). Insulators do not prevent binding of activators to the blocked enhancer or recruitment of basal machinery by activators. Insulators interfere only with enhancer-promoter communication, and therefore it is likely that they target proteins that act between enhancers and promoters. Significantly, many insulators cannot block certain gene-specific facilitators of enhancer-promoter communication. For instance, the Abd-B PTS allows an enhancer to bypass various intervening Drosophila insulators, including the gypsy insulator, to activate a target promoter (65).

To test the idea that there are factors that act broadly to support long-range enhancer-promoter communication in many genes, our laboratory conducted genetic screens for factors needed for transcriptional activation of the Drosophila cut gene by a wing margin enhancer located some 85 kb upstream of the promoter (38, 39, 48). Gypsy transposon insulator insertions at cut reduce activation by this enhancer and cause nicks in the adult wing margin (27). The screens detected mutations that increased the number of nicks caused by a weak gypsy insulator. It was expected that in addition to cut-specific activators, these screens would also identify genes encoding factors that facilitate long-range activation of many genes. Two potential general facilitators, named Chip and Nipped-B, were identified. Chip and Nipped-B are essential, have human homologues, and participate in remote activation of the cut and Ultrabithorax genes. Chip also facilitates the activation of many genes during embryogenesis, interacts with diverse DNA binding proteins, and appears to act as a cross-linker that promotes cooperative binding of homeoproteins to DNA (39, 58).

Reducing Nipped-B dosage reduces activation of a wild-type cut gene by the remote wing margin enhancer (48). Most cut-activating genes, such as scalloped, which encodes an enhancer-binding factor, are most limiting for cut expression when there are lesions in the wing margin enhancer (38). In contrast, Nipped-B is most limiting when there is a gypsy insulator insertion, suggesting that it plays a role in enhancer-promoter communication (48).

The sequence of the Nipped-B protein is similar to those of yeast adherins, Scc2 of Saccharomyces cerevisiae and Mis4 of Schizosaccharomyces pombe, which are required for mitotic sister chromatid cohesion (17, 36). Nipped-B is also related to Rad9 of Coprinus cinereus, required for DNA repair and meiotic chromosome pairing (50). The Scc2 and Mis4 yeast adherin homologues of Nipped-B are required for the Cohesin protein complex that mediates sister chromatid cohesion to associate with chromosomes, although direct interactions between the adherins and Cohesin have not been detected (10, 57). Consistent with the idea that the yeast adherins and Nipped-B may be chaperone-like factors, they contain tandem HEAT repeats, which are thought to form a scaffold for interactions with other proteins (41).

Cohesin is a protein complex conserved in structure and function from yeast to mammals. Cohesin contains a heterodimer of the structural maintenance of chromosome (SMC) proteins Smc1, Smc3, Scc1, and Scc3 (7, 23, 28, 40, 59). Scc1 interacts with the terminal domains of Smc1 and Smc3, and it is proposed that Cohesin forms a ring that circumscribes the sister chromatids (21, 22). The Drosophila complex consists of Smc1, Cap (Smc3), Rad21 (Scc1), and stromalin (SA; Scc3) (61). RNA interference (RNAi) knockdown of Rad21/Scc1 in cultured Drosophila cells causes mitotic delay and cohesion defects (61).

It has become apparent that Cohesin, and the structurally related Condensin SMC protein complex, plays multiple roles in chromosome structure and function (23, 28). The similarity of Nipped-B to yeast adherins suggests that, in addition to facilitating gene activation, it might also be required for sister chromatid cohesion. Here, we show that Nipped-B mutants display precocious sister chromatid separation (PSCS), indicating that Nipped-B is a functional homologue of the adherins. In most organisms, Cohesin associates with chromosomes during all of interphase, and we therefore tested whether, similar to Nipped-B, Cohesin also participates in the activation of cut. Using RNAi, we found that Rad21/Scc1 and SA/Scc3 are essential and that reducing the Nipped-B gene dosage sensitized flies to the lethal effects of Rad21/Scc1 RNAi. Unexpectedly, however, we found that opposite to the effect of Nipped-B mutations, SA/Scc3 RNAi reduced the effects of a gypsy insulator insertion at cut.

MATERIALS AND METHODS

Anti-Nipped-B antibodies.

A His6- Nipped-B fusion protein with Nipped-B residues 1511 to 1981 was expressed in Escherichia coli [BL21(DE3)/pLysS] using the pET15b vector (Novagen). The fusion protein was purified by nickel chromatography under denaturing conditions as recommended in the Novagen pET system manual (fifth edition) and renatured by dialysis at 4°C in storage buffer (10 mM Tris-HCl, pH 7.5, 25 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonylfluoride, 10% [vol/vol] glycerol) containing 2 M urea and two additional times in storage buffer without urea. The renatured protein was used to immunize rabbits at the Pocono Rabbit Farm and Laboratory (Canadensis, Pa.) according to their suggested protocol. Anti-Nipped-B antibodies were purified from rabbit serum using a Western blot of purified antigen as described by Harlow and Lane (24). The purified antibodies were dialyzed overnight at 4°C in Tris-buffered saline (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA) containing 10% glycerol and were stored at −80°C.

Immunostaining.

S2 cells were grown on polylysine-treated coverslips at 25°C, fixed in 8% paraformaldehyde for 10 min, and washed in methanol at −20°C for 6 min and then three times in phosphate-buffered saline (PBS) for 10 min each time at room temperature. The fixed cells were stained with anti-Nipped-B antibody and mouse anti-lamin antibody diluted in PBS containing 1% bovine serum albumin for 60 min at 37°C in a humid chamber, washed three times in PBS for 10 min each time at room temperature, and incubated with various fluorescently labeled secondary antibodies (anti-rabbit and anti-mouse; Jackson Laboratories) at 37°C for 45 min. The stained cells were washed three times in PBS at room temperature, mounted in Prolong Antifade (Molecular Probes) containing 1 μg of DAPI (4′,6′-diamidino-2-phenylindole) per ml, and photographed using a digital camera on a Nikon Microphot epifluorescence microscope with a 60× objective. Several focal planes were photographed and deconvoluted using Northern Eclipse software (Empix Imaging, Inc.).

RNAi in S2 cells was performed as described by others (11). Double-stranded RNA was prepared by in vitro transcription of two different template DNAs prepared by PCR from the Nipped-B cDNA (accession no. AF114160) (48) using primers containing the T7 polymerase promoter and Nipped-B coding sequences. One template containing bp 198 to 908 was amplified using the primers 5′-TTAATACGACTCACTATAGGGAGACCAACCGTACCTGTAACAACA-3′ and 5′-TTAATACGACTCACTATAGGGAGAATTGGCTTCTTGTTGCTGAATAAA-3′; the other, containing bp 543 to 1091, was amplified with the primers 5′-TTAATACGACTCACTATAGGGAGAGACCAACATACTGCTAATGCT-3′ and 5′-TTAATACGACTCACTATAGGGAGATGAAGAGTGGTGTGCTTGAGT-3′. Templates were transcribed in vitro using the Ambion Megascript kit. S2 cells were stained with anti-Nipped-B 48 h after treatment with double-stranded RNA. Both double-stranded RNAs reduced Nipped-B staining.

