Proc. Natl. Acad. Sci. USAVol. 93, pp. 9378-9383, September 1996Colloquium Paper

This paper was presented at a colloquium entitled "Biology ofDevelopmental Transcription Control," organized by EricH. Davidson, Roy J. Britten, and Gary Felsenfeld, held October 26-28, 1995, at the National Academy of Sciences inIrvine, CA.

Genetic and molecular analysis of the gypsy chromatin insulatorofDrosophila

(transcription/enhancer/retrotransposon)

DAVID A. GDULA, TATIANA I. GERASIMOVA, AND VICTOR G. CORCESDepartment of Biology, The Johns Hopkins University, 3400 North Charles Street, Baltimore, MD 21218

ABSTRACT Boundary or insulator elements set up inde-pendent territories of gene activity by establishing higherorder domains of chromatin structure. The gypsy retrotrans-poson of Drosophila contains an insulator element that re-presses enhancer-promoter interactions and is responsiblefor the mutant phenotypes caused by insertion of this element.Thegypsy insulator inhibits the interaction ofpromoter-distalenhancers with the transcription complex without affectingthe functionality of promoter-proximal enhancers; in addi-tion, these sequences can buffer a transgene from chromo-somal position effects. Two proteins have been identified thatbind gypsy insulator sequences and are responsible for theireffects on transcription. The suppressor of Hairy-wing[su(Hw)] protein affects enhancer function both upstreamand downstream of its binding site by causing a silencing effectsimilar to that of heterochromatin. The modifier of mdg4[mod(mdg4)] protein interacts with su(Hw) to transform thisbi-directional repression into the polar effect characteristic ofinsulators. These effects seem to be modulated by changes inchromatin structure.

Expression of eukaryotic genes depends on the activation ofthe transcription complex by factors bound to enhancer se-quences. These enhancer elements act over long distances andin an orientation-independent manner to orchestrate thecomplex spatial and temporal patterns of gene expressionrequired for the proper development of eukaryotic organisms(1). The distance and orientation independence of enhanceraction afford a degree of flexibility in their interaction withpromoter elements that is critical for their role in controllingtranscription. But this flexibility raises the possibility of im-proper activation of genes by enhancers located in nearbytranscription units. Mechanisms must then exist that confineregulatory sequences within specific domains of gene expres-sion to avoid promiscuous interactions between enhancers andpromoters. Primary candidates for sequences charged with thefunction of establishing and delimiting these domains areboundary or insulator elements (2).Domain boundaries or insulator elements have been defined

by two characteristic effects on gene expression: they conferposition-independent transcription to transgenes stably inte-grated in the chromosome (3-5) and they buffer a promoterfrom activation by enhancers when located between the two (6,7). Attempts to define the nature of regulatory sequencespresent in eukaryotic genes by examining their expression afterstable transformation into cultured cells or integration into thegerm line of transgenic animals have concluded that the

expression of the transgene is determined by its site ofinsertion within the genome (8). In some cases, the variegatedpattern of expression of the transgene is reminiscent of effectson transcription caused by the proximity of heterochromaticsequences (9). This finding suggests that the chromatin struc-ture of sequences adjacent to the insertion site determines thelevel of activation of the transgene, and that the eukaryoticgenome contains regions of "condensed" chromatin unfavor-able to gene expression and regions of "open" chromatinpermissive to transcription. These higher order domains ofchromatin structure were postulated to be defined by bound-ary elements, DNA sequences with a peculiar chromatinstructure that separate regions with different degrees ofpermissiveness to gene expression (10). These boundary orinsulator elements could then inhibit cross-regulatory inter-actions between transcription signals located in adjacent do-mains. A consequence of this property is that insulators shouldbe able to inhibit the interaction of an enhancer with apromoter when located between the two without affecting theintegrity of the enhancer. This property has been exploited toidentify and functionally dissect boundary elements (11).

