Cell, Vol. 85, 745–759, May 31, 1996, Copyright 1996 by Cell PressPerturbation of Nuclear Architectureby Long-Distance Chromosome InteractionsAbby F. Dernburg,*§Karl W. Broman,†transcription will often be affected in a curious manner:Jennifer C. Fung,‡Wallace F. Marshall,* thegeneisexpressedinsomecellsbutissilentinothers,Jennifer Philips,* David A. Agard*resulting in amosaicpatternthatcan bereadilydetectedand John W. Sedat*if the gene affects, for example, tissue pigmentation.*Department of Biochemistry and BiophysicsThe prevailing model for this phenomenon invokes aUniversity of California, San Francisco“spreading” mechanism, in which the condensed tran-San Francisco, California 94143-0554scriptionally silent state of heterochromatin extends†Department of Statisticsalong the length of the chromosome into neighboringUniversity of California, Berkeleygenes. The extent of this spreading is proposed to varyBerkeley, California 94720on a cell-by-cell basis, accounting for the variegated‡Graduate Group in Biophysicsphenotype.University of California, San FranciscoHowever, there are particular examples of PEV thatSan Francisco, California 94143-0554do not appear to mesh with this general model. Themost deviant example is probably the dominant PEVinduced by certain alleles of the brown (bw) gene. TheSummarybrownDominant(bwD) allele is a null mutation caused by aninsertion of a large block of heterochromatin into thePosition–effect variegation (PEV) describes the sto-coding sequence of the gene (Slatis, 1955), which lieschastic transcriptional silencing of a gene positionednear the end of the right arm of chromosome 2 (Figureadjacent to heterochromatin. Using FISH, we have1). It has the unique property of being able to causetestedwhether variegatedexpression of theeye-colorvariegated inactivation of a normal copy of the genegene brown in Drosophila is influenced by its nuclearpresent on a homologous chromosome. The geneticlocalization. In embryonic nuclei, a heterochromaticdominance is dependent on physical pairing betweeninsertionat the brownlocusis alwaysspatially isolatedthe bwDchromosome and its wild-type homolog (Dree-from other heterochromatin. However, during larvalsen et al., 1991), analogous to a number ofother knowndevelopment this insertion physically associates withgenetic phenomena (Tartof and Henikoff, 1991).other heterochromatic regions on the same chromo-A model has been proposed by Henikoff (1994, 1996)some in a stochastic manner. These observationsto explain the “trans-inactivation” caused by bwD. Ac-indicate that the brown gene is silenced by specificcording to this view, the insertion of heterochromatincontact with centromeric heterochromatin. Moreover,into one copy of bw (the bwDallele) causes it to movethey provide direct evidence for long-range chromo-toaheterochromatic compartmentofthenucleus.Whensome interactions and their impact on three-dimen-this mutant allele is paired with a wild-type copy of thesionalnuclear architecture, whileproviding a cohesivegene,it dragsthe intactgenealongwithittoanabnormalexplanation for the phenomenon of PEV.nuclear position.In this new environment, thetranscrip-tional activity of the wild-type allele is repressed. Theprimary evidence leading to this model is that theIntroductionstrength of trans-repression bythebwDallele isstronglyinfluenced by its chromosomal position. Its effect canModelsforthe regulationofgenetic activitybytheglobalbe suppressed by chromosome rearrangements thatarrangement of chromosomes in interphase nuclei havemove the region containing bwDto a position more dis-proliferated in recent literature (cf. Manuelidis, 1990;tant from centric heterochromatin (Talbert et al., 1994).Cremeretal.,1993;Palladinoand Gasser,1994).TestingConversely, translocation of bwDtoa more centromere-such models has proved challenging because of theproximal position strengthens its silencing ability (Heni-difficulty ofobserving decondensed chromosomesdur-koff et al., 1995). The crux of this hypothesis is thating the crucial stage at which transcription occurs.the bwDallele inactivates transcription through physicalHowever, the development of chromosome in situ hy-association with centromeric heterochromatin. Thisis abridizationmethods, particularlyfluorescenceinsitu hy-provocative model in that it postulates at least threebridization(FISH),hasmadeitpossibletolocalizepartic-ideas for which direct evidence is currently scant orular portions of the genome within intact nuclei, evenlacking: that the position of a gene within the nucleusduring interphase. Here we have combined FISH withcan be influenced by neighboring sequences, that thisthree-dimensional microscopy and statistical analysischange in localization can affect a homologous copy ofto testa structural model for position–effectvariegationthe gene in trans, and that the resulting mislocalization(PEV) in Drosophila.can, in turn, lead to altered expression.PEV (reviewed by Spofford, 1976; Weiler and Waki-Here,wetest thismodelfor regulationofgeneexpres-moto, 1995) is an epigenetic phenomenon associatedsion in diploid nuclei. Using FISH, we hybridized in-with heterochromatic regions of the genome. When aterphase nuclei with probes to the bw locus and togene is moved to a position near ablock of centromericspecific blocks of heterochromatin. We show that theheterochromatin by chromosome rearrangement, itsorganization of chromosome 2 is dramatically alteredby insertion of heterochromatin at a distal position:§Present Address: Department of Developmental Biology, B300Beckman Center, Stanford University, Stanford, California 94305.whereas normally the bw locus shows no tendency toCell746Figure 1. Genomic Positions of the Loci Examined in This Study(A) Diagram of the four Drosophila chromosomes (our experiments are restricted to female nuclei, so the Y chromosome is not shown). Thechromosome-specific loci are labeled, and the distribution of the AAGAG satellite, which is present on all four chromosomes, is marked bysolid green boxes (adapted from Lohe et al., 1993). The colors used for each locus are consistent throughout the subsequent figures.(B) and (C) Mitotic chromosomes from heterozygous bwDfemale larvae (bwD/1). In (B), the bwDinsertion, which hybridizes with the AAGAGsatellite probe, is detected at the bw locus on one of two copies of chromosome 2.associate with centromeric heterochromatin, in bwDnu- that this phenomenon is due to specific