53

Bmch~mtca et Bmphystca Acta, 1032 (1990) 53-77 Elsevmr BBACAN 87221

Negative regulation of transcriptional initiation in eukaryotes Stephen Goodbourn Gene Expressmn Laboratory, lmperml Cancer Research Fund, London (U K ) (Recewed 5 September 1989)

Contents I

Introduction

53

II

General repression A Inactive chromatm B Chromatm loops and scaffold attachment regmns C The role of DNA methylatmn D The yeast sdent mating type loci - an example of sequence-specific general repression9

54 54 54 56 56

III Specific repression A Lessons from prokaryotes B The evidence for specffm repression mechamsms m eukaryotes C Potentml mechanisms for specific repression D Eukaryouc'paradlgms' for negative regulation E Other eukaryoUc systems under negative regulatmn

58 58 62 62 63 67

IV Conclustons

73

Acknowledgements

73

References

73

I. Introduction Organisms complete defined developmental programmes, respond to hormonal signals, and adapt to changes m their enwronment by the differential expression of genes from their genonuc repertoire Although studies on these processes have concentrated upon the mechamsms of actwatlon, ~t ts becormng increasingly clear that negative regulation plays an ~mportant part m controlhng gene expression In a d d m o n to m h l b m n g specific gene expressmn before or after the appropriate developmental stage, negative regulatory mechamsms are revolved In m a l n t m m n g genes in a 'poised' but reactive state, and in shutdown or feedback controls whtch play a part m cellular homeostasis

Abbrevmtlons PCR, polymerase cham reactmn, SARs, scaffold attachment re~ons, UAS, upstream actwator sequence, EC, embryonal carcinoma Correspondence S Goodbourn, Gene Expression Laboratory, Imperial Cancer Research Fund, Lincoln's Inn Fields, London WC2A 3PX, U K

In higher eukaryotes there are several points at which expression can be controlled Over the last 10 years or so, the majority of research has concentrated upon the most energeucally conservatwe step, namely the mttmuon of transcnptmn Control of this step can be aclueved by modulating the accesslbdlty of genes to the transcriptional apparatus at the level of c h r o m a t m structure (general regulation), or by regulating access to a gene at a fixed site in ' o p e n ' c h r o m a t m (specific regulation) As negative regulatory mechanisms, the former allows whole blocks of genes to be repressed, since 'macttve' chromatln occurs m relatively large domares The latter mechanism allows exquisite regulation of individual genes wlttun linked complexes Because the orgamsaUon of c h r o m a t m into active and mactwe domains may involve sequence-specific events, the dastmcuon between general and specific mechamsms may be artlficml I will therefore discuss potential mechanisms for achieving both specific and general repression at the level of transcriptional tmtlatlon In discussing specific repression I have drawn heawly upon models estabhshed from work on Eschertchta cob and Saccharomyces cerevtstae, and I try

0304-419X/90/$03 50 © 1990 Elsevier Science Pubhshers B V (Biomedical Division)

54 to indicate parallels with systems m ingher eukaryotes Since S cerevtstae seems to regulate gene expression m a s~mllar fashion to mammalian cells, such comparisons may allow insight into more comphcated systems

1I. General repression H-A Inactwe chromatm The concept of general repression arises from studies winch demonstrate that genomes are apparently compartmentahsed into active and inactive chromatin (rewewed m Ref 1) Genes winch are located Wltinn an mactwe chromatln domain are not capable of being expressed Chromatin domains are often large and can contain multiple transcripUon units, leading to the possibility that groups of genes could be co-ordinately regulated The mechamsms winch control the chromatm state of domains are currently poorly understood, but the decision as to whether a domain ~s actwe or reactive may represent one of the key points in gene regulation A great deal of cytologml evidence is consistent with large regions of the eukaryotic genome being condensed in areas of 'inactive chromatln' The most dramatic example of tins ~s seen m the mammahan dosage compensation of X-linked genes, winch is acineved by random inactivation of one X chromosome, the mactwe chromosome forms a characteristic densely staining 'Barr body' In cases where only part of a chromosome is inactivated, chromatin appears to be organised into domains w~th fixed borders, as indicated by the reproduclble staining pattern of the polytene chromosomes of the diptera In Drosophda the regions of densely staining heterochromatm contain relatively few transcription umts In both the mammahan X chromosome and the polytene chromosomes the pattern of condensat~on is heritable, suggesting that the repressed state is actively maintained through cell division The inactivity associated with heterochromatin is not simply a result of an absence of active genes from certain regions of the genome This is borne out by the phenomenon of position effects In Drosophda, rearrangements that bring a normally acuve gene into the wcimty of heterochromatln, often Inactivate the translocated g e n e m a stably inherited manner These classic genetic studies have been complemented by studies using marker genes on P elements in Drosophda Depending on the site of integratmn there is a marked effect on expression, genes integrated into heterochromatin are usually only weakly active in contrast to the activity observed when integrated into euchromatin Posmon effects are also observed in mammahan cell transfectlon experiments D N A introduced into cells using standard transfection procedures appears to integrate at random The expression of a given transfected