Embryos and third-instar larval tissues (imaginal disks, salivary glands, and fat body) were fixed and immunostained as described previously (3, 39) using various fluorescently labeled anti-rabbit and anti-mouse secondary antibodies (Jackson Laboratories). Samples were mounted in Prolong Antifade containing 1 μg of DAPI per ml. Storage of tissues in methanol at −20°C after fixation strongly reduced Nipped-B staining; all samples were processed immediately. Samples were photographed as described above or with a Zeiss confocal microscope. The micrograph of the fat body nucleus in Fig. Fig.11 was not deconvoluted.

FIG. 1.
Nipped-B protein is widely expressed and is in the nucleus. Cultured S2 cells, blastoderm embryos, third-instar salivary glands, and third-instar fat bodies were immunostained for Nipped-B, DNA (DAPI), and in some cases nuclear lamin. The third-instar ...

Neuroblast metaphase spreads.

Nipped-B mutant second-instar larvae were generated by crossing y w; Nipped-B*/CyO Kr y+ males to y w; Nipped-B*/CyO Kr y+ females (the asterisks indicate various Nipped-B mutant alleles) and were identified among the progeny by mouthpart pigmentation (y mutant). Brains of the Nipped-B mutant second-instar larvae and y w second-instar larvae as a control were dissected, treated with colchicine, and scored for PSCS, hyperploidy, and hypoploidy as described for third-instar larval brains (63).

RNAi P element constructs and germ line transformation.

To construct a P element vector for RNAi directed against Nipped-B, a 589-bp BglII fragment (nucleotides [nt] 5066 to 5654) of the Nipped-B cDNA (GenBank accession no. AF114160) (48) was cloned into the BglII site of the P{Sym-pUAST} vector (20). To construct an RNAi vector against Rad21/Scc1, a 686-bp sequence of exon 4 of the Rad21/Scc1 gene (nt 1152 to 1837 in the cDNA sequence; accession no. AF109926) (62) was amplified by PCR from Oregon R wild-type genomic DNA with primers containing BglII and EcoRI sites (5′-ATTAAGATCTTGTATTGGAAAGAAACTGGAGGTGTC-3′ and 5′-ATTAGAATTCTTTTGAAACGCCATTGTCACTTAG-3′). The product was cloned into the BglII and EcoRI sites of P{Sym-pUAST}. In the same manner, an RNAi vector was constructed using 700 bp of exon 6 of the SA/Scc3 gene (nt 1516 to 2215 of the cDNA sequence; accession no. Y14277) (primers 5′-ATTAGGATCTTCCACCCAGAGATTTTGGCTG-3′ and 5′-ATTAGAATTCGCACAAGTTTCCAGAACCTCGC-3′) (60). To prepare an RNAi construct against the CG4203 gene, a 790-bp SalI-to-XhoI fragment of a cDNA clone was cloned into the XhoI site of P{Sym-pUAST}. Intron-exon boundaries for each of the genes were obtained from the Drosophila genome sequence (1). BLAST analysis (2) of the Drosophila genome sequence for short, nearly exact matches using the National Center for Biotechnology Information web server did not reveal any potential unintentional targets for the RNAi constructs. The P{Sym-pUAST} transposons were introduced into the germ line of a y w stock by P element-mediated transformation (52).

RNAi crosses.

Newly eclosed y w; P{Sym-pUAST}/P{Sym-pUAST} males with the various RNAi insertions and newly eclosed y w ctK; P{hsp70-Gal4} or P{Act5c-Gal4}/CyO y+ virgin females were collected from uncrowded cultures and aged 2 to 3 days. Gal4-producing P insertions were provided by the Bloomington stock center (stock 2077, donated by N. Perrimon, and stock 4414, donated by Y. Hiromi). Control crosses used y w males without an RNAi insertion. Crosses were performed with five males and five females in 23- by 95-mm plastic vials with yeasted Drosophila media (64) at the appropriate temperatures. Crosses were transferred every 3 days, and the adults were discarded after the third vial. Separate crosses with the same genotypes were set up to harvest third-instar larvae for RNA for Northern blots (see below). To score adult viability and the ctK mutant phenotype, vials were scored for 9 days of eclosion at 27°C and for 10 days of eclosion at 25°C. Progeny from overcrowded vials (indicated by developmental delays and small size of larvae and adults) or undercrowded vials (determined by dry food and poor yield) were excluded from analyses. Viability was calculated from the ratio of the number of Gal4+ (y; Cy+) adult female progeny to the number of Gal4 (y+; Cy) adult female progeny.

Northern blots.

Crawling third-instar larvae were harvested from RNAi crosses and scored for the presence or absence of the Gal4 transposon by pigmentation of the larval mouthparts (y mutant Gal4+; y+ Gal4). Total RNA was isolated and analyzed by Northern blotting using single-stranded [32P]RNA probes as described previously (16), except that the hybridization solution was obtained commercially (ULTRAhyb; Ambion, Inc.). Blots were probed sequentially with the appropriated probes, imaged by autoradiography, and quantified using a PhosphorImager (Molecular Dynamics). The probes were removed by incubation in 80% formamide (Fluka) and 10 mM sodium phosphate buffer (pH 7.0) at 80°C overnight. Statistical analysis of transcript ratios was performed using Statview software (SAS Institute, Inc.).

Plasmids to generate antisense RNA probes for Nipped-B, Rad21/Scc1, SA/Scc3, and CG4203 mRNAs and transcripts of the P{Sym-pUAST} RNAi constructs were made by cloning the fragments used for the RNAi constructs into pGEM-1 (Promega). Rad21/Scc1 mRNA migrates like the 2.2-kb major RNAi product, and the SA/Scc3 mRNA is virtually the same size as the 4-kb minor RNAi product. Other probes were made for these mRNAs that did not hybridize to the RNAi transcripts. For Rad21/Scc1, a 226-bp fragment (nt 6914 to 7140 of the cDNA) was amplified by PCR from Oregon R genomic DNA with primers containing BglII and EcoRI sites (5′-ATTAAGATCTGCAAGTGTGTCCGAAAATGT-3′ and 5′-ATTAGAATTCGGTGGGGCTAAATCAAGTGT-3′) and was cloned into the BamHI and EcoRI sites of pGEM-1. The same strategy was used to clone a 501-bp fragment of SA/Scc3 (nt 527 to 1028 of the cDNA; primers 5′-ATTAAGATCTGATACAGAGTGAGCGGGAGA-3′ and 5′-ATTAGAATTCAACCAATAGGGCGACATCCA-3′). The rp49 probe is described elsewhere (29).

Quantification of ctK wing margin nicks.

Nicks in the wing margin were counted as previously described (14). Box plots were made, and paired t tests were performed using Statview software.

RESULTS

Nipped-B is widely expressed and is in the nucleus.

