Summary Numerous genes contain regulatory elements located many tens of kilobases away from the promoter they control. Specific mechanisms must be required to ensure that such distant elements can find and interact with their proper targets but not with extraneous genes. This review explores the connections between transvection phenomena, the activation of domains of homeotic gene expression, position effect variegation and silencers. These various examples of long-distance effects suggest that, in all cases, related forms of chromatin packaging may be involved.

over to contact the proteins bound at the promoter(’). Direct evidence for stable interactions between enhancer factors and the promoter complex is lacking but, mechanistically, a substantial amount of evidence has built up to support the looping model, at least for activation over relatively short distances. In vitro, promoter activation works well for trans-acting factors bound within a few hundred nucleotides of the promoter. Over longer distances, regulated transcription is more difficult to reconstruct. Presumably, higher order organization, chromatin structure and additional factors are involved. In vivo, enhancers can act over distances of several kilobases and, in the case of distant regulatory elements, over several tens of kilobases. Although in chromatin the intervening DNA may be packaged in a much more compact form, it is difficult to imagine regulation over distances that are comparable to or greater than distances between genes without a mechanism to select and bring together the appropriate sequences. Some of the components of such a mechanism might be tissue-specific enhancer binding factors while others may be general and play a role in different processes and affect many genes. This article reviews some instances of long-distance gene regulation, examines a possible model and argues that similar proteins or processes affecting chromatin organization underlie a number of apparently disparate genetic phenomena. Most of the examples are taken from Drosophila although similar explanations can be applied to vertebrate examples, such as the control of the p-globin gene cluster(*).

Introduction The genetic analysis of many complex genes, i. e. genes that have complicated and detailed patterns of expression, shows that their regulatory regions can be very extensive. Molecular analysis has confirmed that such regions contain a large number of regulatory elements, sometimes located many tens of kilobases from the promoter they control. This fact immediately poses the question of how factors binding to such distant regulatory regions efficiently communicate with their target promoters. The complementary problem is raised by the very existence of mechanisms that allow such long-distance regulation: these must be prevented from functioning or not be available in cases where different genes with very different temporal or spatial patterns of expression lie in close proximity of one another. The now axiomatic view of eukaryotic gene regulation is based on the concept of enhancer elements. These are binding sites for transcriptional regulatory proteins which can be shown to stimulate transcription initiation by RNA polymerase at a nearby promoter. The detailed molecular mechanism of promoter activation is not entirely clear but the most reasonable and widely accepted model envisions a ‘looping’ interaction through which the factor bound at the enhancer loops

Transvection Effects A possible model for long-distance looping is suggested by the phenomenon of transvection in Drosophila. Transvection, first discovered by Lewd3) in the Bithorax Complex ( B X - C ), is a pairing-dependent complementation between certain alleles of a given locud3). Transvection effects have now been reported in several different of which the white, Ultrabithorax (Ubx), decapentaplegic (dpp) and yellow loci are the best characterized both genetically and molecularly. In general this complementation occurs between certain regulatory mutations or between regulatory and structural mutations. Where sufficient genetic and molecular information is available, it indicates that transvection can be understood as the ability of regulatory elements on one chromosome to control the expression of the corresponding gene on the paired homologous chromosome (Fig. 1).For example, at the Ubx gene, the dominant Cbx mutation causes the expression of the Ubx gene in cells where it is not normally expressed(’’). When paired with a wild-type Ubx gene, the mutated regulatory element confers the abnormal expression pattern on the normal copy of the gene. Transvection effects require that the two transvecting alleles be brought in close physical

‘There appears to be some necessary correlation between hippophagy, pogonotrophy and perhaps paganism’ Rolleston. Archaeologia, XLVII, 1883

Fig. 1. Schematic illustration of transvection. Two copies of a gene residing on homologous chromosomes, are shown in a paired configuration. Each includes a promoter, P, and a distant regulatory element, DRE. A typical transvection effect is detected when the coding region of one gene is inactivated by a mutation (upper copy), while the other gene has a mutation in the regulatory element. Transvection is detected as the pairing-dependent ability of the intact DRE on one copy to regulate the promoter of the other copy of the gene.