gene can vary greatly, with many integrants falling to express at all In almost all cases expression is lower than that from the original chromosomal copy of the test gene To date, it has not been techmcally feasible to assess to what extent this is due to the lack of regulatory elements, or due to the negative influence of sequences flanking the integration site Tins IS because the procedures for stably introducing reporter D N A into mammahan cells are lneffloent and require selectable markers to identify transfectants The need for expression of the selectable marker itself probably prevents the identification of integration events in truly inactive chromatm Microlnjection of D N A into mammahan cells offers one way of circumventing this problem, since most cells stably integrate injected D N A Expression obtained from such cells is duly often lower than that seen in lines derived by transfection methods The difficulties of obtaining large numbers of independent lines using n'ucrolnjectlon makes this approach rather tedious for analysing the phenotypes of random mtegrants Recent advances in polymerase chain reaction (PCR) technology should enable cell hnes carrying integrated probe D N A to be rapidly identified, allowing large numbers of hnes derived from transfection methods to be obtained without selection The molecular basis of the organisatlon of chromatm into active and inactive domains is poorly understood (see Ref 2 for review) The repression seems to be a consequence of the exclusion of the transcriptional apparatus from regulatory regions, and tins appears to be a function of the ingher order structure of the nucleosomes In inacuve chromatin adjacent nucleosomes are held together m a tightly packed array by histone H1 [3], m active chromatm, tnstone H1 is still present but cannot tightly pack the nucleosomes The alteration m the properties of H1 winch causes these differences is not understood, although it is known that H1 can be extenswely modified and is highly polymorphlc Active chromatln is also relatively rich in nonhlstone proteins, such as H M G 1 4 and HMG17, and contains modified forms of other Instones

H-B Chromatm loops and scaffold attachment regions Hlstone H1 binding to nucleosomes to form the tightly packed multimer appears to be co-operative [3] As a result of tins, it might be envisaged that the entire genome should fall into the repressed state We have already discussed above that chromosomes show a highly organised domain structure, and there must presumably be specific 'organiser' sequences present in the genome winch act as barriers to prevent the spread of the inactive domains In fact, in chromosomal preparations that have been carefully stripped of their Instones, a higher order structure can be seen (reviewed in Ref 4) Chromosomal D N A is organlsed into loops of 30-100

55 kb winch are attached to a 'scaffold' structure that hes along the chromosomal axas The points of attachment appear to be umque, the so called scaffold attachment regions (SARs), suggesting specific D N A - p r o t e m mteractxons Although the Instone extractmn procedure reqmres a heat-shock step, raxsmg the possibility of artefacts, the SARs have a number of properties cons~stent w~th a role in determining the domain structure of chromatln (see below) The SARs appear to be excluded from transcribed regmns, m contrast to the D N A in the loops between them There is also ewdence that genes Wltinn loops can be orgamsed into groups that exhibit s~rmlar patterns of regulation For example, the five Drosophila instone genes are all present within one loop [5] One nught therefore envisage a model m winch SARs functmn to estabhsh loops of chromatm as rather active or mactwe, any formgn D N A inserted into the latter would also be mactwated as ~s seen m positron effects In at least two cases [6,7], the SARs mark boundaries between condensed and loose chromatm, suggesting that SARs do define chromatm domains Many, although not all, SARs contain sequences that have been ldenufied as having regulatory functmns [8-15], raising the possibility that some regulatory elements may functmn as chromatin orgamsers However, since the defined SARs are often relatwely large the attachment regmns and regulatory elements may be distract Each of the identified regulatory elements has a positive functmn suggesting that a loop may be reactive because of the lack of appropriate regulatory factors The existence of reactive c h r o m a t m as a dominant 'ground state' would ensure tight repression of the bulk of cellular genes w~thout the need for specific regulatory molecules A possible exception to tins, where mactwe chromatm is estabhshed m response to specific factors, will be &scussed subsequently The model described above suggests that chromatln loops are maintained by being isolated from other sequences m the chromosome via attachment to a scaffold, the scaffold attachment reqmres sequence-specffm D N A - p r o t e m interaction, and each loop may contain its own regulatory s~gnals One possible consequence of tins ~s that the scaffold attachment regmns and sequences located between these elements could comprise an autonomous positron-independent unit Using P element transformatmn, Hlrorm et al [9] have shown that constructs contmmng the Drosophila ftz gene and both 5' and 3' SARs do functmn m a posmon-mdependent fashion, whereas m the absence of the 3' SAR, tins property is not maintained Recently it has been shown that a D N A fragment from remotely flanking sequences from rather end of the human fl-globm complex can also confer posmon-mdependence to a us-hnked human fl-globm gene m transgemc mace [16] These sequences function in an erythrold-spec~fic manner, and

