Position Effects on Eukaryotic Gene Expression

ANNUAL REVIEWS

Annu. R ev

Cell Bioi. 1990. 6: 679-714 Copyright © 1990 by Annual Reviews Inc.

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POSITION EFFECTS ON Annu. Rev. Cell. Biol. 1990.6:679-714. Downloaded from www.annualreviews.org by University of Michigan - Ann Arbor on 11/03/14. For personal use only.

EUKARYOTIC GENE EXPRESSION Clive Wilson Department o f B rain and Co gnitive Sciences, Massach usetts I nstitute of T echnology, 77 M assach usetts Avenue, Cambridge, Massach usetts 02139

Hugo J. Bellen Howard Hughes Medical Institute, Institut e f or Molecular Genetics, B aylor College of Medic ine, Houst on, T exas 77030

Walter

J.

Gehring

Depar tment o f Cell B iolo gy, B iozentrum, University o f B asel, Klingelbergstrasse 7 0, CH- 405 6 B asel, Switzerland KEY WORDS:

enhancer detection, developmental gene regulation, chromatin, cis-acting regulatory elements, Drosophila

CONTENTS INTRODUCTION..............................................................................................................

680

TRANSCRIPTIONAL GENE REGULATION IN EUKARYOTES....................................................

68 1

POSITION EFFECTS ASSOCIATED WITH CHROMOSOMAL REARRANGEMENTS ............. ........ . ....

Positions Effects Induced by Enhancer, Silencer, or Promoter Elements ...... ...... ..... Position Effects Induced by Heterochromatin ....................... . .......... ....... . ............ .

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POSITION EFFECTS IN TRANSGENIC ANIMALS ....................................................................

Production of Transgenic Animals ........................................................................... Position Effects Induced by Heterochromatin .............................................. ............ .

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683 683 684 685 686 687

679 07 43-4 634/ 90/ 1 115-0 67 9$02 .00

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WILSON, BELLEN & GEHRING

Dosage Compensation and Genomic Imprinting ................................ . . . . . . . . . . . . . . . . . . . . . . . . Position Effects Induced by Enhancer, Silencer, or Promoter Elements ............ ......... Regulatory Sequences Conferring Position-Independent Expression .......................... Position Effects on Transgenes with Deleted Regulatory Sequences ... . . . . . . . . . . . . . . . . . . . . . . .

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ENHANCER DETECTION IN DROSOPHILA.......................... ................................................

P-Element-Mediated Enhancer Detection ............... . ................................................. Constructs and Generation of Insertion Strains ..... . .............. .................................... Staining Patterns.................................................. . .............. . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Identification of Genes .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Insertion Site Specificity of Enhancer Detectors ............ . . .. ........................ ..... ........ Analysis of Developmentally Regulated Genes ....... ..... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Further Applications of Enhancer Detection ......... ................................................... The Regulatory Architecture of the Genome ..... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Gene Regulation and its Evolution .................. ...................................... . . . . . . . . . . . . . . . . . . . . .

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Annu. Rev. Cell. Biol. 1990.6:679-714. Downloaded from www.annualreviews.org by University of Michigan - Ann Arbor on 11/03/14. For personal use only.

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687 690 691 693 694 694 694 695 697 699 700 701 702 703

ENHANCER DETECTION IN THE MOUSE ... . ........ . ................. ................................. . . ..... . . . . .

705

PROSPECTS....................................................................................................................

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INTRODUCTION

Scientists h ave known for many years th at the expression of a euka ryotic gene can be affected by its chromosomal position (see Sturtevant 1925; Dobzh ansky 1936; Lewis 1 950). Classically, this phenomenon is referred to as a position effect only ifit is not associated with an intragenic mutation; in molecular terms the transcription unit and at least the most proximal transcriptional control sequences of the gene should remain intact. Under this definition, regulatory sequences responsible for position effects must all sh are one common feature: they must be able to influence the transcriptional apparatus at some distance from a gene's transcription initiation site. This review is concerned with the transcriptional regulatory elements that cause classical position effects. Position effects th at infl uence the expression of genes relocated by ch romosomal rearrangements and pos ition effects on transgenes introduced into eukaryotic genomes are dis cussed in the context of established molecular models for the regulation of gene activity. Particular emphasis is given to examples th at provide information about gene regulatory mechanisms th at at present are poorly understood. In the final sections, we consider a new tech nique, called enhancer detection (or enhancer trapping), in which a minimally regulated reporter gene is introduced at random locations in the genome of an animal and is used as a sensor for genomic regulatory elements (O'Kane & G eh ring 198 7 ; A llen et al 1988; G ossler et al 1989). Th is method is proving to be particularly informative in the study of development and developmentally regulated genes (e.g. see Bier et al 1 989; Bellen et al 1989; Wilson et al 1 989) . Analysis of position effects on enh ancer detectors also promises to shed ligh t on more general questions concerning transcriptional control in complex organisms.

POSITION EFFECTS ON GENE EXPRESSION

681

TRANSCRIPTIONAL GENE REGULATION IN


EUKARYOTES

Activatio n of eukaryo tic gene expressio n and initiatio n o f gene tran scription by RNA po lymerase I I are contro lled by several different classes of cis-acting regulatory elements, no t all of which h ave probably been defi ned. The structure and function of some of these elements and the regulato ry f acto rs to which they bind h ave been reviewed extensively elsewhere (see Atch ison 1 988; Dynan 1989; Mitchell & Tjian 1 989; Johnso n & McKnight 1989; Ptashne 1988; Levine & Manley 1989). O nly a brief summary is pro vided h ere. Regulato ry elements may be classified into two groups, although the distinctio n is becoming increasingly blurred. The first group co nsists of elements that can o perate o nly when in clo se proximity to the transcriptio n initiation site. A goo d example of such an element is the T AT A box, which plays a role in defining the po sitio n of the tran scription start sites in many genes (Beno ist & Chambo n 198 1 ) . These elements are compo nents of a gene' s promo ter, a regio n extending for abo ut 100 base pairs (bp) immediately upstream of the transcriptio n unit. However, some sequences in promo ters can also act at several h undred base pairs from the transcriptio n start site (e.g. Bohmann et al 1987; Parslo w et al 1987; see also B ienz & Pelham 1986). They may be classed in the second group o f regulato ry elements, which includes all sequences that can regulate promo ters at a distance. The best- ch aracterized members o f th is group are the enh ancers and " negative enhancer" elements called silencers, which bo th act in an orientatio n-independent fashio n (fo r reviews, see Serfling e t a1 1985; Atch iso n 1988). Enhancers, silencers, and pro mo ters can individually impo se precise spatial and tempo ral restrictio ns o n gene expression. These elements are compo sed of a number of short sequence mo dules, each of which interacts with o ne or mo re transcriptio nal regulatory pro teins (e.g. O ndek et al 1 988). Clustered combinatio ns of these modules, which to gether may bind a variety of stimulato ry and inh ibito ry f acto rs, f requently functio n as auto nomous regulatory elements (see Dynan 1989). In o ther cases, the multicomponent elements may need to interact with each o ther to pro duce their regulatory specificity, e.g. enhancer/ promo ter interactio ns (Johnso n e t a l 1 989; Fisch er & Maniatis 1988). This co mbinato rial contro l mech anism is extremely flexible, since a limited range o f regulatory modules and developmentally regulated transcriptio n facto rs can to gether direct many different spatial and temporal patterns of expression. The po tential fo r h etero lo go us co ntro l elements to cooperate with or o verride each o th er in such a scheme is th e basis for many of the po sition effects described in this review.


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Several lines of evidence suggest that chromosomal regions containing actively transcribed genes are structurally different from transcriptionally inactive regions. In some organisms, light microscope analysis has revealed that transcriptionally active chromosomal domains are specifically decon densed in certain specialized interphase nuclei (e.g. see Callan 1 982; Ash burner et al 1 974). Indeed, biochemical studies using the nuclease DNase I suggest that chromatin may generally assume a more accessible active configuration in and around transcribed regions (Weintraub & Groudine 1 976; Garel et al 1 977; reviewed by Yaniv & Cereghini 1 986). Often these active domains also contain sequences that are hypersensitive to DNase I digestion; such sites frequently reflect structural changes associated with the binding of regulatory factors to enhancer-like sequences and, in some cases, are thought to mark the boundaries of the active chromosomal domain (e.g. Fritten et al 1983; Grosveld et al 1987). Gene activity also correlates with binding of specific chromosomal proteins to the active region (e.g. Allis et al 1 986; Dorbic & Wittig 1 987) and with decreased methylation of cytosines in the DNA itself (see Cedar 1 988). The correlation between specific structural properties and gene activity has led to the proposal that chromosomal domains may represent regu latory domains, which are isolated from regulatory sequences in neigh boring domains and must be initially activated before the expression of genes within them can be turned on by other regulatory proteins. Although decreased methylation of DNA may contribute to the maintenance of a regulatory domain's activity (Cedar 1 988), it has been difficult to dem onstrate that any of the structural changes observed are actually involved in the initial activation process. Furthermore, there is much debate con cerning the DNA sequences that define the extent of chromosomal domains and that might lead to their activation. The nuclear scaffold, or nuclear matrix, (e.g. M irkovitch et al 1984) is the insoluble matrix remain ing after several nuclear extractions designed to remove chromosomal proteins such as histones. It contacts specific sequences spanning several hundred base pairs that often lie near or within genes and are known as scaffold-associated regions (SARs) or matrix-associated regions (MARs) (for review, see Gasser & Laemmli 1 987). These sequences potentially define the attachment sites for looped scaffold-associated chromosomal domains. Because SARs are often located relatively near to enhancer or promoter sequences (Cockerhill & Garrard 1 986; Gasser & Laemmli 1 9 86), it has been proposed that the scaffold may draw regulatory elements into a restricted region in the nucleus and perhaps may isolate the chromosomal domains defined by the SARs from regulatory activity in neighboring domains. Such speculation, however, remains somewhat controversial, since there is no proof that the nuclear scaffold exists as a discrete structure


683

in interphase nuclei or th at SARs are bound to it in vivo (e.g. See Cook 1988; J ackson et al 1990). Recent experiment s in t ransg enic animals also suggest that , in some cases, non-SAR sequences may contribut e to the establ ishment of regulatory domains (see section on t ransg enic animals).