Several sequences with the expected properties of a bound-ary or insulator element have been identified and three ofthemhave been studied in detail: the specialized chromatin struc-tures (scs and scs') found at the boundaries of the Drosophila87A hsp7O genes (4), sequences associated with the 5' consti-tutive hypersensitive site in the chicken ,B-globin locus (7), andthe insulator element present in the gypsy retrotransposon ofDrosophila (12). The scs and scs' sequences are associated witha set of hypersensitive sites indicative of an unusual chromatinstructure (10). These sequences can buffer the white gene fromboth positive and negative chromosomal position effects afterP element-mediated germ-line transformation (4); in addition,scs and scs' sequences can insulate the hsp70 promoter fromactivation by the yolk protein yp' gene enhancer when locatedbetween the two in a chimeric hsp70-lacZ gene (6). These twoproperties are also displayed by the chicken 13-globin insulator,which can inhibit activation of the y-globin gene promoter bythe 3-globin locus control region (7). Interestingly, the chickenglobin insulator can also buffer the white gene from chromo-somal position effects when integrated in the Drosophila germline, suggesting that protein components and mechanisms ofaction for these regulatory sequences have been conservedthroughout evolution (7).The gypsy insulator is located in the 5' transcribed untrans-

lated region of this element and its characteristic effects ontranscription are the basis for the mechanism employed bygypsy to affect the expression of adjacent genes upon insertionin a new genomic location: gypsy causes mutant phenotypesthat affect only specific tissues at certain times of development,suggesting an inhibitory effect of the insulator on specificenhancers controlling the spatial and temporal expression of

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Proc. Natl. Acad. Sci. USA 93 (1996) 9379

the gene (12). Here we present an overview of our currentunderstanding of the structure and properties of the gypsyinsulator, with special emphasis on recent results that shedlight on the mechanism whereby insulators affect enhancerfunction.

Gypsy Sequences Display the Properties of InsulatorElements

The gypsy retrotransposon of Drosophila is responsible for avariety of mutations that affect genes located throughout thegenome (13). One of these genes, yellow, is specially useful forstudies of gene regulation because of its simple structure andthe nonessential nature of its product, being merely involvedin the pigmentation of embryonic, larval, and adult cuticularstructures (14). Expression of the yellow gene during Drosoph-ila development is controlled by a series of tissue-specifictranscriptional enhancers responsible for yellow expression inspecific tissues and at different times of development. Enhanc-ers that control yellow transcription in the wings, body cuticle,and larval pigmented structures are located in the 5' region ofthe gene, whereas those controlling yellow expression in thebristles and tarsal claws are located in the intron (15). Insertionof gypsy in the 5' region of yellow inhibits the interactionbetween the upstream wing and body cuticle enhancers butdoes not affect the larval enhancer located proximal to thepromoter (Fig. 1B). Analysis of revertants of gypsy-inducedmutations, as well as studies of defined in vitro-constructedmutations, has allowed the identification of a specific regionwithin gypsy responsible for this effect (16, 17). This regioncontains a repeated sequence homologous to the octamermotif present in various vertebrate enhancer and promoterelements flanked by AT tracts (16, 18). This sequence isnecessary and sufficient to recapitulate the effect of gypsy ongene expression. When the sequence is inserted anywhere inthe 5' region or the intron of theyellow gene, enhancers locatedmore distally with respect to the promoter, either upstream ordownstream, fail to activate transcription (Fig. 1 C andD) (19).This effect is not specific toyellow and it also affects regulatorysequences in the Drosophila hsp70 and cut genes (20, 21). Inaddition, these sequences can buffer the expression of a whitetransgene from chromosomal position effects when present onboth sides of the transgene (22). These two properties indicatethat these sequences have the characteristics expected of aboundary or insulator element, suggesting that gypsy mightcause mutations by separating enhancers and promoters intodifferent higher order domains of chromatin structure.

