the virtuoso of versatility: pou proteins that flex to fit

doi:10.1006/jmbi.2000.4107 available online at http://www.idealibrary.com on J. Mol. Biol. (2000) 302, 1023±1039

REVIEW ARTICLE

The Virtuoso of Versatility: POU Proteins that Flexto Fit

Kathryn Phillips* and Ben Luisi*

Department of BiochemistryUniversity of Cambridge, 80Tennis Court Road, CambridgeCB2 1GA, UK

E-mail addresses of the [email protected]; ben@

0022-2836/00/051023±17 $35.00/0

During the evolution of eukaryotes, a new structural motif arose by thefusion of genes encoding two different types of DNA-binding domain.The family of transcription factors which contain this domain, the POUproteins, have come to play essential roles not only in the developmentof highly specialised tissues, such as complex neuronal systems, but alsoin more general cellular housekeeping.

Members of the POU family recognise de®ned DNA sequences, and awell-studied subset have speci®city for a motif known as the octamerelement which is found in the promoter region of a variety of genes. Thestructurally bipartite POU domain has intrinsic conformational ¯exibilityand this feature appears to confer functional diversity to this class oftranscription factors. The POU domain for which we have the most struc-tural data is from Oct-1, which binds an eight base-pair target and var-iants of this octamer site. The two-part DNA-binding domain partiallyencircles the DNA, with the sub-domains able to assume a variety of con-formations, dependent on the DNA element. Crystallographic and bio-chemical studies have shown that the binary complex provides distinctplatforms for the recruitment of speci®c regulators to control transcrip-tion. The conformability of the POU domain in moulding to DNAelements and co-regulators provides a mechanism for combinatorialassembly as well as allosteric molecular recognition. We review here thestructure and function of the diverse POU proteins and discuss the roleof the proteins' plasticity in recognition and transcriptional regulation.

# 2000 Academic Press

Keywords: DNA-protein interactions; transcription regulation;POU domain; folding-induced recognition; Oct-1
*Corresponding authors
Introduction

With the recent advances in whole-genome ana-lyses, the molecular details that underlie the diver-sity of life are emerging. One salient exampleconcerns the complexity of modern multi-cellularorganisms, which appears to have arisen in concertwith the fusion of simple building-blocks to createsophisticated regulatory components (Rubin et al.,2000). POU domains are a classic example of sucha modular molecular construction, taking ancientfolds used in transcriptional control in prokaryotesand early eukaryotes and adapting them to regu-lation in highly specialised tissues associated withcomplex organisms. The modular structure impartsa functional versatility that allows the domain to

ding authors:cryst.bioc.cam.ac.uk

participate in transcriptional regulation of a varietyof ubiquitous and tissue-speci®c genes.

Transcription is a highly organised process thatis tightly regulated by speci®c recruitment of regu-latory cofactors into large assemblies. These in turnaffect transcription rates by interacting, sometimesover several kilobases, with components of the pre-initiation complex (Kornberg & Baker, 1992; Naaret al., 1999; Rachez et al., 1999). The preinitiationcomplex is at the core of the assembly and itrecruits and activates an RNA polymerase mol-ecule at the gene start site to transcribe the DNAtemplate. Structural studies of the preinitiationcomponents is advanced, with crystal complexesavailable for many of them. Now a catalyticallyactive form of RNA polymerase II (pol II) has beensolved to a resolution of 3.5 AÊ (Cramer et al., 2000).This has shed light on the molecular interactionswhich lead pol II to form the transcription

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1024 POU Proteins that Flex to Fit

complex. The complexity of the polymerase, thepre-initiation complex, the associated regulatoryassembly, and in addition the essential role ofchromatin organisation, indicates that transcriptioninvolves many interactions at different structurallevels and that hierarchical organisation is a featureof eukaryotic transcription regulation.

Another hallmark of transcription in eukaryotesis that individual regulatory components have amodular architecture, comprised of discrete func-tional and structural domains. For instance,domains encompassing DNA binding, transcrip-tion activation or repression functions can be fusedtogether to create chimeric transcription factorswith novel characteristics. The family of POUdomain proteins is an example of this principlewith a diverse assortment of domain-de®ned func-tions found outside the POU motif.