POSITION EFFECTS ASSOCIATED WITH


CHROMOSOMAL REARRANGEMENTS

Position Effects Induced by Enhancer, Silencer, or Promoter Elements There are numerous examples of chromosomal rearrangements th at alter the expressi on of a g ene near the chromosomal breakpoint . These rearrangements may be developmentally prog rammed or may simply result from a mutagenic event. Frequently, the affected gene is fused to another

t ranscription unit and comes under the cont rol of the latter' s promoter (e.g . Pays & Steinart 1988; Schneuwly et al 1987 ) . There are also well document ed cases of chang es in expression in which th e normal promoter remains attach ed to its gene, such as those changes induced by the pro g rammed rearrangements of th e immunog lobulin h eavy ch ain g ene and the yeast mating t ype locus (Gillies et al 1 985). Enh ancer or silencer element s, wh ose proximity to the promoter is altered by the rearrang ement , are responsible for these prog rammed ch anges, which represent classical position effects. In a similar way, enhancer-like sequences in certain t rans posable elem ents may also modulate th e activity of promoters near which they insert (e.g . Errede et a1 1 987 ; Geyer & Corces 1 987 ; Geyer et aI 1988). In these examples, as in all cases of position effect s affecting t ranscription, it is essentia l that the i nt erfering cis-acti ng regulat ory elements h ave a dominant effect on any normal cont rol sequences present in the affected gene. In the fruit fl y, Drosophila meianogaster, normally cis- acting sequences on one ch romosome can affect the expression of a g ene on th e homolog ous chromosome (for reviews, see J udd 1988; Wu & Goldberg 1989). This phenomenon, known as t ransvection, is most clearly seen in some mutant fl ies, where sequence ch ang es on the mutant ch romosome affect the expression of a wild-type g ene on the nonmutant chromosome. The pairing of h omologous chromosomes is nondividing somatic cells of Drosophila probably plays an important role in mediating t ransvection effects. Although the t rans-acting sequences responsible may directly modulate the initiation of t ranscription from the affected g ene, it is possible that sequences i nfl uence the production oftranscript s by other less conventional mech anisms (see J udd 1988).

684



Position Effects Induced by Heterochromatin

Long before the molecular structure of the gene had been determined, genetic studies, especially in Drosophila, had already indicated that the expression of a gene could be affected by its location (e.g. see Muller 1 930; Dobzhansky 1 936). In some chromosomal rearrangements, investigators observed that several genetic loci near a breakpoint are expressed in a mosaic fashion in mutant flies and thus reflect inappropriate gene inac tivation in random groups of cells (for review, see Spofford 1 976). This phenomenon of position effect variegation is typically associated with rearrangements that place the affected genes in close proximity to het erochromatin. In many or all eukaryotic cells, extensive regions of the genome, particularly centromeric and telomeric sequences, are packaged into so-called "constitutive" heterochromatin. Heterochromatin consists of highly condensed chromosomal material that is most likely trans criptionally inactive (Brown \966). In variegating mutants, transcriptional inhibitory activity spreads from the heterochromatic region into the trans located regions that are normally active and euchromatic; its most pro nounced effects are on genes adjacent to the breakpoint, but it can act on genes located several hundreds of kilobase pairs (kbp) away. In some cases, it has been demonstrated that the polytene chromosome bands containing the variegating genes become more condensed or more irregular in a proportion of cells in variegating mutant animals (e.g. Hartman-Goldstein 1967; Kornher & Kauffman 1986). Therefore, position effect variegation

is probably caused by a variable degree of packaging of normally euch romatic regions into a transcriptionally inactive heterochromatin-like con formation. On the basis of a molecular analysis of variegating mutations and revertants of these mutations, Tartof et al ( 1 984) have proposed a simple model to explain how heterochromatin domains are formed. They suggest that specific sequences defining heterochromatic boundaries are replaced by normally euchromatic regions in variegating mutants and that this substitution allows the nondelimited heterochromatic domain to extend for variable distances from its normal initiator sites. Recent studies of position effect variegation promise to provide an entry point for the molecular analysis of heterochromatic structure. An estimated 20 to 50 essential genetic loci can dominantly modify variegation in Drosophila. In most cases, deleting one copy of these modifiers sup presses the variegated phenotype; deletion of the other modifiers enhances the phenotype (for detailed discussion and review, see Eissenberg 1 989; Tartof et al 1 989). Strong evidence suggests that two of these modifying loci encode structural proteins for heterochromatin (James & Elgin 1 986; see Eissenberg 1 989; Reuter et al 1 990), and it is assumed that several



685

others will also encode such prot eins . Indeed, th e fact that ch anging the dosage of so many loci can affect variegation is consi stent with a model in which assembly of the struct ural unit s comprising h et eroch romatin is a complex multicomponent process (Locke et al 1988). Cent romeric and t elomeric regions in the genome of all mammalian cells are packaged into constitutive h eterochromatin. I n addition, one of the two X chromosomes in females is randomly selected in each somatic cell t o be packaged int o inactive facultative h et eroch romatin during early development (see G arder & Riggs 1983). In cert ain t ranslocations of auto somal regions to the X chromosome, autosomal genes in proximity to the breakpoint may be inactivat ed together with the adjacent X chromosomal genes (Cattanach 1963; Russell 1 963; for review, see Eich er 1970). As in Drosophila, the inhibit ory effect spreads from the normal h eteroch romatic domain and decreases with distance from the breakpoint . Whether or not all mammalian h et eroch romatin is struct urally related t o heteroch romatin in Drosophila is not clear, h owever. Although constitutive centomeric and telomeric h et erochromatin of both mammals and flies is mainly assembled around sh ort , repetit ive sat ellit e sequences, these sequences are a minor component of facultat ive X ch romosome h et erochromatin. Furthermore, the maint enance of h eterochromatic regions in mammals may be partly mediated by meth ylat ion (see Lock et al 1987); by cont rast , few, if any, cytosines are methylat ed in the Drosophila genome (Urieli-Shoval et al 1 982). Indeed, apparent ly similar reg ions of h eteroch romat in may differ at the structural level even within the same organism; for example, some evidence indicates that not all regions of h et eroch romatin in Drosophila are st ructurally identical (see Locke et al 1988) and th at the inhibitory activity of facult ative h et erochromatin may differ in different cells (Krum lauf et a1 1986; see section on t ransgenic animals). Screens for mammalian genes h omologous t o any newly identifie d h eteroch romatic struct ural proteins should help to identify any features th at are common t o all forms of eukaryotic h eterochromatin. POSITION EFFECTS IN TRANSGENIC ANIMALS

S ince cloned DNA sequences introduced into eukaryotic cells or whole animals are generally no more than 1 0 to 20 kbp in length , the gene promoters within these sequences would seem favorably positi oned t o be affected by any dist antly acting genomic regulat ory element s at th e site of integration. Although expression levels of transgenes at different chromo somal locations may vary great ly, however, frequently the expression pat tern of a transgene consistently resembles the expression pattern of its endo genous counterpart , provided that flanking


686


enhancer, silencer, and pro mo ter elements of the gene are included in tht co nstruct. In o ther wo rds, the quantity of transcripts in the no rmall) expressing tissues may vary co nsiderably, but the expressio n patterr usually does no t sho w qualitative changes. Po sitio n effects have been described in a number of diff erent cell cultun transfo rmatio n systems and transgenic animals (e.g. see reviews by K uch· erlapati & Sko ultchi 1984; Palmiter & B rinster 1986; Nagy et al 1 98 5 Jones et a1198 5; Scholnick et a1 1 983; Spradling & Rubin 1983; Go ldberg el al 1 983). Here, we co ncentrate on results f ro m experiments with transgeni( animals, since the diverse cell types of the who le o rganism pro vide a mon complete assay system fo r tissue-specific by cultured cells. O ur purpo se i s no t to catalogue all reported po sitior effects, but to review how po sitio n effects can reveal the actio n of differen! classes of eukaryo tic regulato ry elements. To do this, we fir st standard transfo rmatio n techniques that are applied to the eukaryo ti( systems mo st commo nly used as transgenic ho sts: the mouse and the f ruil fly .

Production of Transgenic Animals Transgenes are f requently introduced into somatic and germ line cells of miCt by micro injectio n of DNA into the pro nucleus of f ertilized eggs (fo r review, see Palmiter & B rinster 1986; fo r alternative techniq ues, see Jaenisch 1988 : Go ssler et al 1989 and the section o n enhancer detectio n in the mo use). In mo st transgenic lines, integrated genes are fo und at a single genomic lo cus, but t hey are usually arranged in t andem head-to-tail arrays, co nsisting oj between two and several hundred co pies. It is unkno wn if the artificial clustering of the transgene' s regulato ry elements in this co ncatenated arrangement can affect the expressio n of the transgene (see discussio n oj the /i -glo bin locus). Certainly in some cases, pro karyo tic sequence5 included in co nstructs can have significant no nspecific inhibito ry effects in this repetitive co nfiguratio n (e.g. Townes et al 1 98 5 ; K rumlauf et al 198 5a), A n additio nal co nsequence is that regulatory elements adjacent to an insertion site o ft en need to act o ver many tens o r even hundreds of kilo base pairs to infl By co ntrast, the standard method fo r producing transgenic Drosophila, P-element-mediated transfo rmation (Spradling & Rubin 1982; Rubin & Spradling 1 982) no rmally generates fl ies carrying single copy insertio ns with defi to new genomic locatio ns by a specific transpo sase; the P-transpo sase gene co mprises nearly the entire extent of a f ull-length auto no mo us P-element. Pro vided the transpo sase activity is supplied in trans, however, any sequence that is fl anked by short cis -acting P-element termini can be


687

integrated into the genome by the transposition mec hanism. In P-element mediated transformation, embryos are typically c o-injected with a P element construct c ontaining the transgene and with a helper plasmid encoding transposase. In som e germ line c ells, the transgene is integrated into the genome; transgenic progeny can be identified using a marker gene carried in the P-element construc t (e.g. see F igure I for a typical c onstruc t).