The su(Hw) Protein Is a Component of the gypsy Insulator

The phenotype of gypsy-induced mutations can be reversed bymutations in the unlinked suppressor of Hairy-wing [su(Hw)]gene, suggesting that this protein plays a central role inmediating gypsy effects on transcription and therefore elicitingthe mutant phenotype (23). This conclusion supports the viewthat the su(Hw) protein might interact with gypsy sequencesand constitute a component of the gypsy insulator. su(Hw) isa nuclear protein present in all Drosophila cells throughoutdevelopment (24). The protein contains 12 zinc fingers in thecentral region, a sequence highly similar to the second helix-coiled coil region of basic helix-loop-helix proteins, and twohighly acidic domains located in the amino- and carboxyl-terminal regions of the protein. su(Hw) interacts directly withthe octamer motif present in the gypsy insulator (25-27), andthe flanking AT tracts provide a bending in the DNA thatallows further contacts of su(Hw) with sequences located onboth sides of the octamer (28). This interaction is mediated bythe zinc fingers, because mutations in these structures interferewith the DNA-binding ability of the protein. Mutations in theleucine zipper have no effect on DNA binding; this result,

together with the observation that purified su(Hw) proteinbehaves as a monomer in solution, suggests that this domain ofthe protein might be involved in interactions with other proteincomponents of the insulator. Finally, the two acidic domainsare dispensable for insulator function (24).As expected from its nuclear localization and its ability to

bind DNA, the su(Hw) protein is present in chromosomes ofDrosophila cells (25). It is present in approximately 200 sites onpolytene chromosomes from third instar larvae and these sitesdo not correspond to locations ofgypsy elements (Fig. 1E). Theobserved sites of su(Hw) localization might thus correspond toendogenous insulators that play a role in the organization ofthe genome into higher order chromatin domains that allow forthe normal regulation of gene expression.

Characterization of mod(mdg4), a Second Component ofthe gypsy Insulator

Mutations in components of the gypsy insulator other thansu(Hw) should alter its ability to interfere with enhancer-promoter interactions and, as a consequence, these mutationsshould result in changes in the phenotype of gypsy-inducedmutations. Isolation of other second-site suppressors or en-hancers of gypsy-induced phenotypes should then aid in theelucidation of the structure and mechanism of action of thegypsy insulator. One such mutation is modifier of mdg4 [mod-(mdg4)], which causes changes in phenotypes resulting fromgypsy insertion at a variety of loci including yellow (29).Mutations in mod(mdg4) cause an enhancement of the y2phenotype, resulting in flies in which every cuticular structureof the larva and adult is unpigmented. This phenotype can beinterpreted as a consequence of the inactivation of the yellowpromoter itself, or inactivation of every enhancer controllingyellow expression (Fig. 1E). The effect of mod(mdg4) onyellowexpression is specific for gypsy-induced mutations and requiresthe presence of the gypsy element, i.e., the effect is notobserved with the wild-type yellow gene or mutations inducedby insertion of other transposable elements.A copy of thegypsyelement lacking insulator sequences is unable to mediate theeffect ofmod(mdg4) onyellow expression, suggesting that thesesequences are necessary for the observed repression ofyellowtranscription in the background ofmod(mdg4). In addition, thegypsy insulator is sufficient to elicit the same effect as thecomplete gypsy element (30). These results suggest that themod(mdg4) gene might encode a protein that binds to gypsyinsulator sequences or interacts directly with su(Hw). The laterpossibility is supported by the observation that mutations inmod(mdg4) fail to enhance the yellow phenotype of y2 in thepresence of null mutations in the su(Hw) gene, suggesting thatthe presence of the su(Hw) protein is required for repressionof yellow transcription.