confer Ingh-level, tissue-specific expression on a heterologous gene [17] The fragments contain a number of erythrold-speclflC D N a s e I hypersensmve sites, and it has been shown that subsets of these confer some degree of posmon-mdependence and Ingh-level expression, although tins is not absolute [17-20] The fragments spanning the hypersensmve s~tes contain SARs [15] Physlologmal ewdence for the importance of the remote flanking regmns of the fl-globm complex comes from the phenotypes of thalassemm mutatmns, m a n y of winch have impaired globm expression without &sruptmn of the regulatory s~gnals adjacent to the globm genes (see Refs 21 and 22, and references m Ref 16 for relevant revmw material) In two mutants the remote flanking regtons are deleted [23,24], leading to a typmal p o s m o n effect phenotype The mutatmns act only m cts and in one case it was shown by D N a s e I hypersenslnvlty that the mutant fl-globm gene was m an mactwe chromatln conformaUon [23] The existence of a d~stlnct chromatln domain around the fl-globm gene complex has also been demonstrated m cell-fusion experiments m winch re-actwatlon of the human adult/3-globm gene is accompamed by a re-estabhshment of the erythro~dspecific pattern of D N a s e ! hypersensmvlty [25] The failure to express any class I M H C genes m the mouse fibrosarcoma hne, IC9 may represent another example of the mact~vatmn of a loop orgamser In these cells, the H - 2 K b, K a and D k genes all appear to be unrearranged, and functmn normally when cloned and reintroduced into IC9 or other cell hnes [26], consistent w~th tins, IC9 cells contain all D N A - b m d m g proteins thought to be necessary for H-2 t r a n s c n p h o n Nuclease mapping experiments have indicated that the promoter regmn of the silent D k gene lacks the hypersensmve site normally assocmted w~th actwe transcrlptmn of tins gene The s~mplest mterpretatmn of these data ~s that the genes have been turned off by creating an enwronment of mact~ve chromatm in thmr ~mmedmte WCmlty Although the globln remote flanking regions confer both tlssue-specffmlty and p o s m o n independence to hnked genes, and the D N a s e I h y p e r s e n m w e s~tes that reside Wltinn these sequences are also tissue-specific, there ~s no ewdence that scaffold attachment shows tissue-specificity In fact, the fl-globm SARs can be detected m non-erythrold cells [15]. and both xlmmunoglobuhn hnked SARs [12] and Drosophtla hlstone-hnked SARs [8] fail to show tlssue-speclfimty Tins may be an artefact of the procedure used to detect SARs, m that the extractmn of Instones and other proteins may cause all potentml SARs to 'collapse' onto the scaffold regardless of the t~ssue of o n g m Alternanvely, the lack of txssue-spemfimty may ensure that SARs maintain loops m all tissues, but that other regulatory elements and factors functmn to determine the achvlty or otherwise of the loop

56 11-C The role of D N A methylatton Considerable evidence suggests that methylat~on of D N A may also play a part in the repression of gene expression Methylatlon of cytosine m the dmucleotlde C p G is frequently observed in genes that are not expressed For example, globm genes are hypermethylated m fibroblast ceils, and yet are undermethylated in retlculocytes, unmethylated cop~es of the human 3'globm genes are also transcriptionally active when introduced into flbroblast cells [27] Many tlssue-speofic genes are hypermethylated in sperm, and the pattern of methylatlon is faithfully inherited In fact, the pattern of methylat~on of specific genes differs between male and female gametes and tins correlates with the relative contributions to early embryogenes~s of the mdwldual parental genes, a phenomenon called genormc ~mprmtlng (reviewed in Ref 28) These genes only become undermethylated m the tissue of their specific expression (see Ref 29 for review) Inappropriate gene expression can often be obtained by treating cells with 5azacytldme, a cytosine analogue winch cannot be methylated, tins analogue presumably gets incorporated at rephcat~on Methylatlon has been shown to prevent interaction with D N A of some sequence-speofic factors [30-32], but not others [33,34], suggesting that repression could be brought about by bloclong the binding of essentml transcription factors Becker et al [35] have demonstrated that C p G methylatlon m fibroblasts prevents the association of a sequence-specific DNA-blndlng protem with the regulatory region of a hver-speclfiC gene both in vitro and in wvo In contrast, the same sequence is unmethylated m hepatomas Both fibroblasts and hepatomas possess the binding factors, suggesting that differential binding is indeed mediated by tissue-specific methylat~on Recent experiments suggest that methylatlon may have a second role in repression Antequera et al [36] have shown that the majority of methylated D N A is m fact bound by proteins in VlVO, and two factors have been identified winch bind to methylated D N A with relaxed sequence specificity [37,38] These results are consistent with the bulk of methylated D N A being protected by non-speoflc binding factors, and Antequera et al [36] have proposed that the interaction between methylated D N A and protein may increase the stainhty of reactive chromatm Methylatlon ~s unhkely to be a primary regulator of expression Speofic methylatlon of lndlwdual sites on a regulated basis to estabhsh repression would reqmre a series of sequence-specific methylases, expressed in a tissue-specific manner, winch so far have not been d~scovered A model of general methylatlon is consistent with the observauons that C p G regions within coding regions and regions not known to be revolved in regulation are often methylated Studies on the methylat~on