Position Effects Induced by Heterochromatin K rumlauf et al (1986) have investigated the expression of a- fetoprotein transgenes within an X c hromosomal inse rtion in transge nic mice. T he y found no expression i n embryonic c ells in which the X c hromosome was inactivated and thus demonstrated that fl anking heterochromatin can have a dominant inhibitory effect. S urprisingly, this result did not apply to the inactive c hromosome in extraembryonic c ells, which therefore may employ a somewhat different mechanism for sile ncing gene s. In f act, other e xperi ments in c ell c ulture suggest that the inhibitory effect of heterochromatin on transgenes is neither absolute nor irreversible. I n these experiments, transformed c ells are selected on the basis of marker gene expression, so that totally silenc ed, heterochromatic transgene insertions c annot be identifie d. However, insertions in highly repetitive, heterochromatin associated satellite DNA are found, and they often express the marker gene thymidine kinase (tk) in an unstable fashion (Butner & Lo 1986; T alaric o et al 1988 ) ; tk� revertants and tk+ rerevertants are p roduced at high frequency even under nonselec tive c onditions and are usually not associated with obvious c hromosomal rearrangements. T hese results may refl ec t the fact that not all heterochromatin is transcriptionally inactive (Hilliker et al 1980), although transgenes inserted within it c learly behave differently from those inserted in euchromatin. P- elements seem to show a strong preferenc e to integrate into euch romatin (Eggleston et a11988; for review, see Engels 1989). F or this reason and perhaps because , like transformed c ells, transformed Drosophila are identified on the basis of marker gene expression, few P-element- assoc iate d transgene insertions are located near or in heterochromatin. There are, howeve r, a few reports of inhibition and variegation of expression of a transgene inte grated near c entrome ric or telomeric sequences (e.g. Gehring et a1 1984; Steller & Pirrotta 198 5).

Dosage Compensation and Genomic Imprinting Other f orms of gene reg ulation have bee n de scribed that, like the inhibitory effects of heterochromatin, may affect large c hromosomal domains and transgenes within those domains. F or example, in order to c ompensate for the difference in gene dosage, most X c hromosomal genes in male Dro-

688


A E.coU P·lacZ

m"'�IIUII"

eye color marker

ori

AmPR

IlS.l-

o

PL1

gene X


B.

-

-

Elx

E2x

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JJ

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detector

r--

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The principles of enhancer detection. (a) Schematic diagram of a P-element

enhancer detector. The enhancer detectors used by Bellen et al (1989) and Bier et al (1989) are of a similar design. They contain an eye color marker gene (rosy+ and white+ respectively)

and the P-lacZ reporter gene first used by O'Kane & Gehring (1987). The E. coli origin of replication

(ori) and ampicillin resistance gene (AmpR)

allow genomic sequences near to an

insertion to be selectively cloned in bacteria (see Pirrotta 1986); this DNA is surrounded by polylinkers (PL I and PL2) containing different restriction enzyme cutting sites. If a fragment containing these

E. coli sequences and adjacent genomic DNA is generated by digestion of

DNA from an insertion strain with a restriction enzyme that cuts in PLi (or PL2), only this fragment will produce ampicillin-resistant bacterial colonies when the digestion products are circularized and transformed into E. coli. The enhancer detector is flanked by the cis-acting sequences required for P-transposition (striped boxes; 5' P and 3' P).

(b) Gene detection with

enhancer detectors. If an enhancer detector inserts near or within a gene, the expression of the reporter gene may come under the control of some or all of that gene's regulatory sequences. In this case, the enhancer detector has disrupted the promoter of gene X, perhaps affecting the function of the gene (for similar characterized examples, see Wilson et al 1989 and text). Expression from the P-transposase promoter of the P-lacZ fusion gene is controlled by the two enhancers of gene X (EI and E2) and possibly sequences within its promoter; the p-galactosidase staining pattern of the insertion strain therefore reflects part or all of the expression pattern of gene X.

are transcribed at twice the rate of their duplicated counterparts in females (for review, see Jaffe & Laird 1 986). Autosomal transgenes integrated into the X chromosome can become dosage compensated (e.g. Scholnick et al 1 983; Spradling & Rubin 1 983). Evidence relating to the

sophila



689

chromosomal structure at the breakpoi nt of an X-autosome translocati on suggests that the sequences medi ati ng dosage compensati on do not act over such extensi ve distances as those that direct heterochromatizati on (Lakhoti a 1970). In fact, P- element-mediated transformati on experi ments have shown that for a number of X-li nked genes dosage compensati on i s mediated by sequences lyi ng i n close proximity to the gene (e.g. see Hazelri gg et al 1984; Pi rrotta et al 1985; Levi s et al 1985 b). Therefore, al though dosage compensati on affects a broad chromosomal domain, i t may b e i mposed b y relati vely short-range elements, such a s enhancers, each acti ng on only one or a few genes. Experiments usi ng diff erent chromosomal translocati ons in the mouse (e.g. Cattanach & Kirk 1985; Cattanach 1986) show that one copy of some chromosomal regi ons must be derived from a specific development to proceed (e.g. one copy of chromosome six must be of paternal origi n). These regi ons, which consti tute up to one quarter of the genome, are sai d to be i mpri nted (f or reviews, see S olter 1988; S api enza et al 1989). They are thought to contai n one or more i mportant devel opmental genes whose expressi on i s reversi bly i mpai red, perhaps by methylati on, when they are deri ved from the germ line of the nonpermi ssive parent. Experi ments usi ng the large translocati ons presently available can not defi ne th e specific S everal transgene i nserti ons reflect i ng. They are differentially methylated according to parental ori gi n (for review, see Surani et al 1988). In most cases, these di fferences i n methyl ation do not correlate wi th altered expressi on of the transgene. O ne excep ti on i s an i nserti on of a fusi on gene controlled by the R ous sarcoma virus long terminal repeat (R SV LTR) sequences; only paternally i nherited transgene copies, which are hypomethylated, are expressed ( S wain et al 1987). In thi s case, the parental germ line provi des a domi nant swi tch to acti vate or r epress the gene, but the spati al specifi to be controlled by the R SV LTR . At present i t i s not known whether these effects on transgenes reflect i nserti on site i mposed by the parental germ line. In fact, the observati on that the expressi on of some transgenes in certain genetic backgrounds may be irreversi bly i nacti vated in all future generati ons (Hadchouel et a11987) suggests that i mprinting i s not necessari ly i mposed only by the parental germ li ne. Allen et al (1990) recently demonstrated that a particular trans gene i nsertion may become progressi vely more methylated over several generati ons i n an appropri ate genetic background and thi s methylati on may finally lead to i rreversi ble i nacti vati on of gene expressi on; these experi ments may gi ve some i nsi ght i nto the grandparental effects observed in some human genetic di sorders (Reik 1989).

690



Position Effects Induced by Enhancer, Silencer, or Promoter Elements l aeni sch et al (1981) first showed t hat chromosomal position can affect eukaryotic t ransgene expressi on when t hey analyzed viral gene expressi on and vi ral activati on i n di fferent Moloney leukemi a provi ral i nserti ons in t he mouse. The majority of positi on effect s modulating t he expressi on oj both t hese viral genes and other t ransgenes probably reflect t he action oj si lencer- or enhancer-like activities at t he site of i nt egrati on. Si nce t hese effect s are basi cally a product of t wo vari ables t hat are not well defined, t he potency of t he site-specific regulat ory element s and t he suscepti bility of t he t ransgene' s cont rol sequences, it i s not surpri si ng t hat t he range of positi on effect s on different t ransgenes can be quite diverse. O ne general observation can be made, however. When a t ransgene cont ai ns sufficient regulatory i nformation t o di rect significant rates of cell type-speci fic t ran scri ption, t he type of cells i n t he ani mal i n which t he t ransgene i s expressed (it s " spati al pattern of expression") i s not generally affected by t he chro mosomal location of t he t ransgene (e.g. see Palmiter & Bri nst er 1986; for excepti ons, see Chada et a1 1985; Shani 1986) . In other words, the cont rol sequences of transgenes are usually domi nant over t he adjacent genomic regulat ory element s at t he i ntegrati on sit e i n determi ni ng t he cells in which t he t ransgene is expressed. At least t wo mechani sms may be i n volved. Fi rst, elements i n t he transgene wit h regi on-specific acti vity may suppress ect opic t ranscri pti on; element s wit h such acti vities have now been found in many genes (e.g. Wi not o & Baltimore 1989; for review, see Atchi son 1988). Second, many cont rol element s are more active in combination wit h t hei r own promoters t han wit h a heterologous promoter; conversely, complex promoters or promoter/enhancer com bi nati ons are frequently refractory t o t he acti on of foreign regulat ory sequences (e.g. Kolli as et al 1987; Fi scher & Mani ati s 1988; J ohnson et al 1989). Indeed, in yeast and perhaps i n Drosophila even TA TA box bindi ng protei ns may respond selectively t o t ranscri pti on factors (Chen & Struhl 1988; Corbi n & Mani ati s 1989). Despite t he restrictions on ect opi c t ransgene expression, sit e-specifi c regulat ory element s can produce consi derable modulations i n t he level of properly locali zed t ranscri pts; thus t he t ransgene i s not i solated from t he cooperative or i nhi bit ory regulatory effects of nei ghbori ng sequences. Such regulat ory effect s are usually so ext reme i n mi ce t hat t here i s no correlati on between expressi on level and transgene copy number (see Palmiter & Bri nster 1986). Certai n observati ons suggest t hat many of t hese " quan titative positi on effects" are probably i mposed by developmentally regu lated cont rol sequences. For example, t he different spatial component s of



691

the co mplex a -feto pro tein tra nsgene ex pre ssio n patter n are differentially mo dula ted by neighboring regulatory e le me nts at an insertio n site , but the relative e ffect o n each compo ne nt varie s with c hromo somal location (Krumla uf et al 1985b). This phe no me no n is consistent with the actio n o f spatially regula ted, insertio n site-specific ge nomic contro l e lements o n t he a-fetoprotein gene 's pro moter . Similarly in Drosophila, the expression of the white gene , whic h is nor ma lly transcribed thro ugho ut the e ye, is restric te d to specific (e .g. Levis et aI1985a). Distinc t spatial restrictio ns o n gene expressio n are see n in different strains. So me , but no t a ll, o f these po sition effects o n the white gene may reflect impo sitio n o f a temporal rather than a spatial restr iction on transge ne ex pressio n (see Levis et al 1985a) . One interpret atio n o f all t he se o bserva tio ns is t hat genes are normally buffered fro m qua ntitative po sitio n e ffec ts b y ex te nsive interge nic spacer regio ns missing in transgenic constr ucts. A n a lternative ex planation is that discrete seque nce s specifying a define d regulatory domain are absent. Recent re sult s support the latter hypothe sis.