The POU domain was ®rst identi®ed 12 yearsago and takes its name after the ®rst four transcrip-tion factors in which it was found: mammalianPit-1, Oct-1, Oct-2 and Caenorhabditis elegans Unc86(Clerc et al., 1988; Finney et al., 1988; Herr et al.,1988; Ingraham et al., 1988; Sturm et al., 1988). Asub-region of the domain was shown to containthe previously well-characterised DNA-bindingmotif known as the homeodomain. Homeodomainproteins comprise a superfamily of highly con-served DNA-binding factors that are involved inthe transcriptional regulation of key developmentalprocesses. The POU domain was distinguishedfrom other homeodomains by a region foundN-terminal to it, which when ®rst identi®ed hadno known homologues. The two sub-domains areseparated by a segment of variable sequence(Sturm & Herr, 1988) suggesting that the domainwas bipartite in nature.

Today, there are over 150 entries for POUdomains in the SMART database of proteindomains involved in signalling events (Schultzet al., 2000). Although this is a mere fraction of the2000 homeodomain proteins identi®ed so far, theimportance of the POU proteins is undeniable, asthey are known to regulate many fundamentaldevelopmental and homeostatic processes, such asembryogensis and histone gene expression.

POU domain proteins have been groupedaccording to properties of the linker that separ-ates the two DNA-binding domains (Ryan, 1997;Wegner et al., 1993; Xi et al., 1989). At present,seven distinct classes have been elaborated onthe basis of linker length and composition(Spaniol et al., 1996). The neuronally expressedPit proteins de®ne the type I class, and are thesole example. The ubiquitously expressed Oct-1and tissue-speci®c Oct-2 are representative of thesecond category. Brn1,2,4 and Tst-1, which areinvolved in neural development, comprise athird category, while the Brn3.x class withUnc86, also involved in development, comprise afourth category. Type V POU domains tend tobe involved in early embryogenesis and type VIare expressed in the central nervous system,

while type VII POU proteins are critical forearly developmental stages (Ryan & Rosenfeld,1997; Spaniol et al., 1996). Despite this classi®-cation scheme, the evolutionary relationshipbetween the different POU families is not clear.

The pivotal role that the relatively small domainplays in transcriptional regulation has been dis-sected by molecular genetics in parallel withstereochemical analysis. Over the last six years, asmall but steadily growing body of structural datahas become available on the POU sub-domains,the complete domains in complex with their pro-moter sites and, more recently, a POU protein andco-activator assembly. This review addressesbrie¯y the particular developmental pathways thatare governed by POU proteins, the underlyingstructural characteristics of the POU domain, andits versatile role in recruitment of other proteins toaffect transcription of different genes.

Oct 1 and 2, two POU archetypes

Both Oct-1 (Sturm et al., 1988) and Oct-2 (Clercet al., 1988) were identi®ed as the proteins whichtarget the ubiquitous octamer regulatory element,ATGCAAAT (Falkner & Zachau, 1984; Parslowet al., 1984). While Oct-1 is found in all tissuetypes, Oct-2 is expressed speci®cally in theimmune system's B cells. Oct-1 is known to interactwith a variety of other transcriptional regulators toactivate the transcription of small nuclear RNA(snRNA) genes (Segil et al., 1991; Yang et al., 1991),histone H2B (Hinkley & Perry, 1992) and immuno-globulin genes (Sturm et al., 1988). It is also appro-priated by the herpes simplex virus (HSV) toactivate expression of the viral immediate early(HSV IE) genes (Cleary et al., 1993). The tissue-speci®c Oct-2, on the other hand, was thought toplay a much more specialised role, and to be solelyresponsible for regulation of immunoglobulin geneexpression (Landol® et al., 1986; Staudt et al., 1986).However, it has since become clear that the role ofOct-2 in immunoglobulin regulation is to someextent redundant and that Oct-1 can functionallysubstitute for Oct-2. In B cells, a co-activatoractivity has been found which can interact withboth Oct-1 and Oct-2 to regulate immunoglobulingene expression (Annweiler et al., 1992; Luo et al.,1992; Pierani et al., 1990). We shall return to thisco-activator in greater detail later.