pattern of wral transcription umts suggests that methylat~on correlates w~th the lnacuvatlon of funcuon, but that methylatxon can take m a n y generations to estabhsh and tends to occur m domains [39] Several reports have demonstrated that genes can become transcrlpUonally inactivated before methylatlon [40-42] The deoslon to repress a gene or a set of genes ~s therefore more hkely to be made at a different level, for example at the gross level of chromatln structure Following an mact~vation of the 'loop' type discussed above D N A could become methylated, thus re-lnforcmg repression Alternatively, C p G dlnucleotldes may become methylated simply because appropriate positive factors are no longer avadable to brad regulatory domains Methylatlon could function to prevent transcription factors recognlsmg regulatory regions during rephcat~on where the instones are probably transiently dissociated from D N A H - D The yeast silent mating type loci - an example of sequence-specific general repression The deternunation of yeast mating types comprises perhaps the simplest example of dlfferentiaUon decisions made by eukaryotlc cells (reviewed m Ref 43) Haploid yeast cells are of two types, designated a and a, winch can mate only w~th cells of the opposite mating type to produce a / a dlplolds The mating type is deterrmned by expression of the specific information present at the mating type locus ( M A T ) The two mating types have a different form of tins locus In a cells, the M A T locus is occupied by the M A T a l and M A T a 2 genes, whereas in a cells, the locus is occupied by the M A T a l and a2 genes All yeast cells possess silent copies of the determlmstlc genes for both a and a mating types stored at transcriptionally inactive loci, called H M R a and H M L a , respectively Yeast are able to switch mating types by a gene conversion event winch replaces the information at the active M A T locus with information from either H M R a or H M L a The mechanism by winch yeast maintain the H M R a and HMLe~ l o o in a transcriptionally reactive form has been extensively studied and offers the first real model for a sequence-specific repression which seems to operate at the level of chromatln inactivation The information stored at the silent mating type loci includes the entire promoter and transcription umts in each case A sequence-specific repression of the promoter element alone could not account for the silencing, since such a system would also shut off the M A T locus In fact, the silent copies of the mating type locx are negaUvely regulated by sequences located outside of the region of homology with the M A T locus Since these sequences act in cls, only the H M R a and H M L a loci are affected Tins system is thus a classic example of a p o s i t i o n effect The sequences essential for repression reside both sides of the H M R a and HMLc~ loci The more

57 important ' E element' lies over 1 kb 5' to the promoter region, and the ' I d e m e n t ' resides 3' to the transcription unit [44,45] The E element has properties that are sirmlar to enhancer elements, since its repression effects can be conferred upon heterologous transcription umts, and these properties are independent of the position or orientation of the element relative to the transcription umt [46] Such an element has been referred to as a ' silencer' A number of yeast mutants cannot efficiently silence the H M R a and HMLOt loci Four complementation groups have been described (the S I R genes, Ref 47) which cause derepresslon of silent loci without being lethal More recently, mutations which alter hlstone structure have also been shown to derepress silent loci Specifically, Kayne et al [48] have demonstrated flus in mutations which remove the N-terminal 25 amino-acids (which are required for acetylatlon) of histone H4, and Mullen et al [49] have shown that strains that lack an N-acetyl transferase actlwty also fall to silence The latter strains contain hlstone H2B in a form that is not N-terminal acetylated These results suggest that silencing requires histones in a form that can be modlhed and imply that a specific chromatm structure ~s obhgatory Additional evidence for the importance of chromatln structure comes from expenments that suggest that D N A rephcatlon is required to silence transcription Miller and Nasmyth [50] demonstrated that following a shift to a non-permasslve temperature in a temperature sensitive mutant for silencing, repression can only be re-estabhshed after the completion of S phase The simplest interpretation of this is that a specific regulatory structure needs to be assembled at the silent loci and that this can only happen when D N A is free to bind cellular factors Consistent with this, silencers contam sequences that can function as onglns of rephcation (ARS elements) in yeast [44] Silencers also appear to possess some of the features of centromeres in that they can confer some mitotic stability upon plasnuds [51] A fine dlssecnon of the H M R silencer by Brand et al [52] revealed a functional redundancy, the element containing three subregions (in order 5' to 3', A, E and B) Complete silencer activity was observed when the E element was examined in the presence of either the A or the B regions The E region contains a binding site for a protein called RAP-1 [53,54], which also binds to the silencer at the H M L locus The binding site for RAP-1 is also found in some yeast enhancer-hke elements, mchidlng the MA Tot genes This suggests that this sequence element may function positively in some circumstances, and when the E region alone was linked to a test gene it did indeed stimulate transcription [52] The A region bears homology to the ARS consensus sequence [55] which is required for rephcatlon The A region will function as an ARS element when examined