Regulatory Sequences Conferring Position-Independent Expression Distant regulatory seque nce s have bee n ide ntifie d gene locus t hat stimulate e rythroid-specific f3 -glo bin expression a nd make it almo st to tally refractory to e ve n quantitative po sition e ffects in tra ns genic mice (Gro sveld e t a l 1987; Talbo t e t a l 1989) . These seque nces are located more t ha n 50 kbp upstream o f t he f3-glo bin gene a nd include cl us tered erythroid- specific DNase I super-hyper sensitive site s; some frag ments co ntaining o nly o ne of these sites a lso confer po sitio n indepe ndence in transge nic mice (F . Gro sveld, per so nal communicatio n; see a lso Tua n et al 1989; Collis et a l 1990 for a na lysis i n c ultured cells) . L ike many other se quence s conta ining such tissue-specific f3-glo bin re gulatory re gio n beha ve s as a stro ng e nha ncer . U nlike many o ther e nhancer s, however , this domina nt co ntro l regio n (DCR) can impo se its activity u po n co mplex he terologous pro moter s that wo uld nor ma lly o nly direct a completely differe nt developmenta lly regulate d expre ssio n pat tern (Blom van Asse nde lft e t al 1989). Intere stingly, all or par t of the DCR is deleted in so me forms o f yt5f3-thala ssaemia in which the f3 -glo bin gene itself remains intact but tra nscr iptio na lly silent (Taramelli et al 1986; Dr iscoll e t al 1989) . Recent ex perime nts indicate tha t t he human CD2 gene, which e ncodes a T cell-specific sur face marker, also co ntains a DCR in its 3' flanking regio n (Greave s e t al 1989). The DCR ha s similar pro p ertie s to the {i-glo bin DCR , but its activity is T cell specific.


692


Some of t he propert ies of DCRs, suc h as t heir position-independen regul ation of gene expression and t heir eff ect s on het erologous p romot en suggest that they are not simpl e enhancer el ements. Alt hough experiment by one group hav e indicated t hat t he f3-g10bin O CR does not c onsistentl: c onfer position independence (Ryan et aI1 989), it has been suggested t ha many of t he mice anal yzed in t his rep ort were mosaic s and t hus t h transgene c op y number c ould not be est imated accurately (for discussion see C ollis et al1 990). Other groups (Curtin et a11 989; F . G rosveld, persona communicat ion) observ e t hat the l ev el of O CR-induced t ranscription p e gene c op y i s rel ativ el y c onst ant i n diff erent mice and drop s signific antl: onl y when many c opies are integrated. This dec rease perhap s reflect interference between artificiall y clustered t andem arrays of regul at or: element s t hat may alt er c hromatin structure at t he insertion site. Therefore alt hough it is difficult t o rule out t he p ossibilit y t hat DCRs merely rep resent an extremel y p ot ent form of enhancer, an alt ernat iv e and appeal inJ hyp ot hesis is t hat , in addit ion t o t heir enhancer-like act ivit y, t hey in som way define an isolated regul atory domain (Grosveld et al 1987). In this regard, a region c ontaining t he 5' SAR of t he c hicken l ysozym gene has recently b een shown to impose a limited degree of position indep endent exp ression on t he lysoz yme gene in stabl y t ransfo rmed cell (Stief et al 1 989). Interestingl y, t his region, which does not include t hl c haracterized l ysozyme enhancer, stimul at es as well as stabilizes exp ressiOl level s . Because t he 5' and 3' SARs of t he l ysozyme gene c orresp ond witl the borders of a 1 9-kbp DNase I- sensitive domain (Phi-Van & Stratlinl 1 988), it has been suggested that t hese seq uences flank defined regul at ory domain enc omp assing t he l ysozyme gene. F urthe anal ysis of DCRs will be required to ascert ain whether t hey c ontail element s t hat est abl ish a regul atory domain and whether t hese element: al so define the st ructural borders of t hat domain. In cont rast t o t he mouse, expression l ev el s of many t ransgenes are rel a t iv el y unaffected by c hromosomal l oc ation in Drosophila (e.g. Schol nid et al 1983; Sp radl ing & Rubin 1 983). There are t wo p robabl e explanations F irst , sequences in P-element c onst ruct s may buff er t he eff ects of site specifci p osition eff ect s on a minimal transgene p romot er lying sev eral kil obas( p airs from t he border of a P -el ement c onst ruct are significantly reduce( in c omp arison t o effect s on a t erminal minimal p romoter (Hiromi 8Gehring 1 987; Bellen et al 1 989; Y. Hiromi, personal c ommunicat ion) This phenomenon may refl spacer DNA and the presenc e of sequences in the construct that specificall) inhibit the act ion of external control elements. The second explanation fOI t he limit ed quantitative position eff ect s observ ed is t hat sequences direct-



693

ing positio n-indep endent, developmentally regulated gene exp ressio n in Drosophila are p ro bably clustered into much mo re co ndensed domains than in the mo use as a co nsequence of the small size of the Drosophila geno me. Fo r some developmentally regulated Drosophila genes, 5' and 3' SARs appear to delimit a chromo somal do main enco mp assing the gene. These SARs sometimes map near o r at the site o f a gene regulatory element ( Gasser & Laemmli 1986). Altho ugh absence of the 3' SARs in thefushi tarazu (ltz) gene and the Sgs4 gene may comp romise positio n-independent transgene exp ressio n ( Hiromi & Gehring 1 985; McNabb & B eckendo rf 1 986), o ther exp eriments suggest that positio n-independent co ntro l is no t greatly affected when bo th the 5' and 3' SARs are absent ( Sp radling & Rubin 1 98 3; Dearolf et al 1 989; 1. P ick & A. Schier, p ersonal com munication) . The imp licatio n is that even if some SARs ca n iso late genes within regulato ry domains in Drosophila, they are no t the o nly sequences with this capability. In summary, altho ugh the evidence fo r the existence of eukaryo tic regu lato ry domains is fairly stro ng, it seems likely that diff erent classes o f elements may define diff erent do mains. Fo r example, DCRs may b e rela tively specialized, rare elements that are o nly required to regulate co m plex gene lo ci; this may exp lain why transgenes in the mo use are o nly infrequently exp ressed in ectopic sites, when their specificity would be exp ected to be altered by any neighbo ring DCR. It remains unclear whether elements that iso late regulato ry domains from neighbo ring co n tro l sequences function by defining the attachment points of a looped regulatory do main to the nuclear scaffold or some o ther nuclear structure, o r whether o ther mechanisms can be emplo yed to block the action o f distant regulato ry elements. A s new assays are developed to identify the sequences that define regulatory domains, it is hop ed that a mo re unifie d mo del for higher o rders of gene regulatio n will establish a clearer link between chromo somal structure and gene regulatio n.

Position Effects on Transgenes with Deleted Regulatory Sequences Absence o f specific regulatory regions in transgenes may lead to po sition dep endent ectop ic exp ressio n in transgenic animals ( e.g. P inkert et a1 1 987) and p resumably refl ects the lo ss o f silencers o r o ther elements that restrict the actio n of hetero lo go us enhancer- like sequences. This effect is mo st ex treme in co nstructs carrying minimal p romo ters, where very few, if any, silencing sequences are p resent (e.g. Hiro mi & Gehring 1987; Ko thary et al 19 89). I n this case, the exp ressio n p attern i s determined by the regulatory

694


elements at the site o f insertion. T his is the basis o f the enhancer detectiol technique. ENHANCER DETECTION IN

DROSOPHILA


P-Element-Mediated Enhancer Detection B ecause even complex transgene p ro mo ters can respond to the activity 0 nearby developmentally regulated genomic co ntrol elements, O'Kane � Gehring ( 1 987) reaso ned that a repo rter gene transcribed from a minima p ro mo ter and integrated at random genomic lo cations sho uld act as ; much mo re efficient and faithful senso r o f such elements ( fo r a simila system in cell culture, see Hamada 1986) . They designed a P- elemen construct, which contains a translatio nal fusion between the P- transposas, gene and the E. coli f3-galacto sidase (lacZ) gene. Expressio n o f this repo rte gene, which is routinely visualized by staining with a chromo genic sub strate fo r the bacterial enz yme, is co ntro lled by the weak, but p ro babl: co nstitutive, P- transpo sase p ro mo ter. The p ro mo ter seems ideally p lace( to respond to regulatory sequences located o n the 5' side o f an insertion since i t initiates transcription a t a site about 100 b p from the 5 ' terminu:

of the P-transpo so n ( see Figure I). O'Kane & Gehring ( 1 987) generated by microinj ectio n abo ut 40 trans genic Drosophila lines that showed developmental regulatio n o f lacL exp ressio n in embryo s. Mo st strains stained differently, indicating that th� p attern o f exp ressio n o f the reporter gene was dependen t on the in sertioI site. These results enco uraged several gro up s to p erfo rm mo re extensiv� enhancer detecto r screens using a new metho d fo r generating large nUll bers o f independent insertio n strains. B elow, we discuss the technique am its impact o n the study o f Drosophila development and gene regulatio n.