If Nipped-B supports long-range enhancer-promoter communication in many genes, and sister chromatid cohesion, it should be present in the nuclei of all cells. By immunostaining, Nipped-B was detected in interphase nuclei of cultured S2 (Fig. (Fig.1)1) and Kc (not shown) Drosophila cells. RNAi against Nipped-B in cultured cells substantially reduced nuclear staining, demonstrating the specificity of the antisera (Fig. (Fig.1).1). All interphase nuclei in embryos (Fig. (Fig.1),1), third-instar imaginal disks (not shown), and polytene tissues, such as the salivary glands and fat body (Fig. (Fig.1),1), also stained for Nipped-B. In most cases, staining was not homogeneous but granular. Staining was much less in the nucleolus, as shown by the fat body nucleus in Fig. Fig.11.

There was no tissue examined in which Nipped-B was not in the nuclei, indicating that it is likely present in all cells. In embryos or cultured cells at mitotic stages that lack a nuclear membrane, however, it was not possible to distinguish potential authentic Nipped-B staining from background. An example of a blastoderm embryo at metaphase is shown in Fig. Fig.1.1. Because we cannot determine whether the overall low level of staining is authentic or background, it is unknown if the Nipped-B protein disappears or if it is evenly dispersed.

We did not observe staining of chromosomes with anti-Nipped-B. Indeed, Nipped-B staining anticorrelated with DAPI staining of DNA. This is displayed in the high-magnification confocal micrograph of the third-instar salivary gland nucleus in Fig. Fig.1.1. Although the nucleus is not squashed, some polytene chromosome bands are visible as bright bands of DAPI DNA stain, but there is no band pattern to Nipped-B staining. We also did not detect Nipped-B staining on spread salivary gland polytene chromosomes, and nucleoplasmic Nipped-B was lost during the chromosome-squashing procedure (not shown). Although we used polyclonal antibodies from two rabbits, we cannot rule out the possibility that the epitopes recognized by the antibodies are masked if Nipped-B is bound to the chromosomes, that only a small amount of Nipped-B is chromosomal, or that Nipped-B associates only very briefly with the chromosomes, as might be expected if it acts as a chaperone.

Nipped-B mutants display PSCS.

Homozygous Nipped-B mutants or organisms heteroallelic for two mutant Nipped-B alleles survive to the second-instar stage but die prior to molting to the third instar (48). Nipped-B mRNA is present in embryos prior to the onset of transcription, and thus, the mutants likely survive to the second-instar stage by using the maternal contribution.

We examined metaphase nuclei in brains of second-instar Nipped-B mutants. Some 60% of the Nipped-B mutant metaphases displayed PSCS, in which the centromeric regions of the sister chromatids are somewhat detached from each other (Fig. (Fig.2).2). In most mutant nuclei displaying PSCS, at least two chromosomes were affected (Fig. (Fig.2).2). This was true for all Nipped-B mutant combinations. In contrast, some 10% of the wild-type control metaphase nuclei displayed PSCS, and in these, only one chromosome was affected. We conclude that Nipped-B is required for mitotic sister chromatid cohesion.

FIG. 2.
Nipped-B mutant second-instar larval neuroblasts display high levels of PSCS. The photographs are examples of wild-type and Nipped-B mutant second-instar brain metaphases. The Nipped-B mutant metaphases range from those that have no visible defects (left) ...

We examined the Nipped-B mutant metaphases for hyperploidy or hypoploidy, which is expected if the Nipped-B mutant cells with PSCS complete mitosis. The low number of aneuploid mutant nuclei, however, was not statistically higher than in the wild-type control.

Nipped-B RNAi reduces viability and Nipped-B mRNA levels.

The finding that Nipped-B is required for both sister chromatid cohesion and long-range gene activation in cut raised the possibility that Cohesin may also be involved in long-range gene activation. There are no Drosophila Cohesin mutants, so we tested RNAi as a means of reducing Rad21/Scc1 and SA/Scc3 expression to determine if Cohesin affects the long-range activation of cut. We transformed flies with Sym-pUAST P elements (20) that bidirectionally transcribe segments of Nipped-B or Cohesin subunit gene sequences when activated by the yeast Gal4 protein. This produces double-stranded RNA that induces an RNAi response (20). Three independent insertions of each RNAi P element were chosen for use. The insertions were autosomal and homozygous viable and did not cause apparent mutant phenotypes in the absence of Gal4.

Prior to testing the effects of Nipped-B and Cohesin RNAi on cut expression, we determined the effects on viability and target mRNA levels. Nipped-B RNAi, as expected, reduced viability. At 25°C with an Act5c-Gal4 driver, the three Nipped-B RNAi insertions reduced viability to 0, 6, and 7% of that of the control lacking an RNAi insertion (Fig. (Fig.3).3). Lethality occurred during pupal development; survival to the late third-instar larval stage was essentially wild type. Thus, RNAi-induced lethality occurred substantially later than with homozygous Nipped-B mutants.

FIG. 3.
Nipped-B and Cohesin RNAi reduce adult viability. Crosses with Act5c-Gal4 females were conducted at 25°C, as diagrammed above the graph, with y w control males and males with the P{Sym-pUAST} RNAi inserts indicated at the bottom. ...

Brain squashes of third-instar larvae expressing Nipped-B RNAi did not reveal an increase in PSCS, but as shown in Fig. Fig.4,4, Nipped-B mRNA in third-instar larvae was reduced only by some 50%, and thus, PSCS should be much rarer than in homozygous Nipped-B mutant second-instar larvae, which show a much greater reduction in Nipped-B mRNA and die much earlier (47, 48). Western blots of nuclear extracts comparing Gal4+ and Gal4 third-instar larvae from the Nipped-B RNAi crosses revealed decreases in the Nipped-B protein similar in magnitude to the decreases in mRNA (not shown). It is possible that lethality with Nipped-B RNAi is caused by changes in gene expression and not by PSCS, although PSCS in a small population of critical cells, which could be difficult to detect, could also cause lethality. It is unlikely that lethality is caused by reducing a target mRNA other than Nipped-B, because the sequences used in the RNAi construct do not have sufficient homology to any other known Drosophila genes.

FIG. 4.
Nipped-B RNAi reduces Nipped-B mRNA levels. Crosses were conducted with three independent insertions of the P{Sym-pUAST-Nipped-B} RNAi transposon at 25°C and with an Act5c-Gal4 driver, as shown at the top. (Top) Northern blot of ...

The overall 50% reduction in Nipped-B mRNA in response to RNAi may be an average of a large reduction in some cells and a small reduction in others. Nonetheless, the observation that an overall 50% reduction in Nipped-B expression was lethal raised the question of why heterozygous Nipped-B mutants are viable. We found in multiple experiments, however, that Nipped-B mRNA is present at some 75 to 80% of the wild-type level in third-instar larvae heterozygous for the Nipped-B407 mutant allele (not shown). The Nipped-B mRNA level is strongly reduced in second-instar larvae homozygous for this allele (47, 48), and thus, we conclude that Nipped-B mRNA levels are not directly proportional to gene dosage.

Rad21/Scc1 and SA/Scc3 Cohesin RNAis reduce viability with small reductions in target mRNA levels.