in isolation from other silencer sequences Surprisingly, region B also has ARS activity, although it is not especially homologous to the ARS consensus sequence However, the B region contaans a sequence which is found in the vicinity of several other ARS elements, and is a binding site for the protein, SBF-B [54,56] Inactivation of the SBF-B binding site next to an ARS element in the I region of H M R abolishes ARS activity [44] The genetic analysis of the H M R silencer suggests that the only requirements for function are an origin of replication and a binding site for R A P - l , although it has not been demonstrated that this is sufficient to repress a heterologous gene Since it can also be a transcriptional activator the properties of RAP-1 must somehow be influenced by D N A replication In the silencing role, RAP-1 may interact with some component of the rephcatory apparatus, or replication may disrupt the local chromatm organlsatlon so that additional factors may gaan access to the target D N A region, in this context it is noteworthy that none of the 4 S I R genes appear to encode D N A binding proteins [56] The products of these genes would seem to be excellent candidates for factors which would interact with RAP-1 to cause it to function as negative regulator Buchman et al [54] specifically propose that there is an Interaction between RAP-I bound at the silencer and RAP-I bound at the enhancer region of the H M L copy of MA Tot In this model, the intervening D N A is looped out, and the interaction stabillses the gene in an inactive configuration In fact it has recently been shown that the silencer at H M L is bound to the nuclear scaffold [57,58] as are sequences from the I element (a sequence 3' to the coding regions which is also involved in silencing) and the promoter region RAP-1 has also been shown to be a component of the nuclear scaffold [58] Purified scaffold-silencer complexes can arrange cloned H M L D N A fragments in vitro into a loop which can be detected by electron microscopy, the loop formatlon is dependent on the presence of RAP-1 and the cognate binding site [58] It is tempting to speculate that in the presence of the appropriate cellular factors the association of the RAP-1 binding site with an ARS element creates a strong scaffold attachment site which would interact with adjacent sites to form a chromatin loop, the loop itself would create a self-contamed reactive chromatln domain propagated by the essential specific chromatm structure (see above) The I element, which is involved in silencing and resides 3' to the H M L locus, also contains an ARS element and may represent the other boundary to the loop The association of the promoter region with the scaffold Is probably due to it containing an RAP-1 binding site and being in physical proximity to a scaffold-bound RAP-1 binding site at the silencer It should be emphaslsed that the interaction with the promoter is dlspenslble for

58 silencing because the silencer will function to repress heterologous promoters which lack RAP-1 binding sites

III. Specific repression The synthesis of m a n y studies indicates that for all genes examined, tissue-specific or stimulus-specific expression is conferred upon the gene by cis-actmg sequence elements These elements fall into two broad classes (for review see Ref 59), namely the promoter element (taken here to mean sequences within 100 base-pairs or so 5' to the m R N A startpomt), and the enhancer elements (winch are positioned more remotely from the m R N A startpomt, and can be either 5' or 3' to the gene, and indeed can even reside Wltinn mtron sequences) These elements serve as recognition elements for sequence-specific DNA-binding factors, winch facilitate the assembly of transcription complexes and recruit R N A polymerase II to the desired gene (see Ref 60 for review) By virtue of the way in which the ors-acting elements have been analysed, most of the data reflects the nature of sequences needed to give expression under a given set of circumstances, i e, positivelyacting elements More recently it has become apparent that sequence-specific negative regulation is a commonplace event, although the underlying mechanisms are not well understood Since prokaryotes and eukaryotes regulate gene expression using similar means ( i e , sequence-specific interactions of DNA-binding proteins and specific protein-protein interactions) I will briefly consider some model systems for negative regulation in prokaryotes IH-A Lessons from prokaryotes

The lac operon is the original para&gm for gene regulation, and the mechamsm of its regulation well understood However, the traditional views of tins apparently simple system have been challenged by recent work which appears to have been inspired by the discovery of eukaryotic regulatory elements that can act to stimulate or repress transcription at a distance The lacZ gene, winch encodes fl-galactosidase is controlled primarily by sequences winch reside directly 5' to the m R N A startpoint Like many genes m E coh, this region contains two sequence elements required for recognition by the R N A polymerase o TM holoenzyme (see Ref 61 for review), these sequences which are centred around - 1 0 (i e , 10 base-pairs 5' to the startpoint of transcription) and - 3 5 are referred to as the promoter region R N A polymerase binding to the lacZ promoter occupies a region of about 40 base-pairs, and the 3' end of the binding site physically overlaps a sequence called the operator winch functions to bind the lac repressor Lac repressor supposedly inhibits lacZ expression by preventing access of R N A polymerase to