Constructs and Generation of Insertion Strains B ellen et al ( 1 989) and B ier et al (1989) have emp lo yed two redesigned p. element constructs to perfo rm l arge-scal e enhancer detecto r screens ( set also Wilso n et al 1989 and Figure I). These co nstructs contain the P-lac2 fusion gene used by O 'Kane & Gehring ( 1 987) and an eye color markel gene fo r selectio n o f transfo rmed fli es. rep licatio n for E. coli and the f3-lactamase gene, which confers ampicillir resistance in bacterial cells. These E. coli sequences allo w genomic regio n! flanking ( Perucho et a1 1 980; Steller & Pirro tta 1 986; Pirro tta 1986; see Figure I). The generatio n o f fl integrated in the genome ( Ro bertso n et al 1988; Coo ley et al 1 988) ha� allo wed the development o f a convenient alternative technique to micro·



695

injection f or the p roduction of la rge numbers of transgenic lines. In this "jump start" technique ( Cooley et a l 1988), a P-transp oson, such as a n enhancer detector, is first introduced into fl ies by microinjection. I t may then be effi ciently excised a nd, in some cases, reinserted a t new genomic loca tions in the germ line of fl ies that contain both the P-tra nsp oson a nd the stable source of tra nsp osa se. Those p rogeny, which carry a new insertion but have not retained the transposa se gene, a re selected using a combination of genetic markers ( e.g. for diff erent schemes see Bier et a l 1 989; Bellen e ta I 1 989). Therefore, the genera tion o fnew enhancer detector strains is reduced to a simp le series of genetic crosses a nd scr eens. Since in the p resence of a highly active transposa se source, most individua ls produce at lea st one fl y with a single novel insertion, the technique makes feasible the genera tion of hundreds or thousa nds of lines with diff erent insertions (called tra nsp osants).

Staining Patterns Enhancer detector strains have been stai ned f or p-galactosi dase activity a t diff erent developmental stages. Embryogenesis has been most extensively studied. Bier et al ( 1 989) have exa mined embryos from over 3500 inde pendent strains, a nd Bellen et a l ( 1 989) have stained more than 500. Subsequent cytological mapping of about 200 insertions has suggested tha t most enhancer detectors a re loca ted a t unique sites, a lthough there a re some "hot sp ots" for integration ( Bier et a1 1 989; Bellen et a I 1989). If one a ssumes a n other wise relatively random distribution of P-elements in euchromatic DNA (reviewed by Engels 1989), the two experiments to gether genera ted about one insertion in every 70 kbp of the euchroma tic genome. Two genera l p oints should be made concerning the staining of trans formed lines. First, the p-galactosidase activity is normally nuclea r ( see Grossniklau s et al 1989); the P-transposase coding sequence in the P -IacZ fusion gene presumably encodes a nuclea r loca lization signal. Second, the half-life of the p-galactosida se p rotein is severa l hours; persistent staining may theref ore not reflect persistent tra nscriptional activity. Of course, this p roperty ma kes it difficult to recogniz e regula tory elements with rap idly cha nging spatial domains of action, but one advantage is that t he resulting accumulation of rep orter gene product increases the sensitivity of the enhancer detection technique. The sta ining data reported in the two embryonic screens a re broadly similar. Bier et a l ( 1 989) f ound a lower p roportion of sta ined lines ( roughly 65 compared to 85%), but this finding might be accounted f or by the diff erent cla ssifica tion of weakly staining embryos, since the sta ining con ditions used by Bellen et a l ( 1 989) a re p roba bly more sensitive. O f a ll the


696


lines generated, a remarkably high p ropo rtio n ( 60-65%) are stained in a sp atially restricted p attern. No mo re than 5% of strains exp ress {3galacto sidase at high levels in every embryonic cell; a further 25-30% exhibit weaker ubiquitous staining, although many of these transpo sants also have a sup eri mpo sed regio naliz ed staining p attern. Of the spatially restricted p atterns, o nly 15-25% are exclusively localized to a single tissue or cell type. Certain regio ns are stained frequently. Fo r instance, at least 50% o f all restricted p atterns include nervous system exp ressio n. Tissues with many fewer diff erent cell types, such as muscle, stain much less o ften. In so me lines, all cells within a marked tissue synthesize {3- galacto sidase; in many o thers, especially tho se stained in co mp lex o rgans like the nervo us system, o nly sp ecific subsets of cells express the repo rter gene (see Figure 2jfo r an ex treme examp le) .

Figure 2 Staining patterns of Drosophila enhancer detector strains. Strain A 1 89.2F3 is stained for /I-galactosidase activity during embryogenesis (a) staining salivary glands (SG); pharynx (PH); midgut (MG); and some cells in the CNS that are not visible here; (stage 16 according to Campos-Ortega & Hartenstein 1985); and in the third instar larval brain

(b)

staining neural cells in midline and other scattered cells in the brain. Strain A4 1 8 . l M2 is stained in the foregut (FG) during embryonic development (e) (stage 1 5). Part of the midgut is also dark (but not stained) due to the refractory nature of this tissue. This strain is also stained in anterior follicle cells of the egg chamber during oogenesis, (d) (see lower egg chamber). Later, at stage 1 0, the nurse cells are stained weakly (see upper egg chamber). (e) and (f) show two other strains expressing /I-galactosidase during embryogenesis; one strain is stained in a segmentally repeated pattern in mesodermal cells of unknown function, whereas the other unusual strain displays a segment-specific staining pattern in one thoracic neural cell on each side of the embryo (arrow).



697

Bot h studi es have p ro du ced a set o f t ranspo sa nt s i n which a range o f diff erent embryo nic ti ssu es a nd cells a re i ndependently stai ned. These strains p ro vide u sefu l cell lineage markers a nd allo w t he cell typ e-specific effect s of t he numerou s embryonic let hal mutations i n Drosophila to be t ested (e.g . Ghysen & O'Kane 1989) . Some lines a re stained i n cells t hat have not previou sly been recog nized a s diff erent from t heir neighbors by other criteria (see Figure 2e,f) (Bell en et al 1989); it shou ld be po ssi ble to di ssect certai n a spects o f complex developmental pathways more ea si ly wit h t hese markers. The 550 enhancer detecto r strai ns g enerated by Bellen et al (1989) have also been examined at other stages o f development . The larval brains a nd imagi nal di scs (the primo rdia of mo st adult st ru ctu res i n t he fl t he adult o va ri es o f t hese strai ns have been t ested fo r fJ-ga lacto si da se exp ressio n (Gi bso n & Gehri ng 199 0; Gro ssni klau s et a11989; Bellen et al 1990b) . A n additio nal 200 li nes have been stai ned du ri ng oogenesi s i n a separate stu dy (Fa sa no & Kerridg e 1988). At least 50% of a ll strai ns exp ress fJ-gala ctosi da se in a spatially regulated pattern, both in t hi rd instar larvae a nd duri ng oogenesi s . Furthermo re, many t ra nspo sant s pro vi de hig hly specific structures exami ned, t hereby demonst rating t he potential of enhancer detection for stu dying a ny stage o f development . Such markers a re par ticu larly u sefu l i n imagi nal di scs becau se t he o rganization of t hese p ri mo rdia is relatively poorly defin ed.

Identification of Genes Enhancer detectors i denti fy g enomic regulatory elem ents wit h su ch sur p ri si ng effi ciency t hat o ne might suppose t hat many o f t hese elem ents a re cryptic, i ntergeni c co ntro l sequ ences, whi ch do not normally regu late Drosophila g enes. O nly when a no nregulated t ra nsgene was insert ed nea rby, wou ld t hese sequ ences i nfluence exp ression from a gene' s p romoter. T here i s now good evidence, however, t hat i n many t rans posant s t he regu latory sequ ences revealed by t he enha ncer detecto r do no rma lly contro l Drosophila g enes. T he insertions i n about 200 t ra nspo sa nt s, mo st of whi ch have relatively specific logica lly (see Bier et a11989; Bellen et aI1989) . A search has been co nducted for known genes t hat map to t he sam e locatio n as each i nsertion a nd t hat have a simila r pattern o f em bryo nic exp ressio n to t he fJ-galacto si da se exp ressio n pattern o f t he co rresponding enha ncer detector strain. Thi s search ha s been successfu l fo r at least 15 insertions (see Table 1), mo st o f which do not p ro duce a n o bviou s recessi ve mutant p henotype. Since t he exp ressio n patterns o f o nly about 1% of a ll Drosophila g enes a re known

698


Table 1

Genes identified by enhancer detection in Drosophild'

Mapping

Embryonic staining

positionb

pattern

IB

All CNS and PNS

24D

14 ectodermal stripes

25B/C Hemocytes, fat body 28A

Correlation in larval

Gene'

Referenced

elav sloppy paired collagen type I V wingless

3

Recessive mutant phenotype'

I of 2 inserts 1, 2 3


staining 33A

Gnathal/posterior

Yes

spalt

abdominal regions 35BC

Pole cells

36E

CNS, visceral mesoderm, etc

47A

All embryonic cells, neurons

3

vasa fasciclin III misguided

1 , 2, 5 3, 6

?

strongest 48A

Segment polarity

49D

Correlation with larval

62A

Complex

66D

Seven ectodermal stripes

68E 80A

Neurons AlI PNS

3

engrailed scabrous

7

expression/phenotype 3

Yes

8

Yes

3 9

Yes

No name

2, 1 0

Yes

rhomboid hairy cyclin A

83B

Head/midgut

No name

85B

All embryonic cells

I

87B

Neuroblasts

90D

AlI PNS

Drasl seven-up stripe

91F

Midline neurons, glial cells

No name

12

?

92A

All eNS, proventriculus

No name

2, 1 3

Yes

5, I I

Yes

2, 3

Some of inserts

(mitotic mutant)

(larval imaginal discs and CNS) 94B/C Glial cells of CNS

No name

97D

Toll

Complex

a This table was to our knowledge complete in

2, 14

I

March, 1 990.

b Cytological mapping positions of enhancer detector insertions in which reporter gene expression

reflects at least part of the expression pattern of a Drosophila gene lying adjacent to the insertion. C

Most of the named genes were first identified by standard genetic approaches.