proximity by the homologous pairing of the two copies of the gene and, for several but not all of these loci, they require the activity of the zeste gene(12). The roduct of the zeste gene is a DNA binding proteine3) that recognizes sequences at at least 60 sites on the polytene chromosomes(14). I n vitro, it binds to multiple sites to the DNA of the white, Ubx and dpp genes(”). In all three cases, zeste protein binds to sites in the immediate vicinity of the promoter and to other sites that frequently correspond to genetically identified regulatory regions. This distribution suggested a unitary mechanism to account for promoter regulation by distant elements and the interchromosomal promoter activation that occurs in transvection. According to this model, the ability of distant elements to come in contact with the promoter is dependent on proteins such as zeste binding near the distant element and near the promoter. Simultaneous binding of zeste protein at these two sites would then draw them together and permit the activation of the promoter. In transvection, a similar looping occurring between two homologously paired chromosomes could juxtapose the regulatory element of one copy of the gene with the promoter of the second copy (Fig. 2). Evidence supporting this model is that zeste protein in vitro can bind simultaneously to two DNA molecules(15), probably because it forms very large aggregates consisting of several hundred monomers. Recent evidence indicates that the ability to form such aggregates is essential for its role in mediating transvection effects(16). Mutations that delete the part of the protein required for this aggregation abolish transvection effects while mutants in which aggregation is decreased are defective in transvection but can be restored to normal by overexpression of the mutant protein. These results give a picture of zeste as a sticky protein that either exists in vivo in the form of extensive agglomerates that can bring together two binding sites on the DNA, or can form such agglomerates by sticking together lower oligomers bound to two sites independently.

Fig. 2. Long-distance gene regulation mediated by zeste compared to zesre-mediated transvection. This model envisions zeste binding sites flanking a distant regulatory element (DRE) and the promoter (P). The large, rnultimeric zeste protein aggregate (Z) binds and brings together the two regions, allowing the DRE to activate the promoter (flash). In transvection, the zesre aggregate brings together the DRE from one copy of the gene and the promoter from the other copy.

Transvection as a Model for Long-Distance Regulation The long-distance looping model suggested by transvection in essence proposes that a class of proteins with properties similar to those of zeste act as linkers between distant regions and promoter, supplementing the ability of enhancer-binding factors to form loops between enhancer and the promoter complex. The large, multimeric zeste species would facilitate the finding of the promoter by the distant enhancer and would stabilize the loop formed by the enhancer-bound factors with the promoter complex. For this model to work, inappropriate loops, e.g. made between zeste bound at different enhancers, would need to be prevented in some way. A simple extension of the longdistance looping model could be made by including additional levels of control to account for the functioning of complex genes that have multiple regulatory elements. If the binding of zeste near any one regulatory element were itself regulated, the zeste protein would take on the function of a switchboard, selecting one out of many possible regulatory regions to be brought in contact with the promoter. That potential zeste binding sites are not always available for binding is shown by the fact that sites in the white gene that we know are functional in the eye are not occupied by zeste in salivary glands, where the white gene is not active(14). The accessibility could be controlled by the positioning of nucleosomes or by the antagonistic binding of other factors or by altering the chromatin structure of the region (e.g. the higher order folding of the nucleosome string). Many essential developmental genes such as the genes of the BX-C, engrailed, dpp in Drosophila

depend on intricate and extensive regulatory regions that stretch over many tens of kilobases. If proteins such as zeste are necessary for long-distance gene regulation, mutations that inactivate them would be expected to be lethal or at least to produce a variety of developmental defects. Instead, mutations or deletions of the zeste gene are not lethal although they are unable to produce transvection effects at Ubx, white or dpp (ref. 17 and V. Pirrotta and E. McGuffin, unpublished). Such mutant flies are indeed sickly and many appear to die during development but the survivors are viable and fertile without notable abnormalities except that they have a brown eye color, a phenotype that probably indicates insufficient expression of the white gene. It appears therefore that, in the absence of zeste, gene expression may not be optimal but is sufficient for normal development. If long-distance promoter regulation requires the action of proteins that facilitate the looping, there must be other proteins that can substitute for the zeste gene product. That other proteins can play a role similar to that of zeste in certain transvection effects is indicated by the fact that at least one other transvection phenomenon is not zeste dependent("). Many transcription factors may, in fact , be able to act like zesre, though to a more limited extent. Several reports in the recent literature describe promoter activation by enhancers present not on the same DNA molecule but on a second molecule connected with the first by a protein bridge('') or on a second plasmid concatenated with the first('932").These results imply that factors bound to an enhancer on one DNA molecule can scan a second DNA molecule for a promoter with which to interact, provided this second molecule is constrained topologically in the same vicinity. Many transcription factors may therefore be able to form reasonably stable loops over reasonably long distances, if guided by some physical constraints such as the appropriate packaging of the surrounding chromatin. Such physical constraints might be supplied by the anchoring of chromatin domains to some feature of the nuclear architecture. Nuclear matrix or scaffold attachment regions (MARS or SARs) have been identified as DNA fragments that remain associated with a rapidly sedimenting, residual nuclear structure after histone depletion and digestion with endonucleases. Sites that remain bound or that become bound under such conditions have been found in the vicinity of many genes, both in their 5' regulatory regions and in the 3' regions, formin loo s that may reflect a functional organization(21- '). Such attachment sites could constitute a constraint analogous to the concatenation of plasmids and facilitate the juxtaposition of control element and promoter or of two transvecting alleles. It is interesting to note therefore that zeste protein cosediments with nuclear matrix preparations from Drosophilu cells(16). Unfortunately, this does not necessarily mean that zeste is bound to the matrix since zeste protein expressed in E. coli has the same