the promoter region Ligand binding to the lac repressor disrupts D N A binding of repressor and allows R N A polymerase to interact with the promoter region The basis for proposing a steric occlusion mechanism IS the physical overlap of the operator sequences and the region occupied by R N A polymerase Direct evidence that binding of the two factors is mutually exclusive has been difficult to obtain, Majors [62] measured the kinetics of binding of R N A polymerase to the promoter in in vitro transcription experiments, and concluded that exclusion was the most likely possibility However, DNase I footprlntlng experiments [63] suggest that a larger region of D N A is occupied when both factors are present than when either factor is examined in isolation Thas experiment could be interpreted to indicate that both factors bind simultaneously to the lac p r o m o t e r / o p e r a t o r region Alternatively, since D N a s e 1 footprlntlng is essentially an averaging technique, it is possible that the footprint seen is a ' h y b r i d ' of two independent footprints resulting from independent occupations With the recent development of the 'gel-shift' technique [64,65] it has been possible to investigate the occurrence of complexes involving more than one protein bound to a specific D N A sequence, since such complexes have altered mobility compared with the shift caused by binding of a single protein species Using this approach, Straney and Crothers [66] have demonstrated that complexes containing both lac repressor and R N A polymerase can be formed, suggesting that binding is not mutually exclusive The contacts with D N A are different for this ternary complex than for either protein alone The affinity of R N A polymerase for the lacZ promoter IS much higher when the repressor is already bound, indicating that a specific interaction between the proteins can occur The excised complexes can be analysed for the ability to stimulate synthesis of a short transcript in the presence of nucleotide trIphosphates, in the absence of repressor, productive transcripts are efficiently synthesized However, complexes which also contain repressor have significantly reduced transcription, although repression is released by addition of the reducer These data clearly show that the in VlVO pattern of regulation can be reproduced by the isolated complexes and demonstrate that repressor must prevent R N A polymerase activity by a mechanism which is distinct from sterlc occlusion The recent observations that the relative positions of operator and promoter can be varied without loss of repression [67,68] are consistent with tins Idea The mechanism by which R N A polymerase initiates transcription in E ~oh has been extensively studied Following promoter binding, R N A polymerase forms a 'closed complex', so-called because the duplex D N A does not appear to unwind Upon unwinding of the DNA, the R N A polymerase forms an 'open complex' winch can be converted to a productive and stable

59

I,

A

I

-35 box

I--I

I

I

-10 box

operator

Sequencesoccupmdby RNA polymera~sealone Reptessor binding site

E

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( ( F~g 1 R N A polymerase and lac repressor interact to repress the lac operon (A) Binding sites m the lacZ promoter for R N A polymerase and lac repressor (B) Pnor to mducuon, R N A polymerase (shaded) and lac repressor (indicated as a homotetramer of open circles) specifically interact to form a stable complex b o u n d to the promoter In th~s form R N A polymerase is unable to mlttate transcription (C) Following addition of reducer lac repressor dlssocmtes, leavmg R N A polymerase free to mltmte transcnpUon (D) R N A polymerase forms a stable mltmted complex (E) Transcription inmates

'~mtiated complex' upon the addition of nucleot~de trlphosphates The repressor could mediate its negative effects by 'tying-up' R N A polymerase m such a way as to prevent the formation of the stable mmated complex Straney and Crothers [66] have demonstrated m lonet~c experiments that the conversion to an mmated complex is indeed much slower m the presence of repressor Surprlsmgly, upon a d d m o n of an reducing hgand, the rate of formation of the productive complex is faster than in the absence of repressor, suggesting that the repressor can act as a transient activator of transcription This modified view of lac repressor interaction is dlustrated in Fig 1 The second challenge to the traditional view of the regulation of the lac operon is more directly related to studies on eukaryotlc elements In a d d m o n to the lac repressor binding site located adjacent to the promoter region discussed above, two other repressor binding sites have been Identified in the v l o m t y of the l a c Z gene (see Ref 69 for review) The function of these

sequences was not understood until work on eukaryotes, and on the E coh ara operon (see below) demonstrated the importance of action at a distance by a regulatory element The stronger of the two additional repressor binding sites, 02, is located within the coding reg0on of lacZ, and this element can co-operate with the operator site to maximlse repression [70] In addition, synthetic copies of the lac operator have been shown to interact m cts with the natural [71] or mutant [72] operator to enhance repression Since lac repressor ~s known to tetramerlse, even though dlmers brad to target D N A sequences, it nught be imagined that hawng a second binding site m the v l o m t y of the promoter could act to load the template with repressor m a form which has a 'spare' dimenc D N A - b m d m g surface available, with the resulting high local concentrahon of a potential D N A binding form ensuring rapid occupation of an unbound site In fact, rather than simply acting as a 'sink' for lac repressor, the conformatlonal flexlbdlty of D N A seems to allow both sites to be occupied simultaneously by a