& c. Goodman, personal (7) M. Mlodzik & G. Rubin, personal communication; (8) Fasano et al 1 988; (9) H. Bellen, unpublished data; ( 1 0) U. Grossniklaus, personal communication; ( I I ) Mlodzik et al 1 990; ( 1 2) S. Crews, personal communication; ( 1 3) M. Goldberg, personal communication; (14) C. Klambt & c. Goodman, personal communication. d ( l ) Bellen et a1 1 989; (2) Wilson et a1 1 989;

communication;

(3)

Bier et a1 1 989;

(6) E. Giniger, L. Jan & Y. N. Jan,

(5) Y.

Hiromi

personal communication;

' Insertions producing an obvious recessive mutant phenotype, e.g. lethality.

a nd ca n t herefo re be co nsidered in these screens, the resu lt s sugg est that a sig nificant p ropo rtio n o f enhancer detecto rs respond to the regu la to ry elements of genes in thei r vici ni ty. In a seco nd approach to estima te this p ropo rtion, g enomi c sequences adjacent to the 3' end of insertions i n 2 3 transpo sa nts were clo ned by



699

p la sm i d rescu e (Wilso n et al 1989 ; see Figure l a). No rthern a nalysi s revealed that 1 2 o f these sequ ences encoded detecta ble transcripts. In seven ca ses, i n si tu hybri dizatio n to embryo sections with p ro bes complementary to the 12 transcripts pro du ced a signa l above backg round levels. Fou r o f the expression patterns resembled the sta i ni ng pattern o f the co rrespo ndi ng tra nspo sant. These limi ted data i ndicate that at lea st 25% o f all develop mentally regulated enha ncer detecto rs respond to the co ntro l elements of neig hboring genes. Gi ven the lim i tatio ns of the experiment, i n which only a sho rt g enom i c fragment on one side of the i nsertion was u sed as a p ro be, the actua l p ropo rtion i s p ro bably much clo ser to 50% (fo r discu ssion, see Wilso n e t a l 1989).

Insertion Site Specificity of Enhancer Detectors Between 1 0 and 20% o fau to somal enhancer detecto r insertions a nd abou t 5% of X chromo some i nsertio ns a re a sso ciated wi th a recessive lethal pheno type. Presumably, many of these disruptive i nsertio ns are well p laced within the mutated gene to respond to at lea st some of i ts regula to ry elem ents. In support of this hypo thesis, Gro ssni klau s et a l (1989) have shown a goo d co rrelation between a small number of stra i ns carrying i nsertio ns disrup ting genes i nvo lved in oogenesi s a nd early g erm line stai n i ng o f these stra i ns. Since i t i s estimated that abou t two thirds o f a ll g enes in Drosophila are no t essential for viabi li ty (fo r discu ssion, see Wi lso n et a I 1989) , the to tal frequency o f di srup ti ve i nsertio ns i n an enhancer detecto r screen may be a s high a s 30-60% ([1 0-20%] x 3); hence these i nsertio ns alo ne cou ld i nclu de a signific enhancer detecto rs that a re co ntro lled by gene regu la to ry elements a t the site o f i nsertio n. The p recise loca tions of severa l enhancer detecto rs that revea l the exp ressio n pa ttern of a nearby g ene have been mapped at the mo lecu lar level. Remarkably, a lmost a ll lethal a nd nonlethal i nsertions exami ned a re posi tioned wi thi n a few hundred base pai rs o f the g ene's tra nscrip tio n start site, usually in the p romoter region (Wilson et al 1989 ; Bier et a l 1989 ; Fa sano et al 1988; Y. Hiromi & M . Mlo dz i k, perso na l commu nica tions) . These resu lts co rrela te with p reviou s P-element mu tagenesi s experiments i ndicati ng tha t disruptive i nsertio ns a re frequ ently located near the 5' end of a gene (fo r review, see Engels 1989) . Fu rthermo re, P-elements a nd enhancer detecto rs with su btle or no eff ects o n gene exp ression may also be p referentially loca lized i n 5' regu lato ry sequ ences (Tsubo ta et a l 1985; the elav g ene in Bier et al 1989 ; the scabrous gene, M. Mlodzik, p erso nal commu nication). Therefo re, i t is no t u nreaso nable to p ropose that many enhancer detecto r i nsertions a re posi tioned in clo se p ro xim i ty to the p ro mo ter o f a Drosophila g ene. Thi s i s p ro bably the ideal site to respond to


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all of the gene's distantly acting control sequences and possibly even short range regulatory sequences within its promoter (see Figure 1). Conse quently, even the most complex gene expression patterns are often quite faithfully reproduced by the fj-galactosidase staining pattern of a strain carrying an insertion within the gene locus (e.g. insertions in the Toll, fasciclin III, and collagen type IV genes; Bellen et al 1 989; Wilson et al 1 989). In light of the insertion site specificity, it is also not surprising that transposant staining patterns, even at different stages of development, generally reflect part or all of the expression pattern of a single gene (see Wilson et al 1 989; Mlodzik et al 1 990; M . Mlodzik, Y. Hiromi & J. Bernholz, personal communications). A nalysis of Developmentally Regulated Genes

We have already indicated the applications of enhancer detection in gen erating markers for all stages of development (see also Bellen et al 1 989) . The evidence discussed above suggests that enhancer detection also pro vides a powerful gene detection system that is particularly useful for studying genes expressed in complex organs like the nervous system. Sequences flanking an insertion in any strain with an interesting staining pattern can be rapidly cloned using plasmid rescue (see Figure 1 ) ; standard techniques can then be used to identify the appropriately regulated gene, which will frequently lie near the enhancer detector (see Wilson et aI 1 989). Since hundreds or even thousands of transposants may express fj-galac tosidase in the tissue being studied by an investigator, one of the most difficult tasks may be selection of priority strains (see discussion in Bellen et al 1 990a). The most conservative option is to stain and analyze only those insertion strains associated with a recessive mutant phenotype. In this regard , enhancer detection is preferable to standard P-element mutagenesis because it often reveals the expression pattern of the mutated gene. Since the visible phenotype (if any) of most recessive lethal mutations does not indicate the function of the essential gene (e.g. see Wieschaus et al 1 9 84), the information from staining can prove vital in assessing the role of the gene both before and after the lethal period of the mutant (e.g. the seven up gene; Y. Hiromi, personal communication; Mlodzik et al 1 990). In this regard, even quite general staining patterns may give some indication of the developmental function of the disrupted gene (e.g. expression of fj galactosidase in dividing cells may suggest that the detector has inserted within a gene involved in mitosis; M . Goldberg, personal communication; see Table 1). There are two reasons why nonmutant strains should also be examined in comprehensive enhancer detector screens. First, just the staining pattern of a nonmutant strain may indicate that the corresponding insertion is



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near a developmenta lly impo rtant g ene (e.g . the spall a nd sloppy paired g enes in Bellen et a1 1989) o r nea r a g ene acting downst ream o f a n impo r tant reg ulato ry pro tein (J . Bernho lz, perso na l communication). S eco nd, it is speculated that many g enes invo lved in the diff erentiation of complex structures a re no nessential and would not be identified in a p urely g enetic screen (e.g . t he fasciclin III g ene; C. Goo dman, perso na l communica tion; Wilso n et a1 1989 ; see also A nderso n 1988). Perhap s the g reatest a sset o f enhancer detection i s t hat no nmuta nt strains i n these two catego ries can be selected purely o n the ba sis of (3-galacto sida se expressio n pattern a nd can subsequently be subjected to g enetic a nalysis. In m utagenesis experi m ents, t he marker gene(s) in t he enhancer detecto r a llo ws flies to be identified in which the insertio n has been lo st. Deletions o f the enhancer detecto r a nd adjacent g enomic sequences can be generated by standard x ray or chemica l m utagenesis techniques, o r simply by excision o f the P element wi th transposase, which in some cases leads to deleti on of flank sequences or to rearrang ement s (e.g . Bellen et a l 1989; fo r review, see Bellen et a l 1990a). Because t he staining pattern o f a strain indica tes t he tissues in which a gene may be active, muta nt flies can be examined in detail i n the region in which the g ene is exp ressed, a nd thus even subtle p heno types can be detected. This approach also t ests t he relevance o f diff erent a spects o f a complex exp ressio n pattern at a functional level (see sectio n on gene regulatio n a nd its evolution).

Further Applications of Enhancer Detection A t a time when i ncreasing numbers of Drosophila labo rato ries a re beg in ning to use enhancer detectio n, i t i s i nteresting to speculate abo ut novel ways in which the technique might be emp lo yed. Fo r example, the regu latory elements identifie d development . They may be characteriz ed by subcloning flanking g enom ic sequences into a P-element vector co ntaining a minimal p romoter-repo rter g ene fusion (e.g. p HZ50PL; Hiromi & Gehring 1987). Genes encoding cell-autonomous toxins (e.g . Palmiter et a l 1987; Landel et al 1988) o r pro teins regulating cell determinatio n may b e linked to these newly iden tified reg ulatory elements a nd reintroduced into flies. Particularly in com p lex o rgans, t hese experiment s mig ht a llow the functio n o f specific cell types to be tested by cell ablat io n or by changing the cell's fate. In a n extension o f this approach, it would be co nvenient to design a n enhancer detector i n which the repo rter g ene enco des a function a llowing inducible activa tio n of g enes tha t synthesize p ro ducts such a s to xins. Fischer et al (1988) have shown that the yea st transcrip tio n factor GAL4 can activa te transcrip tio n in Drosophila from a p romoter co nta ining GAL4 bi ndi ng sites. If an enhancer detecto r encoding a functional {3-galac-