With the Act5c-Gal4 driver at 25°C, three Rad21/Scc1 RNAi inserts gave 25, 70, and 108% of control viability (Fig. (Fig.3).3). By Northern blotting, the insert that did not reduce viability (P{Sym-pUAST-Scc1}-2) produced some 70% of the Sym-pUAST-Rad21/Scc1 transcript produced by the most lethal insertion (not shown). Three SA/Scc3 RNAi insertions gave 0, 15, and 37% of the control viability (Fig. (Fig.3).3). As with Nipped-B RNAi, lethality with Rad21/Scc1 and SA/Scc3 RNAis occurred during pupal development. Similar to the results with Nipped-B RNAi, we did not detect significant PSCS in third-instar neuroblasts. The lethality could therefore be caused by PSCS in a small population of critical cells or by the reduction of other functions of the Cohesin subunits. We note that nearly complete depletion of SA/Scc3 by RNAi does not cause detectable PSCS in S2 cells (61).

The reduced viabilities with Rad21/Scc1 and SA/Scc3 RNAis occur with small reductions in target mRNA levels. The amount of SA/Scc3 mRNA (3.8 kb) was reduced by an average of 17% with the three RNAi insertions at 25°C with the Act5c-Gal4 driver (Fig. (Fig.5,5, top), although survival to adulthood was reduced by some 60 to 100% (Fig. (Fig.3).3). Similarly, the amount of Rad21/Scc1 mRNA (2.3 kb) was reduced by an average of 30% by the three insertions (not shown). The insert that did not reduce viability (P{Sym-pUAST-Rad21/Scc1}-2) detectably decreased Rad21/Scc1 mRNA.

FIG. 5.
SA/Scc3 RNAi reduces SA/Scc3 mRNA and increases Rad21/Scc1 mRNA. Crosses to females with the Act5c-Gal4 driver were conducted at 25°C, as diagrammed at the top, with y w control males and males with three independent inserts of the P{ ...

Potential cross-regulation interactions between adherin and Cohesin mRNA levels.

Unexpectedly, Nipped-B, Rad21/Scc1, and SA/Scc3 RNAi increased SA/Scc3 and Rad21/Scc1 mRNAs in some cases. In all experiments, Nipped-B and Rad21/Scc1 RNAi increased the levels of SA/Scc3 mRNA and Rad21/Scc1 RNAi increased the levels of SA/Scc3 mRNA. In contrast, Rad21/Scc1 and SA/Scc3 RNAis did not have detectable effects on Nipped-B mRNA in any experiments. Nipped-B RNAi increased Rad21/Scc1 mRNA levels in some experiments, but not in others.

Some of these effects are illustrated by the experiments shown in Fig. Fig.55 and and6.6. In Fig. Fig.55 (middle), SA/Scc3 RNAi with the Act5c-Gal4 driver at 25°C increased Rad21/Scc1 mRNA levels by 10 to 15%. This was observed with all three P inserts. Although small, these effects are significant. Setting the ratio of SA/Scc3 mRNA to Rad21/Scc1 mRNA in the control to 1, the average ratio observed with the Gal4 siblings with the three SA/Scc3 RNAi crosses in the experiment shown in Fig. Fig.44 was 1.01 ± 0.06 (95% confidence interval), and the average ratio observed with the Gal4+ siblings was 0.78 ± 0.10, which is significantly lower (P = 0.007) (Fig. (Fig.5,5, bottom).

FIG. 6.
Nipped-B and Rad21/Scc1 RNAi increase SA/Scc3 mRNA levels. Control males (y w) or males with the P{Sym-pUAST} RNAi inserts were crossed to females with the hsp70-Gal4 driver at 25°C, as diagrammed at the top. Total RNA was isolated ...

In the experiment shown in Fig. Fig.66 with the hsp70-Gal4 driver at 25°C, the three Nipped-B RNAi inserts increased the level of SA/Scc3 mRNA an average of 2.0- ± 0.1-fold and the three Rad21/Scc2 RNAi inserts increased the SA/Scc3 message an average of 1.4- ± 0.2-fold. Similar effects were observed with the Act5c-Gal4 driver (not shown).

Although there are many potential explanations for the above-mentioned results, a simple possibility is that Cohesin represses transcription of the Rad21/Scc1 and SA/Scc3 genes. RNAi directed against either component would then increase expression of the other. If Nipped-B were required for binding of Cohesin to the chromosomes, this would also explain the effect of Nipped-B RNAi on SA/Scc3 transcripts.

Reduction of Nipped-B dosage increases lethality of Rad21/Scc1 RNAi.

The effect of Nipped-B RNAi on SA/Scc3 mRNA levels suggested a regulatory connection between Nipped-B and Cohesin. To test for a functional link between Nipped-B and Cohesin, we took advantage of the observation that one of the Rad21/Scc1 RNAi insertions (P{Sym-pUAST-Rad21/Scc1}-2) did not reduce viability with the Act5c-Gal4 driver at 25°C. This line produced less RNAi transcript than the other Rad21/Scc1 lines and slightly reduced Rad21/Scc1 mRNA. This suggested that Rad21/Scc1 activity was reduced, but not enough to cause lethality. When combined with a heterozygous Nipped-B407 mutation, however, this insert reduced viability to some 50% of that of controls (Fig. (Fig.77).

FIG. 7.
Rad21/Scc1 RNAi and a heterozygous Nipped-B407 mutation combine to reduce viability. Females of the Nipped-B and P{Sym-pUAST-Rad21/Scc1}-2 genotypes were produced by crossing y w ctK; P{Act5c-Gal4}/CyO y+ females ...

This synthetic lethality indicates that Nipped-B and Rad21/Scc1 are functionally linked in vivo. One common function is sister chromatid cohesion, but with half the animals surviving to adulthood and the rest dying during pupal development, it is unlikely that we would be able to detect PSCS. As noted above, even with Nipped-B and Cohesin RNAi that caused a higher frequency of pupal lethality, we did not detect PSCS in third-instar neuroblasts. Because we could not be sure that loss of sister chromatid cohesion is responsible for the lethality and because Nipped-B participates in long-range activation of cut and Ultrabithorax, we also considered the possibility that Cohesin might be needed for long-range gene activation.

Nipped-B RNAi increases severity of the ctK mutant wing margin phenotype.

To determine if Cohesin components participate in cut activation, we tested the effect of RNAi directed against Nipped-B, Rad21/Scc1, and SA/Scc3 on the wing-nicking mutant phenotype displayed by the ctK gypsy insertion. We chose ctK because the mutant phenotype is exquisitely sensitive to the dosages of several genes that regulate cut, including Chip and Nipped-B (18). We reasoned that this might allow us to detect effects on cut expression, although the effects of RNAi on Cohesin mRNA levels in the whole organism are small, particularly under conditions that do not reduce viability. In ctK, the gypsy insertion near the cut promoter partially blocks activation by several enhancers, including the wing margin enhancer. Other gypsy insertions in this region are lethal, but the ctK gypsy insulator is weak. The cut gene is on the X chromosome, and the viability of males hemizygous for ctK is some 25% of wild type.