The mod(mdg4) Gene Encodes a Protein That ConfersDirectionality to the gypsy Insulator

The mod(mdg4) gene has been cloned and found to encode atleast three alternatively spliced RNAs (30). The two majortranscripts encode proteins containing a BTB domain in theamino terminal region, whereas the minor transcript lacks thisdomain (30, 31). This motif has been found in numeroustranscription factors including GAGA (32, 33), a proteinthought to play a role in nucleosome remodeling (34, 35), andit is involved in mediating protein-protein interactions. In vitroexperiments using a glutathione S-tranferase-mod(mdg4) fu-sion protein indicate a direct interaction between this proteinand su(Hw); this interaction might take place between theleucine zipper region of su(Hw) and the BTB domain ofmod(mdg4) (30). The mod(mdg4) protein lacks a recognizableDNA-binding domain and is unable to bind to insulatorsequences or gypsy DNA in vitro. Nevertheless, the mod(mdg4)

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FIG. 1. Summary of the properties of the gypsy insulator of Drosophila. (A) Transcription factors that interact with tissue-specific enhancersresponsible for the expression of the yellow gene in various tissues are represented by ovals of different colors. The su(Hw) protein is representedin yellow and contains zinc fingers (green), a leucine zipper region (blue), and two acidic domains (red). The mod(mdg4) protein is representedas a green sphere. (B) Structure of the y2 allele caused by insertion of the gypsy element in the 5' region of the yellow gene. gypsy is representedin orange and is flanked by two long terminal repeats depicted as wide cylinders; arrows on top of the long terminal repeats indicate the directionof transcription. The gypsy insulator is indicated as a cylinder located downstream of the 5' long terminal repeat, with the binding sites for the su(Hw)protein represented in red. The yellow gene is located in the lower part of the panel and contains two exons separated by an intron. Color-codedtranscription factors are bound to enhancer elements located in the 5' region and intron of the gene. A red X on the wing and body cuticletranscription factors indicates that these enhancers are unable to act on the promoter as a consequence of the presence of the gypsy elementand its associated insulator. (C) Symbols are as in B. The gypsy insulator, in the absence of other gypsy sequences, can affect transcription from

(Fig. 1 legend continues on the opposite page.)

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protein is present on polytene chromosomes of third instarlarvae as deduced from immunolocalization experiments usingantibodies against the mod(mdg4) protein (Fig. IF). Thepresence of mod(mdg4) on the chromosomes must then bemediated by its interaction with su(Hw). Interestingly, thenumber of bands of mod(mdg4) localization is approximately2-3 times that of su(Hw), suggesting that mod(mdg4) mightalso interact with other proteins. This conclusion is in agree-ment with the fact that mod(mdg4) mutations have a null lethalphenotype (31), whereas the null state ofsu(Hw) mutants onlyresults in female sterility, suggesting that mod(mdg4) has abroader function than su(Hw). The sites of su(Hw) andmod(mdg4) immunolocalization on chromosomes do not cor-respond to sites of gypsy insertion; rather, they must corre-spond to the location of endogenous insulators that play a rolein the normal expression of the fly genome.The direct interaction between su(Hw) and mod(mdg4)

supports a role for the later as an integral part of the gypsyinsulator. In the presence of both proteins, the insulator affectsenhancer function uni-directionally; only enhancers locateddistally from the insulator with respect to the promoter arerepressed (Fig. 1G). In the absence of mod(mdg4) protein ina mod(mdg4) mutant, the insulator loses its uni-directionaleffect on enhancer function and it acquires properties typicalof a silencer; enhancers located both upstream and down-stream of the insulator are inactivated, thus explaining theenhancement of the yellow phenotype (Fig. 1D). This bi-directional effect on transcription is caused by the presence ofsu(Hw) alone bound to insulator sequences. Thus, the functionof mod(mdg4) appears to be establishment of uni-direction-ality in the su(Hw) silencer.

The su(Hw) Protein Causes Heterochromatization ofAdjacent Sequences

The effect caused by mutations in mod(mdg4) is not simply anenhancement of the y2 phenotype to that of a null allele, butrather it causes a variegated phenotype characterized by smallpatches of wild-type tissue in a background of mutant cells(30). This phenotype is similar to that caused by chromosomalrearrangements that bringyellow or other genes, juxtaposed toheterochromatic sequences near the centromere. It is impor-tant to emphasize that the variegated phenotype is observed inmod(mdg4) mutants, indicating that it is caused by the su(Hw)protein alone, in the absence of mod(mdg4). Thus, su(Hw)protein bound to insulator sequences represses both upstreamand downstream enhancers by an effect similar to that ofheterochromatin, suggesting that su(Hw) might affect thecompaction of adjacent chromatin. This conclusion is inter-esting in view of the presence of BTB domains in bothmod(mdg4) and the GAGA factor. Binding of mod(mdg4) tosu(Hw) inhibits the variegating effect to give rise to aninsulator. This suggests that the mod(mdg4) protein suppressesthe formation of heterochromatin, and therefore, the mod-(mdg4) mutation should behave genetically as a classicalenhancer of position-effect variegation.