Oct-1 can activate transcription by a variety ofdifferent mechanisms. For example, the snRNAgenes, which are activated by Oct-1, aretranscribed by two RNA polymerases, pol II andpol III. It is intriguing that both polymerases areregulated by the same activator complex. Oct-1behaves as an accessory factor to locate this activa-tor complex at the correct regulatory element(Mittal et al., 1999). In other cases, Oct-1 acts insynergy with recruited factors to upregulate tran-scription. For example, the activation domain ofOct-1 was originally shown to have a relativelyweak capacity to stimulate HSV IE gene

POU Proteins that Flex to Fit 1025

expression, but it was shown that its functionwould be enhanced by co-operation with the acti-vation domain of VP16 (Ryan & Rosenfeld, 1997).

Anatomy of the POU domain

After the initial identi®cation of POU domains, itwas already clear that the fold could be dividedinto two distinct sub-domains separated by a vari-able linker. These sub-domains were named thePOU speci®c domain (POUS) at the N terminus ofthe motif, and the POU homeodomain (POUH) atthe C terminus. The larger POUS domain (75amino acid residues) was thought to be largelyhelical, while the POUH domain (60 amino acidresidues) had all the features of the DNA-bindingmotif found in the ubiquitous homeodomain tran-scription factors and was known to contain thecharacteristic helix-turn-helix motif (Sturm et al.,1988; Sturm & Herr, 1988).

The sub-domains were found to be structurallyautonomous and to have separate sequence speci®-cities, although with af®nities reduced comparedto the intact domain (Verrijzer et al., 1992). The ®rststructural data about homeodomains came notfrom POU proteins but from two Drosophilahomeotic transcription factors: Engrailed (Kissingeret al., 1990) and Antennapedia (Otting et al., 1988).Both structures veri®ed that homeodomains weresmall, compact helical bundles which contain ahelix-turn-helix type motif, and the mode of inter-action with DNA was through the third helix,which gained an extra turn compared to the freeform (Kissinger et al., 1990; Otting et al., 1990). The®rst structural information about a POUH domaincame from the NMR structures of the Oct-3 home-odomain (Morita et al., 1993) and later the Oct-1homeodomain (Cox et al., 1995). This was sooncomplemented by the 3D structure of a POUS

domain derived by NMR (Assa-Munt et al., 1993;Dekker et al., 1993). It is striking that the fold ofthe POUs domain was found to be very similar tothat of the well-known prokaryotic bacteriophagelambda repressor, despite the lack of sequencehomology. The NMR structures revealed a bundleof four helices with the second and third helicescomprising a helix-turn-helix motif. Based on thisstructural homology, Assa-Munt et al. (1993)predicted that the interaction of POUS with itsoperator site would be very similar to that foundin the bacteriophage 434 and lambda repressorstructures.

From NMR experiments with a high-af®nity site,Dekker et al. (1993) also proposed a model for theinteraction of the intact POU domain, where thesub-domains bound to opposite faces of the pro-moter with the helix-turn-helix domains from eachmodule inserted in the major groove. This modelhas been corroborated and extended by crystallo-graphy of the full Oct-1 POU domain complexedwith DNA (Klemm et al., 1994).

How does POU recognise the octamer?

The crystal structure of the Oct-1 POU domaincomplexed with the octamer site from the H2B pro-moter has revealed the detailed interactions under-lying recognition of the DNA element (Figure 1)(Klemm et al., 1994). This has shown that the twosub-domains make independent interactions withthe two half-sites of the operator, and that they arebound on opposite faces of the DNA. Only part ofthe linker that joins the sub-domains is visible andthis segment is found to track along the minorgroove. The remaining 25 residues of the linker aredisordered, indicating unrestrained ¯exibility. TheDNA oligomer with which the domain was crystal-lised showed an almost ideal B-form structure,with little local distortion or global curvature.