To evaluate the effects of RNAi on ctK, we crossed y w males homozygous for autosomal insertions of the RNAi P elements to y w ctK females heterozygous for the hsp70-Gal4 P element. The Gal4+ adult male progeny were scored to determine the effect of the RNAi on the ctK mutant phenotype. In such crosses conducted at 25°C, two of the Nipped-B RNAi insertions reduced female progeny viability to some 70% of wild type, and as expected, both increased the severity of the male wing-nicking phenotype, indicating that cut expression was reduced. A typical result is shown in Fig. Fig.8.8. We used two controls in most experiments, one with y w males lacking an RNAi insert and one with males with an RNAi insert against a non-Cohesin gene (CG4203). In several experiments with three different insertions of the CG4203 RNAi P element, we did not observe effects on viability or on cut expression.

FIG. 8.
Nipped-B RNAi increases the severity of the ctK wing-nicking phenotype. Crosses with a y w control and two independent insertions of the P{Sym-pUAST-Nipped-B} transposon were conducted at 25°C with an hsp70-Gal4 driver, as diagrammed ...

SA/Scc3 RNAi decreases the severity of the ctK wing-nicking phenotype.

We tested the effects of RNAi directed against the Rad21/Scc1 and SA/Scc3 Cohesin components with the hsp70-Gal4 driver at 27°C. Higher temperature increased activation of the RNAi insertions by Gal4. At 27°C, Nipped-B RNAi reduced viability to such an extent that we did not recover sufficient males to quantify the phenotype. In contrast, none of the Rad21/Scc1 or SA/Scc3 RNAi inserts discernibly reduced viability at 27°C.

Figure Figure99 shows that four of the six Cohesin RNAi lines tested decreased the severity of the ctK wing-nicking phenotype relative to two controls. This included one of three Rad21/Scc1 lines and all three SA/Scc3 lines. Significantly, none of the Cohesin RNAi lines increased the severity of the phenotype, which would be expected if Cohesin facilitated cut activation. Figure Figure99 shows the most complete experiment, which included all Cohesin RNAi insertions, but each insertion was also used in smaller experiments with similar results. We conclude that RNAi knockdown of SA/Scc3 increases expression of ctK in the developing wing margin and therefore that this Cohesin subunit inhibits cut expression. We consider it likely that the Rad21/Scc1 Cohesin component has a similar effect, but only one of three RNAi lines reproducibly had a statistically significant effect. Although it is improbable, we cannot rule out the possibility that the effect on cut expression occurred with this insertion because the P element altered expression of genes flanking the insertion site.

FIG. 9.
SA/Scc3 RNAi decreases the severity of the ctK wing-nicking phenotype. Crosses were conducted at 27°C with an hsp70-Gal4 driver with three independent insertions of the Cohesin subunit RNAi transposons. Both a y w control lacking an RNAi transposon ...

We did not detect significant effects of Nipped-B, Rad21/Scc1, and SA/Scc3 RNAis on the other cut alleles tested, ct53d and ct2s/+. A lack of effects with these alleles was expected: ct53d is a deletion in the wing margin enhancer that partially reduces cut expression, and heterozygous Nipped-B mutations do not enhance the ct53d phenotype (48). ct2s is a deletion that removes the entire enhancer. ct2s/+ females do not display a mutant phenotype, and thus, suppression could not be detected. Heterozygous Nipped-B mutations cause a weak wing-nicking phenotype with ct2s/+ (48).

DISCUSSION

Our experiments addressed two questions: (i) does Nipped-B, in addition to facilitating remote activation of cut and Ultrabithorax, participate in mitotic sister chromatid cohesion, and (ii) does Cohesin participate in long-range activation of cut? The first was motivated by the sequence similarity of Nipped-B to yeast adherins required for sister chromatid cohesion, and the second was motivated by the published observations that the yeast adherins are required for the Cohesin complex to associate with chromosomes. Our results indicate that the cooperation between adherins and Cohesin that occurs in yeast is conserved in Drosophila. Our findings also indicate, however, that the SA/Scc3 subunit of Cohesin opposes Nipped-B in long-range activation of the cut gene.

Functional connection between Nipped-B and Cohesin.

The high rate of PSCS in homozygous and heteroallelic Nipped-B mutants and the increased lethality of Rad21/Scc1 RNAi in flies heterozygous for a Nipped-B mutation observed here are consistent with the findings that the yeast Scc2 and Mis4 homologues of Nipped-B are required for Cohesin to associate with chromosomes (10, 57). Although we did not detect PSCS in third-instar neuroblasts in RNAi experiments, it is unlikely that the synthetic lethality of a heterozygous Nipped-B mutation and Rad21/Scc1 RNAi is caused by changes in long range gene activation, because as discussed below, Nipped-B and Cohesin appear to have opposing roles in gene activation. In the RNAi experiments, it is possible that the pupal lethality is caused by PSCS in a subpopulation of critical cells, but we cannot rule out the possibility that Nipped-B and Cohesin cooperate with each other in other essential functions.

No physical association between the yeast adherins and Cohesin has been detected, nor do they colocalize on chromosomes. We were unable to detect a chromosomal population of Nipped-B, but if Nipped-B acts primarily as a chaperone for loading Cohesin onto chromosomes, only a small fraction of Nipped-B may be transiently interacting with chromosomes at any time. The mechanisms by which yeast adherins facilitate Cohesin chromosome binding are unclear, but the synthetic lethality between a heterozygous Nipped-B mutation and Rad21/Scc1 RNAi observed here indicates that this functional connection is conserved in metazoans. In budding yeast, Cohesin begins to associate with chromosomes in late G1, while in fission yeast, Caenorhabditis elegans, Drosophila, and mammalian cells it begins to associate in telophase (37, 54, 57, 62). We detected Nipped-B in the nucleus at all stages that have a nuclear membrane, indicating that it could be involved in Cohesin chromosomal association beginning in telophase and thus could influence all potential interphase functions of Cohesin, in addition to sister chromatid cohesion.

Opposing roles of Nipped-B and SA/Scc3 in cut expression.

The finding that SA/Scc3 RNAi reduces the severity of the ctK wing-nicking phenotype indicates that the SA/Scc3 component of Cohesin inhibits cut expression. This is opposite to the role of Nipped-B at cut. Multiple Nipped-B mutations were recovered in a screen for mutations that increase the severity of a wing-nicking phenotype displayed by a cut allele with a weak gypsy insulator insertion (48). Reduced mRNA levels indicated that some of these Nipped-B mutations are loss-of-function alleles, and viability of homozygous Nipped-B mutants was rescued by a transgene expressing a Nipped-B cDNA from a Chip gene promoter (47, 48). Thus, Nipped-B protein facilitates activation of cut by the wing margin enhancer.

The effect of Nipped-B on cut expression is likely direct (48). Nipped-B does not regulate cut by altering the activities of known cut regulators because it is most limiting for cut expression when there is a gypsy insertion at cut while the other known regulators are more limiting with other types of cut mutations. Moreover, heterozygous Nipped-B loss-of-function alleles reduce cut expression, and partial reduction of Nipped-B is unlikely to cause an equal or greater change in the expression of another cut regulator. Although the effects of Nipped-B on gene expression were most apparent with gypsy insertion alleles of cut, a measurable effect was observed in heterozygous females with a wild-type cut allele and an allele in which the wing margin enhancer is deleted. Thus, Nipped-B also facilitates the activation of wild-type cut.