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tosidase-GAL4 fusion was constructed, transposant strains with inter esting staining patterns might then be crossed to other strains carrying a toxin or developmental gene linked to a promoter with GAL4 binding sequences. This approach requires no molecular characterization of the transposant strain and allows any target gene to be expressed in the cells synthesizing the GAL4 fusion protein (see Byrne & Ruddle 1 989, for a comparable system in the mouse). If a temperature-sensitive version of the GAL4 protein could be identified and the corresponding gene expressed in the enhancer detector construct, the role of expressing cells in enhancer detector strains could be analyzed at several different developmental stages. Although this type of approach seems particularly appealing, it may have its problems. For example, it has not been shown that GAL4 is active in all Drosophila cells (see Fischer et al 1 988). The promoter sequences controlling reporter gene expression in the enhancer detector may also be altered so that the resulting constructs identify only a restricted set of gene regulatory elements. For instance, linking the reporter gene to a strong, constitutive promoter should produce a silencer detector. Other promoters containing regions interacting with more specific regulatory factors should restrict the range of genomic COn

trol elements that can influence reporter gene expression. Appropriately designed constructs with these modified detector genes could be used for preferentially generating a set of marker strains for a particular tissue or cell type (Jacobs et al 1 989; N. Perrimon, personal communication) or for a general study of the interactions of specific promoter sequences with different regulatory elements. An analysis of the latter kind might be more easily interpreted if detector transposons contained both the modified reporter gene and a second different reporter gene under the control of a minimal promoter, which would respond to the broad range of regulatory information at each insertion site. It might also lead to the identification of regulatory elements that could override the spatial restrictions of tissue specific control sequences (e.g. elements with some of the properties of DCRs, if such sequences exist in Drosophila). The Regulatory Architecture of the Genome

If enhancer d etectors insert at totally random genomic locations, analysis of large numbers of insertions should provide valuable information con cerning the arrangement of regulatory sequences and domains in the genome. In this respect, the P-element system may not be ideal. The apparent preference for insertions to disrupt the 5' ends of genes means that the majority of staining patterns observed probably reflects the range of Drosophila gene expression patterns and not the spectrum of regulatory information in intergenic regions.



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More detailed analysis of some strains nevertheless indicates that several loci in Drosophila may contain g roup s of coordinately controlled g enes and therefore may represent regulatory domains, For example, reg ulation of expression of an enhancer detector at the spall locus (Bellen et al 1989) is similar to reg ulation of the characterized spalt gene during early development (Frei et aI 19 88). The enhancer detector lies 40 kbp up stream from the gene's known transcrip tion start site, however, and separates the g ene from the sequences of one or more other g enes with the same early p attern of expression (J. Bernholz , R. Schuh, p ersonal communications). Two other enhancer detector insertions in the sloppy paired (sip) locus appear to be reg ulated by some of the sip control sequences (see Bellen et al 1989 ). The inserts are 1 0 kbp apart, however, and the P-lacZ g enes are transcribed convergently; there are also at least two similarly regulated Drosophila genes in the sip locus, one in close p roximity to each insertion (R. K urth-Pearson, personal communication). These data suggest that, in common with other chromosomal regions (Coleman et al 19 87; K nust et a1 1987; Romani et al 19 87), the spall and sIp loci contain coordinately regulated genes and that similar regulatory information is p resent in different p arts of each locus. O ne attractive hypothesis is that the g enes within such a locus are all p artially controlled by a master reg ulator element that p ossibly defines a reg ulatory domain (cf the f3-g10bin DCR; Grosveld et al 19 87). Alternatively, each g ene may have independent regulatory elements derived from a common ancestral sequence. Unless methods can be desig ned to screen for the structural p roperties associated with putative master reg ulator elements (see section on transgenic animals), these alternatives can only be disting uished by scanning each locus for control sequences using minimal p romoter rep orter g ene fusions,

Gene Regulation and its Evolution Because so many enhancer detector strains express f3-g alactosidase and the staining p atterns observed frequently refl ect expression patterns of sing le g enes, the range of different p atterns must bear some similarity to the rang e of expression p atterns of all Drosophila g enes. Althoug h, as we have discussed, there are hot and cold sp ots for insertion, it has been estimated that a saturation P-element mutag enesis screen will produce mutations in more than 50% of all essential genes (see Kidwell 19 86). By extrapolation, in a saturation enhancer detection screen, detector trans p osons should be suitably positioned to identify the regulatory elements of a similar p roportion of all g enes, essential and nonessential. However, it cannot be ruled out that P-elements will fail to insert within genes of a specific reg ulatory class or that certain control elements will fail to regulate


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the enhancer detector's expression. For example, only about 35% of strains express [3-galactosidase in all or most embryonic cells and, of the 550 strains generated by Bellen et al ( 1 989), only one is stained ubiquitously at all three stages of development examined. It is possible that weak, constitutive promoter or promoter/enhancer elements may often fail to regulate enhancer detector expression, thus leading to an artificially low proportion of patterns associated with the transcription of a so-called, housekeeping gene. Furthermore, some ubiquitously expressed genes may be transcribed at very different levels in different tissues (e.g. Shotwell 1 983); strains that contain an enhancer detector responding to the regu latory sequences of these genes may express [3-galactosidase in an appar ently tissue-specific fashion. Even if these arguments are considered, how ever, we still believe that the range of enhancer detector patterns reflects the fact that there are only limited numbers of genes transcribed at all times and in all cells; many other general functions essential to the cell may be provided by families of spatially regulated genes (e.g. Steinert & Roop 1 988) or by a gene that is only expressed in all cells at certain times of development (e.g. during cell division; see Edgar & O'Farrell 1 989). Other interesting observations concerning the range of staining patterns are probably relevant to the mechanisms by which genes are regulated during Drosophila development. For instance, complex organs, like the nervous system, are stained more frequently, often in specific subsets of cells. Such staining presumably reflects the greater number of genes and the greater degree of regulatory complexity required to specify cellular diversity in these regions. Certain patterns of staining are often linked. For example, if embryos are stained at their posterior end, they are frequently stained at their anterior end also. Similarly, most strains that express [3galactosidase in the antennal imaginal disc also express it i n the leg disc (Gibson & Gehring 1 990). These correlations indicate that the co-express ing regions are to some extent specified by common regulatory mechan isms. The fact that almost all staining patterns in the segmented structures of the embryonic thorax and abdomen are segmentally repeated also demonstrates that very few genes or regulatory mechanisms are segment specific (for an example and an exception, see Figure 2e,.n. Despite these interrelationships between some staining patterns, perhaps the most revealing feature of an enhancer detector screen is the complexity and remarkable variety of staining patterns observed in the different strains. This observation suggests that many, if not most, genes are ex pressed in several cell types. However, it does not imply that the gene prod uct is required or even functional in all these cells, since there may be no evolutionary pressure to restrict the expression of the majority of genes precisely to the cells in which they are needed. Even genes playing fun-



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dam enta1 developmental ro les, like t he hom eotic g enes, can be o ver p ro duced ectopically (e.g. Gibso n & Gehring 19 88) with no deleterious effect s in larg e reg ions of t he o rganism . I n fact , i f genes a re co ntro lled by combinations o f t ra nscription facto rs t hat reg ulate many other g enes, t here may be evolut io na ry pressure to retain appa rently superfl inso n 1988) ; a ll t ra nscription facto rs contributing to t his exp ressio n may be required in t he cell fo r essential regulatio n o f other g enes, while t he reg ulatory sequences binding t hese facto rs may be necessary for t ra n scription of t he g ene in tissues in which its exp ressio n is impo rtant . A n a rg ument o f t his kind may exp lain t he considera ble interspecies variatio n in t he exp ressio n pattern o f several no nessential enz ymes in diff erent , but clo sely related, Drosophila species (Dickinso n 19 80) . Fo r instance, in a dditio n to being exp ressed in t he fat body of a ll species, a lco hol dehydrogenase is synthesized at lower levels in a range o f seco nda ry tissues, which vary so much between species t hat it is difficult to conceive t hat a ll features of each pattern confer a selective advantage. Alt ho ug h it is tempting to speculate t hat certain a spect s of such expressio n patterns p lay no adap t ive role in the o rganism , such a hypothesis is very difficult to demonst rate. Perhap s t he best evidence will be o btained when facto rs t hat bind impo r tant reg ulatory sequences in a g ene, but activate potentially no nessential transcriptio n in some cells, a re sho wn to be needed fo r regulation of t he expressio n o f other g enes t hat a re required in t hese sam e cells. ENHANCER DETECTION IN THE MOUSE

Enhancer detectio n may be used to study t he development of a ny o rganism in which t ra nsgenes can be intro duced at relatively random g enomic locatio ns. Two g ro up s have repo rted enhancer detectio n experiment s in t he mouse (fo r a mo re detailed review, see Kothary et a1 l989) . A llen et a1 ( 19 88) m icro injected a lacZ reporter g ene under t he co ntro l o f t he weak herpes simplex virus t hymidine kinase ( HSV TK) promoter into mouse embryo s. Of t he 52 result ing embryos t hat co ntained integrated cop ies o f t he tra nsg ene, 1 1 exp ressed detectable levels o f fJ-galactosida se i n t he embryo ; in 5 cases, t he pattern was complex ( 2 o r more tissues were stained), while in t he other 6, it was tissue specific. In a n a lt ernative approach, Go ssler et a l ( 19 89) used elect ropo ration to t ransform embryonic stem ( ES) cells with a lacZ g ene controlled by a minimal promoter derived from t he mouse heat-shock p rotein 68 (hsp68) gene. These cells may be introduced into early embryo s where t hey can form part o f a chimeric a nimal a nd can co ntribut e to t he germ line. Most enhancer detecto r t ra nsfo rmant s co ntained o nly o ne o r t wo cop ies of t he


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reporter gene construct. As many as one in every seven transformants expressed p-galactosidase in ES cells; two of these expressing lines were used to generate chimeric embryos, which were stained in a spatially restricted pattern. Five ES cell lines that did not stain in culture were also tested. One expressed p-galactosidase in the embryo, again in a restricted pattern. In an interesting additional experiment, Gossler et al ( 1 989) gen erated a construct in which the lacZ gene is part of a translational fusion within an exon containing a splice acceptor site at its 5' end (for related retroviral systems in cultured cells, see Brenner et al 1 989; von Melchner & Ruley 1 989). Since the construct carries no upstream exon and promoter sequences, the reporter gene should only be expressed when it integrates in an intron of a gene, forming an in-frame fusion with that gene. Not surprisingly, only 1 0 of 600 lines that were transformed with this "gene trap" expressed p-galactosidase. Of the seven expressing lines analyzed in embryos, three were stained in all embryonic cells, three expressed the reporter in many cell types, and one exhibited a spatially regulated staining pattern. It is unclear whether the higher proportion of strains with consti tutive expression in this screen relative to the proportion found in the enhancer detector screens reflects a bias in one or the other technique.