This is indeed the case. Mutations in mod(mdg4) enhancethe variegation of the white-mottled 4 allele, which is caused bya rearrangement that brings the white gene next to hetero-chromatin (30, 31). This mutation is not associated with gypsy,supporting a general role, independent of the gypsy insulator,for the mod(mdg4) protein in the regulation of chromatinstructure. In agreement with this conclusion, and as predictedfrom the interaction between mod(mdg4) and su(Hw) protein,mutations in su(Hw) act as suppressors of position-effectvariegation and reverse the enhancing effect ofmod(mdg4) onwm4 (30).

Distinct Domains of the su(Hw) Protein Are Involved inInsulation and Silencing

The role of various domains of the su(Hw) protein in insulatorfunction has been established by analyzing the effect of specificmutations constructed in vitro on the phenotype ofy2 flies after,introduction of the altered su(Hw) genes into the germ line byP element-mediated transformation. Mutations in the 7th and10th zinc fingers interfere with the ability of su(Hw) to bindDNA and result in an inactive insulator that does not repressenhancer function. Deletions of the leucine zipper region, orpoint mutations in the conserved Leu residues, do not affectthe binding properties of su(Hw), but do abolish insulatoractivity (24). Finally, deletion of either of the amino- orcarboxyl-terminal acidic domains, or both simnultaneously,does not affect insulator integrity, judging from the observa-tion that an su(Hw) protein lacking these domains can stillrepress enhancer function in a polar fashion.The role of these domains in mediating the silencing prop-

erties of the su(Hw) protein has been determine by analyzingthe phenotype of su(Hw) mutations carrying specific alter-ations in the background of a mutation in the mod(mdg4) gene(30). In addition to the leucine zipper region, the two acidicdomains are also important in eliciting the variegated pheno-type characteristic of the bi-directional silencing by su(Hw).Deletion of either acidic domain decreases the variegationeffect, whereas deletion of both domains simultaneously givesrise to an su(Hw) unable to repress enhancer function. Theseresults support the following model: the mod(mdg4) proteininteracts with su(Hw), perhaps through the leucine zipperregion, and in so doing occludes the two acidic domains andconstrains their interaction with other proteins. In the absenceof mod(mdg4), these acidic domains can interact with com-ponents of the chromatin or the transcription machinery tocause the variegating bi-directional repression of enhancerfunction.

The Silencing Effect of su(Hw) Can Be Transmitted intrans to the Homologous Chromosome

Flies heterozygous for the y2 mutation display, as expectedfrom the recessive nature of y2, a wild-type phenotype. Nev-ertheless, in the presence of mod(mdg4) mutations, y2/+ fe-males display mutant coloration in the bristles (36). Thisobservation can only be explained if the enhancer that controls