60 I

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-~o

I

lacO

I

I 02

II-

B

I

I 02

C

Fig 2 Repressor binding to two linked sites with looping out of mtervemng D N A stablhses repression of lacZ promoter (A) Arrangement of lac repressor binding sites O and 0 2 m the laeZ promoter (B) Lac repressor homotetramer (open circles) binds to the high affimty O site (C) Lac repressor bound to O causes an increase in the local concentration in the VlClmty of 0 2 , with concomitant binding and looping out of the intervening D N A

tetramer of repressor, w~th the 'looping out' of intervening D N A (Fig 2, see Refs 73 and 74 for reviews, and Ref 75 for specific ewdence of looping of D N A between two lac operators) It has recently been observed that lac repressor binding can bend the D N A molecule, winch could considerably aid loop formaUon over small &stances [76] The formation of a loop tethered at ItS base by the co-operat~ve interaction of repressor protelns would not only enhance repression because the stablhty of the complex would dramatically decrease the rate of &ssocmt~on from the promoter-proximal s~te, but m some c~rcumstances rmght even exclude R N A polymerase or components of the transcriptional apparatus The tturd repressor binding s~te, 03, is located upstream from the lacZ promoter, although lnteracuon w~th tins sequence ~s so weak that ~t ~s not clear what role ff any tins has in negatwe regulaUon A more soptusUcated 'loop-out' repression system exists in the E cob arabmose operon The following model has been proposed based upon the data presented by Huo et al [77] and Harmlton and Lee [78], and previous work from these two groups (dmcussed m the ctted references) Tins operon contains two promo-

ters (called Pc and PBAD) arranged m opposite onentauons which are repressed in the absence of arabmose by the product of the araC gene Three binding sites (O1, 0 2 and I) for the araC product have been identified m the regulatory regions (shown m F~g 3A) In the absence of arabmose, araC binds co-operatively to 0 2 and I, winch are separated by 200 base-pmrs, with the concomitant looping out of the intervening D N A (see Fig 3B) When examined independently, the 0 2 site was found to be much weaker than the I site, and m the absence of the closely hnked I site, would not be expected to brad araC m VlVO The same loop is involved m repressing both promoters, the I s~te resides between the two promoters, whereas the 0 2 site ~s mtragemc Pc is isolated m the loop, whereas PBAD lS d~rectly adjacent to the I s~te The arrangement of the I and 0 2 site w~th respect t o PBAD lS slrmlar to that observed m the lac operon with O and 0 2 (see above), and the mechanism of repression of PBAD may well be eqmvalent Repression of Pc may be brought about by the inability of the looped out conformation to interact with R N A polymerase The a d d m o n of arabmose disrupts co-operatlv~ty between araC molecules, w~th the

61

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PBAD

Pc

~

I

"

site for cI is OR1, and b m d m g to ttus site prevents R N A polymerase from initiating transcription at PR, and thus the cro protein is not made Binding of the cI protein IS co-operative, and involves an interaction between the N-termini of separate molecules, the co-operattvlty is a

I

A

~PRM

R:I I

I

R I ~ i~lymermm mcagnlllon mite

R l ~ Ix)lymermm rel~gnlllon slle

B

el gene

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I

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I

OR3

OR2

OR1

©1 repremmr

© araC

~o gem

I

RI~ polymen,-e

RNA polymerase

IB

' I _

Fig 3 Repression of divergent promoters m the arabmose operon by the araC protein See text for d~scussaon

result that the weak 0 2 site becomes unoccupied This destroys the loop, and presumably alters the conformation of the araC bound at the I site After induction, the araC protein bound at the I site functions as a transcnptional stimulator from P~AD The ability of araC to stimulate transcrlpUon differs from the lac repressor in that its positive activity is not simply transient In the absence of the loop, Pc is also activated However, the product of the gene dnven by Pc is araC itself, with the result that the repressor concentration increases As a result of ttus, the O1 site, winch has been opened up by loop mactlvauon becomes occupied and tbas in turn shuts off Pc, since O1 and the R N A polymerase binding site physically overlap, there is some evidence that araC binding at O1 and 0 2 can form an alternative loop which would enhance repression from Pc [77] The final bacterial system to be considered is the regulation of early gene expression in bacteriophage lambda Thas has been extensively reviewed by Ptashne [79] Essentially, this system consists of a pair of promoters, a leftward promoter, PRM wtuch controls cI repressor synthesis, and a rightward promoter, PR, which controls synthesis of a second repressor molecule, the cro protein The two promoters are overlapped by three elements that serve as binding sites for the repressor molecules These three binding sites are referred to as O R 3 , O R 2 , and OR1 i n a left to right onentat,on (see Fig 4A) In the lysogemc state, the cI repressor is constitutively produced The highest affinity binding

I

I

I

C el gere

I

I

I

I

gene

D

Fig 4 Regulation of davergent promoters m the bacteriophage lambda genome by repressor molecules with overlapping speoficltles (A) Components required to repress to cro gene and actLvate the cl gene (B) Oal and On2 are occupied by low concentrations of cl The bmdmg to these sites ]s highly co-operauve RNA polymerase can brad to PRM when OR3 ~S not occup]ed by cI The presence of cl at OR2 stimulates R N A polymerase activity and consequently cl product]on (C) Excess cl occupies Oa3 with the result that PaM ~s blocked and no further cl synthesis occurs (D) Following reduction, cl is reactivated and the concentration falls allowing RNA polymerase to bind at PR The cro protein thus synthesized can bmd to Oa3 and OR2 and block further cl production