The ATGC subsite of the octamer element (ATG-CAAAT) is contacted by the POUS domain, withan extensive array of direct and sequence-speci®chydrogen bonds from surface-exposed residues onhelix 3. A hydrogen-bonded network of highlyconserved residues orientates Gln44 such that itmakes two hydrogen bonds to A1 (Figure 1(c)). Itis remarkable that the same network of interactionsoccurs in the lambda repressor/DNA complex(Klemm et al., 1994). Residues Thr45 and Arg49also directly recognise T2 and G3/G40, respectively.There is also one hydrophobic interaction betweenLeu55 and the methyl group of T5 (Klemm & Pabo,1996). These direct sequence-speci®c interactionsare augmented by a network of phosphate back-bone contacts which are contributed by residues inall four of the POUS helices and in some of theloops. One strand of the duplex is contacted byresidues Arg20, Thr26, Gln27 and Ser48, whilephosphate groups on the opposite strand are con-tacted by residues Ser43, Thr46, Ser56, Asn59 andLys62. These extend into the AAAT site and theresidues involved tend to be highly conserved inthe POU family.

The details of the POUH interaction are verysimilar to those that have already been observedfrom other homeodomain/DNA complex struc-tures. Helix 3 from the homeodomain rests in theAAAT site major groove. The three base speci®ccontacts which can be seen in this structure arealso a mixture of hydrophobic and electrostaticinteractions. Val47 is in van der Waals contact withT8 and Cys50 sits between the methyl groups ofboth T90 and T100. The only clearly visible sequence-speci®c hydrogen bonding interaction is contribu-ted by Asn51 to A7.

The POUH N-terminal arm, which comprisespart of the ¯exible linker peptide, tracks along theminor groove in a manner similar to that seen inall other homeodomain structures. Arg5 of POUH

hydrogen bonds in the minor groove to A5, and itscomplementary base is contacted by Leu55 of thePOUS domain. This set of interactions distinguishesoctamer position 5 as the only base-pair contactedby both POUS and POUH.

Figure 1. (a) View of the Oct-1/H2B crystal structure (Klemm et al., 1994) looking into the major groove of the pro-moter, showing the POUH and POUS sub-domains bound to the two half-sites. The helices are labelled for the indi-vidual sub-domains and the N and C termini are indicated. This and all subsequent Figures were made by RIBBONS(Carson, 1997). (b) View down the DNA axis showing that in projection, the POU domain wraps around three-quarters of the DNA circumference. (c) Schematic views of three POU domain/DNA complexes. Hydrogen bondedinteractions are shown by unbroken arrows while non-polar interactions are depicted by broken arrows. Interactionswith the minor groove are indicated by green arrows, all other arrows represent interactions with the major groove.The octamer-binding site is highlighted in pink and the DNA numbering scheme used in the text is shown. Comp-lementary bases are indicated in the text with a prime but for clarity are not labelled here. Bob1 is represented as ablue shaded block, and new interactions which are found in the Oct-1/Bob1 complex are highlighted in red. Thebackbone of Va122 makes two hydrogen bonds with A5, while the side-chains of Val22 and Val24 buttress the inter-action through hydrophobic contacts with T50 and T60.



Linker function in promoter recognition andco-operative effects

Although structural biology is an incrediblypowerful tool, providing detailed informationabout stereochemistry, it gives little informationabout conformationally variable regions. Forinstance, the crystal structure of the Oct-1/H2Bpromoter (Klemm et al., 1994) provided no struc-tural information about the role that the linkermight play in sequence-speci®c recognition. Moresubtle approaches had to be used to begin tounravel the linker's contribution to the assemblyprocess (Klemm & Pabo, 1996; van Leeuwen et al.,1997) (Figure 2).

It appears that one of the potential roles of anintra-domain linker is to mimic the co-operativeeffect of dimerisation (Perutz, 1990). By tetheringthe sub-domains, the POU linker increases thelocal concentration of each sub-domain when inthe vicinity of a promoter so that the domainbehaves as a heterodimer (Herr & Cleary, 1995).Although the single-domain homeodomain pro-teins are monomers, they often form co-operativeassemblies to achieve the same effect (Li et al.,1995; Tan & Richmond, 1998).