All three SA/Scc3 RNAi insertions and one of three Rad21/Scc1 insertions reduced the number of nicks displayed by the ctK gypsy insertion. We think it likely that the Cohesin complex, and not just one or two of its subunits, is responsible for reducing cut expression. Scc1 and Scc3 operate together as a unit in both Drosophila and C. elegans (9, 61). Thus, it is unlikely that they work independently of each other in regulating gene expression. Indeed, Rad21/Scc1 RNAi in cultured Drosophila cells reduces both Rad21/Scc1 and SA/Scc3 proteins (61), and data presented here indicate that Rad21/Scc1 and SA/Scc3 may regulate each other's transcript levels. However, we cannot rule out the possibility that Rad21/Scc1 and SA/Scc3 work independently of the Smc1 and Cap/Smc3 Cohesin subunits, which form another stable subcomplex (9, 61). Our initial attempts to reduce expression of the SMC subunits by RNAi were unsuccessful.

Potential roles for Cohesin in long-range gene activation.

It is unlikely that the effects of SA/Scc3 on cut expression occur by reducing the expression of a cut activator. The small reductions in SA/Scc3 expression in these experiments are unlikely to cause equal or larger changes in the activities of other cut regulators. We also did not see effects of Cohesin RNAi on ct53d, which has a small deletion in the enhancer and is affected by all known cut regulators except Nipped-B (38, 48). It is most likely, therefore, that SA/Scc3 acts directly at cut or by reducing the ability of Nipped-B to facilitate activation.

The possibility that the negative effect of SA/Scc3 on cut expression may be specific to gypsy insertion alleles cannot be ruled out, but we think it improbable. The negative effect is likely to be related to the positive effect of Nipped-B, and Nipped-B facilitates the expression of wild-type cut (48). If SA/Scc3 does specifically affect gypsy insertion alleles, however, it may interact with the gypsy insulator and contribute to enhancer blocking. This is consistent with evidence that Cohesin functions at chromosomal boundaries in yeast. Certain Smc1 and Smc3 mutations reduce the ability of a boundary that flanks the HMR silent mating-type locus to block the spread of gene-silencing Sir protein complexes (12), and Scc1 associates with this boundary (31). It has also been proposed that Cohesin binding sites are boundaries that control the extent of chromosome loop formation by Condensin (32). This proposal is based in part on the observation that Cohesin is needed to reestablish chromosome condensation upon returning temperature-sensitive Condensin mutants to the permissive temperature. In Drosophila, the gypsy insulator partially blocks the negative effects of heterochromatin on the expression of a euchromatic gene, suggesting that it has boundary activity (49), and in yeast, the Su(Hw) protein that binds the gypsy insulator also blocks the spread of gene-silencing complexes (13). If SA/Scc3 or Cohesin increases insulation by gypsy, Nipped-B could facilitate activation by reducing their association with the insulator.

We prefer a more general version of the “Cohesin insulator” model, in which native Cohesin binding sites in the 85-kb region separating the wing margin enhancer from the cut promoter act as insulators and impede the formation of structures needed to bring the wing margin enhancer close to the promoter (Fig. (Fig.10).10). In yeast, Cohesin binds every 10 kb or so along the chromosomes (4, 31, 55). The spacing of Cohesin in Drosophila has not been investigated, but multiple complexes could bind in the 85-kb interval between the wing margin enhancer and the cut promoter. If we assume that Nipped-B, perhaps by opening the Cohesin ring, facilitates both the loading and the removal of Cohesin from chromosomes, this could explain how Nipped-B facilitates the activation of wild-type cut. By opening the Cohesin ring, Nipped-B would help achieve equilibrium between the bound and unbound states by providing opportunities to load or remove Cohesin from chromosomes. This mechanism would be distinct from proteolytic removal of Cohesin by separase at the metaphase-to-anaphase transition but could be involved in the removal of Cohesin from chromosome arms in prophase. In heterozygous Nipped-B mutants, which retain substantial Nipped-B activity, the equilibrium endpoint would not be altered, but it might take longer to achieve equilibrium. Thus, we would not expect to see reduced Cohesin binding to chromosomes, but the lower Nipped-B levels would reduce the windows of opportunity for removal of Cohesin needed to allow long-range activation. This model also predicts that Nipped-B does not have to stably associate with chromosomes, which could explain why we did not detect chromosomally bound Nipped-B by immunostaining.

FIG. 10.
Proposed model to explain opposing effects of Nipped-B and SA/Scc3 Cohesin subunit on cut gene expression. The central proposals are that Nipped-B both loads and removes Cohesin from the region between the wing margin enhancer and the promoter and that ...

Finally, in a simple indirect model it could be supposed that, similar to its role in loading Cohesin, Nipped-B could also facilitate chromosomal binding of another protein complex that assists long-range enhancer-promoter interactions. In this case, there would be competition between Cohesin and the long-range activation complex for Nipped-B, and reduction of Cohesin would make Nipped-B more available to facilitate long-range activation. This and the insulator model described above are not mutually exclusive, but both explain how Nipped-B cooperates with Cohesin in sister chromatid cohesion but opposes the effect of Cohesin proteins on cut expression.

Acknowledgments

We are grateful to Byron Williams and Michael Goldberg for teaching R.A.R. how to make larval neuroblast squashes. We thank Paul Fisher for anti-lamin antibody; Joel Eissenberg for the RNAi vector, help with third-instar neuroblast squashes, and comments on the manuscript; Ziva Misulovin for help with RNA isolation; the Bloomington stock center for Gal4 drivers; Yair Dorsett for comments on the manuscript and suggesting the short-sequence BLAST for unintentional RNAi targets; and Amy MacRae, Idriss Bennani-Baïti, and Maia Dorsett for comments on the manuscript.

This work was supported by a grant from the NIH to D.D.