62 function of protein-protein interactions, and slgmficantly the topology of the protein complexes restricts the co-operatwlty of binding to D N A to pmrs of bindmg sites As a result of this, OR2 lS occupied by cI at about the same level of cellular protein as is required for occupation of OR1 Tins necessarily tightens up the repression of PR, but has a second effect As we saw above for araC the cI protean can also function as a transcriptional stimulator, occupation of OR2 posmons cI so that ~t can interact with R N A polymerase to stimulate transcription from PRM (Fag 4B) The regions of the cI molecule required for the DNA-bindmg, cooperatlvaty by protein-protein interaction, and for positive lnteractaon wath R N A polymerase have been mapped to different parts of the cI protein This exqmslte specmlisatlon of a transcriptional regulatory factor may reflect the space limitations of the bacteriophage genome, but as we shall see below, slmdar complexatles may exast m regulatory factor in eukaryotes The sttmulatory effects of cI interaction with R N A polymerase causes even more cI to be made, tins effect is autoregulatory since the excess cI can bind to OR3, which is a weak affinity site, and prevent further cI synthesis b;y blocking banding of R N A polymerase to PRM (Fig 4C) Upon induction, the cI molecule is inactivated by the protease activity of the recA protein This causes the levels of cI to drop to the point where OR2 and OR1 are no longer occupied, with the result that PR can bind R N A polymerase and tins leads to the production of the cro protein Cro protean is itself a repressor, and it also binds to the OR1, OR2 and OR3 s~tes, but with the opposite range of affinities to that seen by cI Thus low levels of cro occupy OR3 and O a 2 to block any further cI synthesis (Fag 4D) The repressor proteins cl and cro have related structures in the regions winch bind DNA, as might be expected for factors that recogmse slmdar sequences Both proteins have a hehx-turn-hehx structure, and mutagenesas experiments have indicated that the subtle but crucial differences in binding speoflCltles are due to differences in the recognition helix

with the subsequent appearance of differentiated functions [81-83] Kdlary and Fournxer [84] have demonstrated that the extraction of hver-specfflC funct,ons occurs at the level of transcriptional repression and can be assigned to a specific locus, called Tse-1 Recent work has shown that Tse-1 is expressed in m a n y nonhepatic cell types [85] The product(s) of the Tse-1 locus are apparently not sufficient to establish full repression of hver-speclfic functions, since hybrids retaining human chromosome 17 or mouse chromosome 11 as their only fibroblast derived chromosome do not shut down hver-speclfiC genes as strongly as hybrids with a larger flbroblast complement [86] Independent evidence for the existence of trans-actmg repressor molecules comes from experiments winch demonstrated that lninbltors of protein synthesis could induce specific gene expression Derepression of gene expression has been observed for fl-lnterferon [87,88], c-myc [89,90] c-fos [90], lnterleukin-1 [91], lnterleukln-2 [92] and the PDGF-responsive genes JE, KC and JB [93] Perhaps even more surprisingly, Ishlhara et al [94] have shown that a transfected human immunoglobuhn 3' chain gene could be transcribed m flbroblasts in the presence of cyclohexxmlde In m a n y cases the actlvataon of gene expression has been shown to be at the level of transcription [88,90] The third hne of evidence for specific trans-actmg repressor molecules comes from cts-actmg sequence mapping approaches In the same way that deletion of key sequences with a corresponding loss of expression imphes loss of posmvely-actlng elements, an Increase m expression is taken as evidence for deletion of a negatively-acting element Many such deletion experiments have indicated the existence of negaUvely-actmg sequence elements, and it appears that m a n y genes are flanked by considerable expanses of alternating positive and negative elements However, most of these studies have only been performed at a low level of resolution (1 e, deletions of hundreds of base pairs), and m only a few cases has the arrangement of these elements been analysed m detail Some examples of this wall be discussed below

I l I - B The evtdence for spectftc represston mechamsms m eukaryotes

I H - C Potential mechamsms for spectfic repression

Some of the earliest evidence for the involvement of diffusible trans-actlng factors in the regulation of higher eukaryotic gene expression came from analysas of experiments involving cell fusions When differentiated and non-differentiated cells are fused, the differentiated functions are usually turned off, a phenomenon known as extmct~on (see Ref 80 for revaew) In an analysis of hver-speofic functions, extraction IS not related to loss of the genes for these speclahsed functions, suggesting specific repression mechanisms exist, continued passagmg of stable fusions can lead to loss of chromosomes

A key point to emerge from the dmcusslon of bacterml paradigms, ~s that negative regulation can be achieved by more than one mechamsm In eukaryotes, this also seems to apply, although m few cases have the molecular detads been established Fig 5 shows four basic models which represent the types of repression so far d~scovered in eukaryot~c systems Panel A indicates repression by stenc occlusion (1 e , a direct competition for overlapping sites between positive and negaUve transcription factors) Tins m e c h a m s m has been clearly estabhshed in prokaryotes (e g , the regulation of lambda

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