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sedimentation properties, due not to association with nuclear matrix but to the size of the zeste-zeste aggregates formed. In view of the aggregation properties exhibited by zeste and some other nuclear factors, the matrix binding experiments may need to be reexamined since DNA fragments bound to fast sedimenting aggregates of zeste or zeste-like proteins will appear in these experiments to be matrix-attached. Nevertheless, the possibility that zeste or other similar proteins might be associated with some nuclear structure has not been ruled out. The C-terminal region of the zeste amino acid sequence contains a long helical domain with heptad repeats highly suggestive of coiledcoil interactions. This might be involved in the zeste-zeste interactions that are responsible for the large-scale aggregation of zeste but it is interesting to note that it might also interact with the nuclear lamina. Similar helical domains have been found in other proteins that take part in gene regulation, such as the SIR4 protein(24325), involved in transcriptional silencing in yeast, or that mediate chromosome pairing in meiosis, such as the RADSO protein of yeast(26). Modifiers of zeste and Polycomb-Group Genes While transvection in many cases requires zeste, the function of zeste in transvection in turn requires the activity of a number of other proteins. These are revealed by second site mutations that are dominant enhancers or suppressors of zeste-dependent transvection effects. These gain-of-function mutations define at least five loci: Su(z)2, Su(z)3,Psc, Su(z)SOI (also called E ( z ) l or pco) and Su(z)302 (also called S C ~ ) ( * ' - ~ ~ ) . Different alleles of the same locus can either enhance or suppress zeste-dependent transvection effects. At present we know little of the products of these genes but three of them have been cloned recently (Psc, Su(2)2 and p c o / E ( z ) l ) and more information on their molecular biology should become available soon. The suppressor and enhancer of zeste mutations are dominant modifiers of transvection phenomena but, when homozygous, they are lethal and in many cases produce homeotic transformations. At least three of the loci are in fact identifiable as members of the Polycomb group, a class of genes known to be required for proper regulation of the Bithorax Complex and Antennapedia Complex (ANT-C)(30). The Polycomb group includes more than 30 different genes which, while not necessarily similar in structure or function, are clearly involved in processes that involve all members of the group. Mutations in one member of the group are greatly enhanced by mutations in another member of the group and duplications of one gene frequently alleviate the effects of mutations in another member. These genes are necessary for the selective inactivation of regulatory domains of the two major homeotic gene complexes during development. Mutations in the Polycomb gene (Pc), for example, result in the apparent derepression