Both enhancer detector studies suggest that a significant proportion of transgene insertions are controlled by insertion site-specific regulatory elements; as with Drosophila, many of these elements direct nervous system expression, and many of the expression patterns are complex (Kothary et al 1 989). Some strains provide useful markers for certain developmental processes in the mouse embryo. Kothary et al ( 1 988) have reported that a multicopy transgene insertion disrupting the neural-specific gene dystonia is expressed specifically in the nervous system; this finding suggests that enhancer detectors in mice can identify regulatory elements that normally control genes in some cases. A correlation with the expression of neigh boring genes, however, has not yet been shown for any of the newly gen erated transgenic lines. Further work will be required to develop a more efficient enhancer detection system in mice. Since transformed ES cells may be stored and expression of the p-galactosidase reporter gene can be tested in micro injected chimeric animals, ES cells should be convenient vehicles for the generation and analysis of libraries of different enhancer detector and gene trap insertions. It remains to be seen whether selection for staining in culture will affect the types of pattern observed in transgenic mice. Fur thermore, it is unclear whether the multiple transgene copies at a single insertion site generated by microinjection of embryos (which may form domains of up to 1 000 kbp or more) will be regulated in the same way as



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a n equiva lent g ene wit hi n the single cop y i nsertions, whi ch a re more frequently generated by transfo rmation of ES cells. Nevertheless, despite t he fact t hat enhancer detectio n in the mouse i s a relatively labo riou s t echni que when compared to the Drosophila syst em and that t he m etho d's effici ency a s a gene detection system is u nclea r, i t seem s likely that it will become wi dely u sed in the stu dy of mouse development. As in Drosophila, t he f3-galacto sidase exp ression pattern i n i nsertio n mu tants should be particularly i nfo rmati ve, a llowi ng t he ro le of developmental g enes to be a ssessed, even i f t he muta nt has no vi sible p heno type. Althoug h p resently it i s u nreali sti c to consi der u si ng enha ncer detecto rs to scan the who le mouse g enom e fo r regulatory elements, enhancer detec tion may pro vi de valuable i nfo rmatio n co ncerning g eneral gene control. One parti cu la rly i nt eresting approach i s to target t ransg enes to specific g ene loci to stu dy t he di stri bu tio n of regu la to ry i nfo rmatio n wi thin and a round t he proposed regu la to ry domain (fo r a n example, see Na ndi et al 19 88). Sing le copies o f t ransgenes can be targ eted to specific sequ ences by homo logou s recom bi na tion (fo r reviews see Cappechi 19 89; Fro hman & Marti n 19 89) . Appropriate po sitioni ng of mo dified detecto r co nstructs i n the f3-g lo bi n locu s shou ld a llow t he domai n o faction of t he f3-g lobi n DCR to be defined and cou ld reso lve questions co ncerning the preferential acti vatio n o f specific of development (see Blom va n Assendelft et a1 19 89; Behri ng er et a I 1990).

PROSPECTS

In t hi s review, we have di scu ssed t he broad range of position effect phenomena o bserved i n eukaryotic systems. Recent evi dence gathered u si ng a number of different exp erimenta l approaches suggest s that po si tion effect s can pro vi de impo rtant clues co ncerni ng the diff erent levels o f eu ka ryotic gene regu lation. In addi tio n, enhancer detecto rs p ro vide powerfu l tools fo r fu rther analysi s o f thi s regula tio n i n many diff erent genes and fo r a systematic approach to the stu dy of development i n any organi sm t hat can be t ransfo rm ed wit h clo ned DNA. Enhancer detectors (and g ene t rap vectors) may also be u sed to i nvestigate specific i n cell cu ltu re (e.g . Bhat et a1 19 88; Brenner et a I 1989) . As the mechani sms underlying posi tio n effects a re fu rther characterized, we will develop a better understandi ng of the different ways by whi ch expression o f different g enes i s co ntro lled i n a p reci se spa tial and tempora l p rog ram and how the coo rdi nate contro l o ft hese g enes can lead to the development of a complex eu karyo tic organi sm.

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ACKNOWLEDGMENTS

We thank Pierre Spierer, Nick Allen and Azim Surani, Frank Grosveld, Marek Mlodzik and Gerry Rubin, Christian Klambt, Yash Hiromi and Corey Goodman, Paul Lasko and Mike Ashburner, Ed Giniger, Lily Jan and Yuh Nung Jan, Stephen Crews, Norbert Perrimon, Mike Goldberg, and also Leslie Pick, Alexander Schier, Juliane Bernholz, Rebecca Kurth Pearson, Greg Gibson, and Ueli Grossniklaus in our own laboratory for allowing us to mention their unpublished data in this review. We are grateful to Deli Grossniklaus and Greg Gibson for providing photographic material for Figure 2 . We thank Anthony Percival-Smith, Markus Affolter, Alexander Schier, Walter Keller, Hermann Steller, and Katharine Lang for critical comments on the manuscript and Erika Wenger-Marquardt for very efficient preparation of the manuscript. W.J.G. is supported by grants from the Swiss National Science Foundation and the Cantons of Basel Stadt and Basel-Landschaft. Literature Cited Abraham, J., Nasmyth, K. A., Strathern, J. N., Klar, A. J . S., Hicks, J. B. 1984. Regulation of mating-type information in yeast: negative control requiring se quences both 5' and 3' to the regulated region. J. Mol. BioI. 176: 307-3 1 Allen, N. D . , Cran, D. G., Barton, S. c., Hettie, S., Reik, W., Surani, M. A. 1 988. Transgenes as probes for active chromo somal domains in mouse development. Nature 333: 852-55 Allen, N. D., Norris, M. L., Surani, M . A. 1 990. Epigenetic control of transgene expression and imprinting by genotype specific modifiers. Cell 6 1 : 853-6 1 Allis, C. D., Richman, R., Gorovsky, M. A., Ziegler, Y . S., Touchstone, 8., et ai. 1 986. hv l is an evolutionarily conserved H2A variant that is preferentially associated with active genes. J. Bioi. Chern. 26 1 : 1 94 1-48 Anderson, H. 1 988. Drosophila adhesion molecules and neural development. Trends Neuro. Sci. 1 1 : 472-75 Ashburner, M., Chihara, c., Meltzer, P., Richards, G. 1 974. Temporal control of puffing activity in polytene chromosomes.

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38: 655--62 Atchison, M. L. 1 988. Enhancers: mech anisms of action and cell specificity. Annu. Rev. Cell Bioi. 4: 1 27-53 Banerji, J., Olson, L., Schaffner, W. 1 983. A lymphocyte-specific cellular enhancer is located downstream of the joining region

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Behringer, R. R., Ryan, T. M . , Palmiter, R. D., Brinster, R . L., Townes, T. M . 1 990. Human y-to tJ-globin gene switching in transgenic mice. Genes Dev. 4: 380-89 Bellen, H. J., O'Kane, C. J., Wilson, C., Grossniklaus, U., Pearson, R . K., Gehring, W. J. 1989. P-element-mediated enhancer detection: a versatile method to study development in Drosophila. Genes Dev. 3: 1 288-1300 Bellen, H. J. , Wilson, c., Gehring, W. 1. 1 990a. Dissecting the complexity of the nervous system by enhancer detection. Bioessays 1 0: 1 99-204 Bellen, H. J., Wilson, C., Gibson, G., Grossniklaus, U., Pearson, R. K . , et al. 1 990b. P-element-mediated enhancer detection allows rapid identification of developmentally regulated genes and cell specific markers in Drosophila. J. Physiol. Paris 84: 1 - 1 0 Benoist, c . , Chambon, P. 1 98 1 . In vivo sequence requirements of the SV40 early promoter region. Nature 290: 304- 1 0 Bhat, K., McBurney, M . W . , Hamada, H . 1 988. Functional cloning o f mouse chro mosomal loci specifically active in embry onal carcinoma stem cells. Mol. Cell. BioI. 8: 325 1 -59 Bienz, M . , Pelham, M. R. B. 1986. Heat shock regulatory elements function as an inducible enhancer in the Xenopus hsp70 gene and when linked to a heterologous promoter. Ce!l 45: 753--60


POSITION EFFECTS ON GENE EXPRESSION Bier, E., Vaessin, H . , Shepherd, S., Lee, K., McCall, K . , Barbel, S., et a!. 1 989. Search ing for pattern and mutation in the Dro sophila genome with a P-lacZ vector. Genes Dev. 3: 1 273-87 Blom van Assendelft, G . , Hanscombe, 0., Grosveld, F., Greaves, D. R . 1 989. The p globin dominant control region activates homologous and heterologous promoters in a tissue-specific manner. Cell 56: 96977 Bohmann, D . , Keller, W., Dale, T., Scholer, H. R., Tebb, G., M attaj, 1. W. 1 987. A transcription factor which binds to the enhancers of SV40, immunoglobulin heavy chain and U2 snRNA genes. Nature 325: 268-72 Brand, A. H., Breeden, L., Abraham, J., Sternglanz, R., Nasmyth, K . 1 985. Char acterization ofa 'Silencer' in yeast: a DNA sequence with properties opposite to those of a transcriptional enhancer. Cell 4 1 : 41-

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ADDED IN PROOF Levis, R., Hazelrigg, R., Rubin, G. M . 1985b. Separable cis-acting control elements for expression of the white gene of Drosophila. EMBO J. 4: 3489-99

Since March 1 990, further examples of enhancer detector strains that identify gene regulatory elements have been communicated to us by several groups. Consequently, Table I includes only about half of all known instances of correlation between gene expression patterns and staining patterns.