REFERENCES

1. Adams, M. D., et al. 2000. The genome sequence of Drosophila melanogaster. Science 287:2185-2195. [PubMed]
2. Altschul, S. F., W. Gish, W. Miller, E. W. Myers, and D. J. Lipman. 1990. Basic local alignment search tool. J. Mol. Biol. 215:403-410. [PubMed]
3. Aulner, N., C. Monod, G. Mandicourt, D. Jullien, O. Cuvier, A. Sall, S. Janssen, U. K. Laemmli, and E. Käs. 2002. The AT-hook protein D1 is essential for Drosophila melanogaster development and is implicated in position-effect variegation. Mol. Cell. Biol. 22:1218-1232. [PMC free article] [PubMed]
4. Blat, Y., and N. Kleckner. 1999. Cohesins bind to preferential sites along yeast chromosome III, with differential regulation along arms versus the centric region. Cell 98:249-259. [PubMed]
5. Burgess-Beusse, B., C. Farrell, M. Gaszner, M. Litt, V. Mutskov, F. Recillas-Targa, M. Simpson, A. West, and G. Felsenfeld. 2002. The insulation of genes from external enhancers and silencing chromatin. Proc. Natl. Acad. Sci. USA 99(Suppl. 4):16433-16437. [PMC free article] [PubMed]
6. Calhoun, V. C., A. Stathopoulos, and M. Levine. 2002. Promoter-proximal tethering elements regulate enhancer-promoter specificity in the Drosophila Antennapedia complex. Proc. Natl. Acad. Sci. USA 99:9243-9247. [PMC free article] [PubMed]
7. Campbell, J. L., and O. Cohen-Fix. 2002. Chromosome cohesion: ring around the sisters? Trends Biochem. Sci. 27:492-495. [PubMed]
8. Carter, D., L. Chakalova, C. S. Osborne, Y. F. Dai, and P. Fraser. 2002. Long-range chromatin regulatory interactions in vivo. Nat. Genet. 32:623-626. [PubMed]
9. Chan, C. C., A. Chan, M. Jeon, T. F. Wu, D. Pasqualone, A. E. Rougvie, and B. J. Meyer. 2003. Chromosome cohesion is regulated by a clock gene paralogue TIM-1. Nature 424:1002-1009. [PubMed]
10. Ciosk, R., M. Shirayama, A. Shevchenko, T. Tanaka, A. Toth, A. Shevchenko, and K. Nasmyth. 2000. Cohesin's binding to chromosomes depends on a separate complex consisting of Scc2 and Scc4 proteins. Mol. Cell 5:243-254. [PubMed]
11. Clemens, J. C., C. A. Worby, N. Simonson-Leff, M. Muda, T. Maehama, B. A. Hemmings, and J. E. Dixon. 2000. Use of double-stranded RNA interference in Drosophila cell lines to dissect signal transduction pathways. Proc. Natl. Acad. Sci. USA 97:6499-6503. [PMC free article] [PubMed]
12. Donze, D., C. R. Adams, J. Rine, and R. T. Kamakaka. 1999. The boundaries of the silenced HMR domain in Saccharomyces cerevisiae. Genes Dev. 13:698-708. [PMC free article] [PubMed]
13. Donze, D., and R. T. Kamakaka. 2001. RNA polymerase III and RNA polymerase II promoter complexes are heterochromatin barriers in Saccharomyces cerevisiae. EMBO J. 20:520-531. [PMC free article] [PubMed]
14. Dorsett, D. 1993. Distance-independent inactivation of an enhancer by the suppressor of Hairy-wing DNA-binding protein of Drosophila. Genetics 134:1135-1144. [PMC free article] [PubMed]
15. Dorsett, D. 1999. Distant liaisons: long range enhancer-promoter interactions in Drosophila. Curr. Opin. Genet. Dev. 9:505-514. [PubMed]
16. Dorsett, D., G. A. Viglianti, B. J. Rutledge, and M. Meselson. 1989. Alteration of hsp82 gene expression by the gypsy transposon and suppressor genes in Drosophila melanogaster. Genes Dev. 3:454-468. [PubMed]
17. Furuya, K., K. Takahashi, and M. Yanagida. 1998. Faithful anaphase is ensured by Mis4, a sister chromatid cohesion molecule required in S phase and not destroyed in G1 phase. Genes Dev. 12:3408-3418. [PMC free article] [PubMed]
18. Gause, M., P. Morcillo, and D. Dorsett. 2001. Insulation of enhancer-promoter communication by a gypsy transposon insert in the Drosophila cut gene: cooperation between Suppressor of Hairy-wing and Modifier of mdg4 proteins. Mol. Cell. Biol. 21:4807-4817. [PMC free article] [PubMed]
19. Geyer, P. K., and I. Clark. 2002. Protecting against promiscuity: the regulatory role of insulators. Cell. Mol. Life Sci. 59:2112-2127. [PubMed]
20. Giordano, E., R. Rendina, I. Peluso, and M. Furia. 2002. RNAi triggered by symmetrically transcribed transgenes in Drosophila melanogaster. Genetics 160:637-648. [PMC free article] [PubMed]
21. Gruber, S., C. H. Haering, and K. Nasmyth. 2003. Chromosomal cohesin forms a ring. Cell 112:765-777. [PubMed]
22. Haering, C. H., J. Lowe, A. Hochwagen, and K. Nasmyth. 2002. Molecular architecture of SMC proteins and the yeast cohesin complex. Mol. Cell 9:773-788. [PubMed]
23. Hagstrom, K. A., and B. J. Meyer. 2003. Condensin and cohesin: more than chromosome compactor and glue. Nat. Rev. Genet. 4:520-534. [PubMed]
24. Harlow, E., and D. Lane. 1988. Antibodies: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
25. Hendrickson, J. E., and S. Sakonju. 1995. cis and trans interactions between the iab regulatory regions and abdominal-A and Abdominal-B in Drosophila melanogaster. Genetics 139:835-848. [PMC free article] [PubMed]
26. Hopmann, R., D. Duncan, and I. Duncan. 1995. Transvection in the iab-5,6,7 region of the bithorax complex of Drosophila: homology independent interactions in trans. Genetics 139:815-833. [PMC free article] [PubMed]
27. Jack, J., D. Dorsett, Y. Delotto, and S. Liu. 1991. Expression of the cut locus in the Drosophila wing margin is required for cell type specification and is regulated by a distant enhancer. Development 113:735-747. [PubMed]
28. Jessberger, R. 2002. The many functions of SMC proteins in chromosome dynamics. Nat. Rev. Mol. Cell. Biol. 3:767-778. [PubMed]
29. Kim, J., J. J. Yim, S. Wang, and D. Dorsett. 1992. Alternate use of divergent forms of an ancient exon in the fructose-1,6-bisphosphate aldolase gene of Drosophila melanogaster. Mol. Cell. Biol. 12:773-783. [PMC free article] [PubMed]
30. Labrador, M., and V. G. Corces. 2002. Setting the boundaries of chromatin domains and nuclear organization. Cell 111:151-154. [PubMed]
31. Laloraya, S., V. Guacci, and D. Koshland. 2000. Chromosomal addresses of the cohesin component Mcd1p. J. Cell Biol. 151:1047-1056. [PMC free article] [PubMed]
32. Lavoie, B. D., E. J. Hogan, and D. Koshland. 2002. In vivo dissection of the chromosome condensation machinery: reversibility of condensation distinguishes contributions of condensin and cohesin. J. Cell Biol. 156:805-815. [PMC free article] [PubMed]
33. LeBlanc, H. N., T. T. Tang, J. S. Wu, and T. L. Orr-Weaver. 1999. The mitotic centromeric protein MEI-S332 and its role in sister-chromatid cohesion. Chromosoma 108:401-411. [PubMed]
34. Lin, Q., D. Wu, and J. Zhou. 2003. The promoter targeting sequence facilitates and restricts a distant enhancer to a single promoter in the Drosophila embryo. Development 130:519-526. [PubMed]