of the BX-C and ANT-C genes, resulting in the abdominal transformation of all body segments(31). In these mutant embryos, expression of the homeotic genes initiates normally and establishes a normal pattern under the control of segmentation genes but then fails to be maintained at later stages, resulting in inappropriate expression. The role of the Pc-group genes appears to be to maintain the state of gene activity that was established in the early embryo and, in particular, to keep in an inactive state those regulatory elements that were not initially activated in each segment. A possible function for Pc gene products might be to package the chromatin in such a way as to ensure that long-range activation is not possible except where desired. In their absence, zeste-like proteins might cause indiscriminate activation of inappropriate regulatory elements in inappropriate segments. Some Pc-group genes are also required for other cellular functions. The pco or E ( z ) gene, for example, is also necessar for cell proliferation and chromosome integrityiiz"32).The Polycomb gene has been cloned and immunostaining reveals the presence of its product bound at at least 50 sites on the polytene chromosome~(~'),including the BX-C and ANT-C loci. Interestingly, many of these sites correspond to the location of many of the other Pc-group genes, suggesting that these genes may be mutually regulating. Boundaries Between Activation Domains Equally important as long-distance promoter activation is the prevention of activation of promoters by extraneous distant regulatory regions. In many cases when genes with different patterns of expression lie in close proximity to one another, some measures would be required to block access to the promoter by the regulatory region of the other gene. This could be imagined to take place by two different mechanisms. One is the barrier or insulator mechanism. Sequences responsible for this kind of block would act as insulators protecting a gene from unwanted regulatory input from more distant elements. Another mechanism would involve the inactivation of a gene or regulatory region in the tissues where it is not needed, by packaging its chromatin in an inactive conformation, similar, perhaps, to heterochromatin. There are several observations that can be interpreted as evidence for elements that act as boundaries or insulators. One of these is the case of the mutational effects produced by insertions of the gypsy transposable element in the regulatory regions of a number of different genes, including the BX-C. These mutational effects are now known to be due not just to the insertion of the transposable element but to the interference with regulatory processes caused by the binding of a protein, the product of the su(Hw) locus, to its target sites within the gypsy element(34).In the absence of su(Hw) product, the gypsy insertion has no phenotypic effects. At the yellow locus, the insertion of gypsy between the enhancers for expression in the

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Fig. 3. Regulatory block circumvented by transvection at the Drosophila yellow locus. The yellow gene includes regulatory elements responsible for pigmentation in the larval mouth parts and denticles (larv), the adult bristles (br), the body cuticle (b) and the wings (w). The insertion of the gypsy transposon prevents the w and b elements from acting upon the promoter, but does not affect the br and larv elements. Pairing with another copy of the gene, in which part of gypsy, including the binding site of the su(Hw) protein, and part of the yellow gene itself have been deleted, allows the activation of the promoter in trans by a zeste-dependent transvection mechanism(lO).

wing and body and the promoter causes the loss of expression in these tissues but not in other tissues in which the expression is controlled by enhancers that lie downstream of the gypsy insertion. The present evidence suggests that the sequences to which the su(Hw) protein is bound act as a block in some way, preventing the access of the enhancer to the promoter. Interestingly, enhancer action in this case can be provided in trans by a zeste-dependent transvection mechanism(") (Fig. 3). Many mutations in the BX-C are caused by gypsy insertions (e.g. most of the bx mutations of the Ubx gene(35)) and many of their phenotypes could probably be better understood if we think of gypsy as interposing a block between regulatory elements and promoter. The Drosophilu BX-C is a fertile ground for examples of long-distance regulatory elements. This complex contains only three genes, Ubx, abd-A and Abd-B but their expression is governed by a number of regulatory domains that specify activity in different have proposed the parasegments. Peifer et existence of parasegmental domains within the complex, which need to be activated in order to allow the regulatory elements residing in them to controI promoter activity in individual sets of cells. Whether this activation takes the form of 'opening' their chromatin structure or suppying them with a means of contacting the promoter by a looping mechanism cannot be determined from the present data. Perhaps these are two ways of saying the same thing. However, if the boundaries between these domains of activation were to be removed, say by a small deletion, the result would be that a regulatory element would be enabled to control the promoter in an inappropriate parasegment. ) in fact, isolated mutations of Gyurkovics et u Z . ( ~ ~ have, this sort. The iab-6 and iab-7 regulatory domains, which control the expression of the Abd-B gene, are functionally separated by a boundary. The Fub-7