promoter-distal enhancers; in this case, only the tarsal claw enhancer is inhibited, whereas the rest are active. (D) Symbols are as in B. When thegypsy insulator is located in the 5' region of the yellow gene, between the wing and body cuticle enhancers, only the former, located distally withrespect of the insulator, is affected. (E) Symbols are as in B. In a mod(mdg4) mutant background, the mod(mdg4) protein is nonfunctional(represented by a red X), and only the su(Hw) protein is present on the gypsy insulator. In this case, all enhancers are inactive and the flies showa variegated phenotype that affects every tissue. (F) Distribution of su(Hw) protein on polytene chromosomes of Drosophila larvae. Blue is theDNA and yellow corresponds to sites of su(Hw) immunolocalization. The strain lacks gypsy elements and each site presumably corresponds to anendogenous su(Hw) insulator. (G) Distribution of mod(mdg4) protein on polytene chromosomes of Drosophila larvae. This protein is present inmany more sites than su(Hw), presumably indicating a more general role of mod(mdg4) in the function of other insulators in Drosophila. (H) Effectof the two-component gypsy insulator. In the presence of mod(mdg4) (green) the bi-directional effect of su(Hw) on enhancer function (E) becomesuni-directional, and only the upstream wing and body cuticle enhancers are affected. (I) Trans-repression of enhancer function by the su(Hw)protein. In the absence of mod(mdg4), the su(Hw) protein bound to the gypsy insulator in one chromosome can inhibit (represented by a red arrow)the interaction between the bristle enhancer and its cis-promoter located in the wild-type homologous chromosome. This trans-repression requiresclose pairing between the two chromosomes.

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yellow gene expression in the bristles is inhibited in thewild-type chromosome. The effect is dependent on the pres-ence of the gypsy insulator on one of the chromosomes, andrequires an intact su(Hw) protein, suggesting that the inacti-vation of the bristle enhancer is caused in trans by the su(Hw)protein present in the gypsy insulator in the absence ofmod(mdg4) (Fig. 11). Furthermore, the acidic domains of thesu(Hw), as in the bi-directional cis-silencing effects, also seemto be crucial in causing trans-repression of the bristle enhancer.The trans-inhibitory effect is most dramatic when the wild-typechromosome contains gypsy sequences other than the insula-tor. These sequences have no phenotypic consequences on theexpression of the yellow gene, but probably allow for tighterpairing of the two homologs, thus increasing the trans-repression of bristle enhancer function. In agreement with this,rearrangements that disrupt pairing of the two copies of theyellow gene interfere with the trans-repressive effect, againsuggesting that tight apposition of both copies of the gene isimportant to allow the su(Hw) protein to interfere withenhancer function in trans (36).The mechanism by which a protein present in one chromo-

some can inhibit promoter-enhancer interactions in the pairedhomolog is difficult to visualize, but other precedents inDrosophila might shed some light on this phenomenon. Thebrown-Dominant (bwD) mutation is caused by a rearrangementthat brings heterochromatic sequences close to the brown gene.These sequences not only inhibit the expression of the bw genein cis, but also interfere with the transcription of the copy ofthe gene in the other homolog (37). By analogy with thissituation, the trans-repressive effect on the bristle enhancercan be interpreted as a consequence of the formation ofheterochromatic sequences upon binding of the su(Hw) pro-tein to insulator sequences and the negative effect of thesesequences on the enhancers located in the homologous chro-mosome.

Conclusions

Boundary or insulator elements have been found separatingregions with different degrees of chromatin compaction, andcan only affect promoter-enhancer interactions when inte-grated in the chromosome, suggesting a role for chromatinmetabolism in the function of these regulatory sequences.Evidence for a role of alterations of chromatin structure ininsulator function has been clearly obtained in the case of theboundary element present in the chicken f3-globin gene locus(7). In this case, insulator activity correlates with alterations inDNA accessibility to restriction enzymes caused by changes innucleosome positioning. Studies on the gypsy insulator supportthese conclusions: two of its protein components so far iden-tified cause changes in chromatin structure, suggesting a rolefor chromatin in insulator function.

Evidence supporting a role for chromatin changes in insu-lator function comes from the structure of the mod(mdg4)protein, a component of the gypsy insulator. The BTB domainlocated in the amino terminal region of mod(mdg4) is alsopresent in various transcription factors including GAGA, atranscriptional activator encoded by the Trithorax-like (Trl)gene that functions by influencing chromatin structure. Mu-tations in Trl result in lower levels of expression of homeoticgenes by a mechanism involving chromatin packaging (33), andthe GAGA factor itself has been shown to remodel chromatinin vitro (34, 35). Further evidence for a role of Trl in chromatinstructure comes from the observation that mutations in thisgene act as enhancers of position-effect variegation (33).Interestingly, mutations in mod(mdg4) display a variegatedphenotype affecting the gene adjacent to the insulator se-quences. In addition, mod(mdg4) mutations act as enhancersof position-effect variegation for genes brought close toheterochromatin by chromosomal rearrangements (30, 31).