35. Mahmoudi, T., K. R. Katsani, and C. P. Verrijzer. 2002. GAGA can mediate enhancer function in trans by linking two separate DNA molecules. EMBO J. 21:1775-1781. [PMC free article] [PubMed]
36. Michaelis, C., R. Ciosk, and K. Nasmyth. 1997. Cohesins: chromosomal proteins that prevent premature separation of sister chromatids. Cell 91:35-45. [PubMed]
37. Mito, Y., A. Sugimoto, and M. Yamamoto. 2003. Distinct developmental function of two Caenorhabditis elegans homologs of the Cohesin subunit Scc1/Rad21. Mol. Biol. Cell 14:2399-2409. [PMC free article] [PubMed]
38. Morcillo, P., C. Rosen, and D. Dorsett. 1996. Genes regulating the remote wing margin enhancer in the Drosophila cut locus. Genetics 144:1143-1154. [PMC free article] [PubMed]
39. Morcillo, P., C. Rosen, M. K. Baylies, and D. Dorsett. 1997. Chip, a widely expressed chromosomal protein required for segmentation and activity of a remote wing margin enhancer in Drosophila. Genes Dev. 11:2729-2740. [PMC free article] [PubMed]
40. Nasmyth, K. 2002. Segregating sister genomes: the molecular biology of chromosome separation. Science 297:559-565. [PubMed]
41. Neuwald, A. F., and T. Hirano. 2000. HEAT repeats associated with condensins, cohesins, and other complexes involved in chromosome-related functions. Genome Res. 10:1445-1452. [PMC free article] [PubMed]
42. Orihara, M., C. Hosono, T. Kojima, and K. Saigo. 1999. Identification of engrailed promoter elements essential for interactions with a stripe enhancer in Drosophila embryos. Genes Cells 4:205-218. [PubMed]
43. Ptashne, M., and A. Gann. 1997. Transcriptional activation by recruitment. Nature 386:569-577. [PubMed]
44. Qian, S., B. Varjavand, and V. Pirrotta. 1992. Molecular analysis of the zeste-white interaction reveals a promoter-proximal element essential for distant enhancer-promoter communication. Genetics 131:79-90. [PMC free article] [PubMed]
45. Ringrose, L., S. Chabanis, P. O. Angrand, C. Woodroofe, and A. F. Stewart. 1999. Quantitative comparison of DNA looping in vitro and in vivo: chromatin increases effective DNA flexibility at short distances. EMBO J. 18:6630-6641. [PMC free article] [PubMed]
46. Rippe, K. 2001. Making contacts on a nucleic acid polymer. Trends Biochem. Sci. 26:733-740. [PubMed]
47. Rollins, R. A. 2000. The genes of the Drosophila Nipped locus participate in activation by remote enhancers and Notch receptor signaling. Ph.D. thesis. Cornell University, Ithaca, N.Y.
48. Rollins, R. A., P. Morcillo, and D. Dorsett. 1999. Nipped-B, a Drosophila homologue of chromosomal adherins, participates in activation by remote enhancers in the cut and Ultrabithorax genes. Genetics 152:577-593. [PMC free article] [PubMed]
49. Roseman, R. R., V. Pirrotta, and P. K. Geyer. 1993. The su(Hw) protein insulates expression of the Drosophila melanogaster white gene from chromosomal position-effects. EMBO J. 12:435-442. [PMC free article] [PubMed]
50. Seitz, L. C., K. Tang, W. J. Cummings, and M. E. Zolan. 1996. The rad9 gene of Coprinus cinereus encodes a proline-rich protein required for meiotic chromosome condensation and synapsis. Genetics 142:1105-1117. [PMC free article] [PubMed]
51. Sipos, L., J. Mihaly, F. Karch, P. Schedl, J. Gausz, and H. Gyurkovics. 1998. Transvection in the Drosophila Abd-B domain: extensive upstream sequences are involved in anchoring distant cis-regulatory regions to the promoter. Genetics 149:1031-1050. [PMC free article] [PubMed]
52. Spradling, A. C., and G. M. Rubin. 1982. Transposition of cloned P elements into Drosophila germ line chromosomes. Science 218:341-347. [PubMed]
53. Struhl, K. 1989. Molecular mechanisms of transcriptional regulation in yeast. Annu. Rev. Biochem. 58:1051-1077. [PubMed]
54. Sumara, I., E. Vorlaufer, C. Gieffers, B. H. Peters, and J. M. Peters. 2000. Characterization of vertebrate cohesin complexes and their regulation in prophase. J. Cell Biol. 151:749-762. [PMC free article] [PubMed]
55. Tanaka, T., M. P. Cosma, K. Wirth, and K. Nasmyth. 1999. Identification of cohesin association sites at centromeres and along chromosome arms. Cell 98:847-858. [PubMed]
56. Tolhuis, B., R. J. Palstra, E. Splinter, F. Grosveld, and W. de Laat. 2002. Looping and interaction between hypersensitive sites in the active beta-globin locus. Mol. Cell 10:1453-1465. [PubMed]
57. Tomonaga, T., K. Nagao, Y. Kawasaki, K. Furuya, A. Murakami, J. Morishita, T. Yuasa, T. Sutani, S. E. Kearsey, F. Uhlmann, K. Nasmyth, and M. Yanagida. 2000. Characterization of fission yeast cohesin: essential anaphase proteolysis of Rad21 phosphorylated in the S phase. Genes Dev. 14:2757-2770. [PMC free article] [PubMed]
58. Torigoi, E., I. M. Bennani-Baïti, C. Rosen, K. Gonzalez, P. Morcillo, M. Ptashne, and D. Dorsett. 2000. Chip interacts with diverse homeodomain proteins and potentiates Bicoid activity in vivo. Proc. Natl. Acad. Sci. USA 97:2686-2691. [PMC free article] [PubMed]
59. Uhlmann, F. 2003. Chromosome cohesion and separation: from men and molecules. Curr. Biol. 13:R104-R114. [PubMed]
60. Valdeolmillos, A. M., R. Villares, J. M. Buesa, S. Gonzalez-Crespo, A. Martinez, and J. L. Barbero. 1998. Molecular cloning and expression of stromalin protein from Drosophila melanogaster: homologous to mammalian stromalin family of nuclear proteins. DNA Cell Biol. 8:699-706. [PubMed]
61. Vass, S., S. Cotterill, A. M. Valdeolmillos, J. L. Barbero, E. Lin, W. D. Warren, and M. M. Heck. 2003. Depletion of drad21/scc1 in Drosophila cells leads to instability of the cohesin complex and disruption of mitotic progression. Curr. Biol. 13:208-218. [PubMed]
62. Warren, W. D., S. Steffensen, E. Lin, P. Coelho, M. Loupart, N. Cobbe, J. Y. Lee, M. J. McKay, T. Orr-Weaver, M. M. Heck, and C. E. Sunkel. 2000. The Drosophila RAD21 cohesin persists at the centromere region in mitosis. Curr. Biol. 10:1463-1466. [PubMed]
63. Williams, B. C., T. L. Karr, J. M. Montgomery, and M. L. Goldberg. 1992. The Drosophila l(1)zw10 gene product, required for accurate mitotic chromosome segregation, is redistributed at anaphase onset. J. Cell Biol. 118:759-773. [PMC free article] [PubMed]
64. Wirtz, R. A., and H. G. Semey. 1982. The Drosophila kitchen-equipment, media preparation, and supplies. Drosophila Inform. Serv. 58:176-180.
65. Zhou, J., and M. Levine. 1999. A novel cis-regulatory element, the PTS, mediates an anti-insulator activity in the Drosophila embryo. Cell 99:567-575. [PubMed]

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