mutation is a deletion of 4 kb that apparently removes this boundary and causes the iab-7 control region to be functionally fused with iab-6 and be activated in parasegment 11 as well as parasegment 12. Gyurkovics et al. suggest that boundary sequences such as these may be the sites of action of the Polycomb-group genes. The products of these genes might assemble on these DNA sequences and keep the regulatory domains inactive by a mechanism similar to that involved in heterochromatin assembly. Heterochromatin Condensation and Position Effect Variegation About one third of the Drosophila genome, mostly residing around centromeres, begins to condense into heterochromatin in the early embryo and remains in this tightly packaged form throughout later development. When genes are placed next to heterochromatin by chromosomal rearrangements, they are subject to inactivation by the condensation effect that spreads out from the heterochromatic region over distances of several hundred kilobases. The effect differs from cell to cell but, once inactivated by heterochromatin, a gene stays inactive in the progeny cells. This phenomenon, called position effect variegation, can be suppressed or enhanced by mutations in a number of Su(var) genes whose products are believed to be involved in the highly cooperative assembly of hetero~hromatin(~~). Like the Polycornb-group genes, the genes involved in heterochromatin formation show dosage effects and duplications in one gene can at least partially substitute for deficiencies in other members of the group. Like the heterochromatin formation responsible for position effect variegation, the establishment of the patterns of homeotic gene expression occurs during embryonic development and is inherited by progeny cells. Results cited by Reuter et indicate that some of the genes involved in heterochromatin assembly also affect the control of BX-C expression. Two Su(var) genes have been cloned and ~ e q u e n c e d ( ~ ~and ' ~ ' ) one of them, Su(var)(3)7, encodes a protein with five widely spaced zinc fingers. Although the DNA binding properties of the two proteins have not yet been examined, the Su(var) (3) 7 sequence is consistent with its having a direct role in packaging chromatin; while many differences in detail can be pointed out, the multimeric assembly of heterochromatin bears many points of resemblance to the behavior of the large zeste aggregates involved in transvection. Silencers The phenomena of heterochromatin formation and of activation/inactivation of regulatory domains are reminiscent of the silencer elements that have been characterized in yeast. Silencers are DNA sequences to which certain specific factors bind, generate a chromatin structure reminiscent of heterochromatin and

repress the activity of promoters one or more kilobases away. Silencing of the mating t pe loci involves two DNA binding proteins, ABFlC4') and RAP1(42),and the products of the four genes SIR1 to SZR4(43),whose functions have many parallels with the action of the Pcgroup genes of Drosophila. Together or separately, the factors involved in silencing are also important for a number of other cellular functions including transcriptional activation, telomere function and DNA replication. Hofmann et U Z . ( ~ ) report that RAPl is associated with the nuclear scaffold and causes the attachment of silencer DNA to the nuclear matrix. However, as in the case of zeste, it is not clear whether RAPl is an integral component of a nuclear substructure or if it simply co-sediments with the scaffold because of a tendency to form fast-sedimenting aggregates. A study of protein-protein interactions between RAPl or zeste and other nuclear proteins should shed light on this. It is significant nevertheless that RAPl binding to the H M L locus DNA can form loops in vitro that are consistent with silencer-silencer and silencer-promoter looping. The similarity in properties and functions between these yeast proteins and the Drosophila zeste, Su(z) and Polycomb-group proteins is compelling and suggests that a more detailed comparison will be rewarding. In conclusion, the precise mechanisms responsible for long distance gene regulation are still unclear. However, the results reviewed in this article strongly suggest that certain common mechanisms underlie a collection of seemingly disparate phenomena. It is likely that transvection, long-distance gene regulation, the control of homeotic genes, transcriptional silencers, heterochromatin assembly and possibly also DNA replication, chromosome pairing and recombination require the activity of the same or related proteins that package the chromatin and arrange its disposition in the nucleus. Such packaging, with the aid of appropriate factors, may specifically favor or prevent looping and the consequent interaction of one part of the chromatin with another, including the interaction between distant regulatory elements and the promoter they control. Acknowledgements I am grateful to Pam Geyer, FranGois Karch, Allen Shearn and Rick Jones for providing me with important information before publication and to Su Qian for a critical reading of the manuscript. References 1 PTASHNE, M. (1988). How eukaryotic transcriptional activators work. Nature 335, 683-689. 2 GROSVELD, F., BLOMVAN ASSENDELFT, G., GREAVES, D. R. AND KOLLIAS, G. (1987). Position-independent, high level expression of the human kglobin gene in transgenic mice. Cell 51, 975-985. 3 LEWIS,E. B. (1954). The theory and application of a new method of detecting chromosomal rearrangements in Drosophila melanogaster. Am. Nar. 88, 225-239. 4 GELBART, W. M. AND Wu, C-T. (1982). Interactions of zeste mutations with

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Pirrotta is at the Department of Cell Biology, Baylor College of Medicine, Texas Medical Center, Houston, Texas 77030, USA.