These results suggest that the bi-directional repression ofenhancer function caused by the gypsy insulator lacking themod(mdg4) protein is the result of heterochromatization ofsequences surrounding the insulator insertion site. This effectis caused by the su(Hw) protein, affects all tissues of the fly,and can be interpreted as a consequence of the inhibition ofpromoter function by heterochromatic sequences or the inhi-bition of all enhancers of the yellow gene. The later possibilityis supported by the enhancer-specific inhibitory effects ob-served when insulator sequences are located -20 kb away fromthe yellow gene (38), or when expression of the wild-type genelocated in the homologous paired chromosome is examined(36). In both cases, the presence of the su(Hw) protein in theabsence of mod(mdg4) has a repressive effect on individualenhancers, suggesting that the silencing effect takes place atthe level of enhancer-promoter interaction rather than on thetranscription complex itself. Since changes in gene expressioncaused by position-effect variegation are concomitant withalterations in chromatin structure (39), the enhancer silencingobserved in mod(mdg4) mutants might also be caused bychanges in the packaging of nucleosomes. The mod(mdg4)protein plays a critical role in modulating these changes andtransforming the silencing effect into the uni-directional re-pression characteristic of insulator elements. Further evidencesuggesting a relationship between the function of the gypsyinsulator and chromatin structure comes from the observationthat the gypsy insulator element can affect dosage compensa-tion in Drosophila. In this organism, equal expression ofX-linked genes in both sexes is due to a 2-fold increase in thetranscription of genes on the male X chromosome. This isaccomplished by the assembly of multimeric male-specificlethal protein complexes on the X chromosome of the malethat remodel nucleosome structure through the specific acet-ylation of Lys 16 on histone H4 (40). Transgenes containingX-chromosome loci inserted in autosomal regions fail todosage compensate completely, suggesting an inhibitory effectof autosomal chromatin on the compensation process. Never-theless, when the copy of the transgene is flanked on both sidesby thegypsy insulator almost 90% of autosomal insertions showproper dosage compensation (41). The mechanism by whichthe gypsy insulator causes this effect is not clear, but it mightact by stabilizing the interaction of the male-specific lethalcomplex with the transgene or it might prevent access ofhistone deacetylases to increase the degree of nucleosomemodification in the inserted sequences.

This modulation of chromatin structure caused by insulatorelements must affect the interaction between an enhancer anda promoter without affecting the functionality of the enhanceritself. When the gypsy insulator is interposed between the twoenhancers that control expression of the even-skipped gene instripes 2 and 3 during Drosophila embryogenesis, transcriptionfrom a promoter located on the other side of the insulator isrepressed, whereas expression from a different promoterlocated on the same side of the enhancer is still active (42). Thesame effect has been observed for the shared enhancerspresent in the intergenic region of the Drosophila yolk protein(yp) genes that control transcription of the divergently ex-pressed yp1 and yp2 genes in the fat body of the fly. When thegypsy insulator is interposed between theyp' promoter and theenhancer, this gene is not expressed but the yp2 gene istranscribed normally (43). This conclusion suggests that thepresence of an insulator does not disrupt the interaction oftranscription factors with enhancer sequences. This require-ment of maintaining enhancer functionality while inhibiting itsinteraction with proximal promoters is central to understand-ing the mechanism by which insulators create and maintainindependent domains of gene activity. Further work on thegypsy insulator, as well as the scs sequences and the boundaryelement of the chicken ,3-globin gene, will help elucidate these

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processes and shed light on the role of these elements inregulating gene expression during development.

This work was supported by U.S. Public Health Service GrantGM35463 from the National Institutes of Health.

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Colloquium Paper: Gdula et al.