The co-operative effect of the Oct-1 POU linkerhas been quanti®ed to show that it increases thelocal concentration to 3.6 � 10ÿ3 M, well above theKd of the isolated domains (Klemm & Pabo, 1996).In addition to the contribution of the linker, a

Figure 2. POU domains can assume a variety of conformaproposed orientations that POU domains assume on differmonomer, as shown by crystallography (Klemm et al., 1994underlined (Chasman et al. 1999). Pit-1 co-operatively dimeritallography (Jacobson et al., 1997). Oct-1 POU binds the ICdomains in alternative orientations as shown by protein/domains are believed to co-operatively oligomerise, which ha

second co-operative effect is associated with POU/DNA interactions. This can be seen in DNA bind-ing by a mixture of free, untethered sub-domains,which surprisingly still bind the octamer elementco-operatively (Klemm & Pabo, 1996) despite thelack of direct protein/protein interactions betweenthe sub-domains (Figure 1) (Klemm et al., 1994). Itseems likely that this second type of co-operativityarises from a type of allostery in which one sub-domain binds and pre-organises the DNA so thatit is ready to accept the second sub-domain. The®fth base-pair (A-T) contributes in some way to theco-operativity of the POU interaction, as it is theonly site where the two sub-domains share a base-pair contact. Isothermal titration calorimetry hasalso shown that this base-pair is important for theOct-1/octamer interaction, as switching the base-pair (to T-A) reduces the binding constant from18.9 � 106 Mÿ1 to 7.5 � 106 Mÿ1 (Chang et al.,1999). The reduction in af®nity occurs despite afavourable increase in enthalpy, suggesting thatthere is some unfavourably compensating entropiccost associated with this mutation, possibly due tothe loss of van der Waals contacts.

The linker also contributes to the DNA inter-action in another way. The most highly conservedresidue in POU class II linkers is a glutamate, fourresidues before the basic patch at the N terminusof the POUH domain. van Leeuwen et al. (1997)mutated this glutamate to lysine and found, quite

tions and oligomerisation states. Schematic views of theent promoter sites. Oct-1 POU binds the octamer as a). Bob1 is shown as a shaded block and the octamer isses on the PrlP promoter, which was also shown by crys-

P0 and ICP4 promoters as a monomer with the sub-DNA crosslinking (Cleary et al., 1997). The Brn POUs been shown by gel shift assays (Rhee et al., 1998).

Figure 3. (a) View perpendicular to the DNA axis ofthe PrlP/Pit crystal structure 39 (Jacobson et al., 1997)showing two monomers of Pit-1 bound to the promoter.One monomer is shown in purple and the other ingreen. The dimer interfaces are shown, as the monomersassociate on one surface of the DNA. (b) View down theDNA axis showing that the doughnut-shaped Pit dimerforms a puckered clamp that partially encircles theDNA surface.


remarkably, a threefold reduction in af®nity for theoctamer site, even though this might seem to bethe more electrostatically favourable match to thecharge of the DNA. This effect must arise fromsome other phenomena, possibly such as pre-organising the DNA binding heads for presen-tation to the octamer site (van Leeuwen et al.,1997). Another possible explanation is that the glu-tamate residue is involved to some extent in target-ing to a complementary electrostatic ®eld in amanner similar to that proposed to explain theeffects of charge mutations of the Trp repressor(Guenot et al., 1994).

Pit-1, POU regulation in pituitary developmentand function

Pit-1 is expressed exclusively in the centralnervous system during development and later inneurons and in the pituitary for regulation ofhormone secretion (reviewed by McEvilly &Rosenfeld, 2000). Once differentiated, the pituitarygland is comprised of six distinct cell types, each ofwhich is dedicated to the secretion of speci®c hor-mones (Dasen & Rosenfeld, 1999). Pit-1 was ®rstidenti®ed as the transcription factor that bound aregulatory region common to the prolactin andgrowth hormone genes (Bodner et al., 1988;Ingraham et al., 1988). However, its role in cellulardifferentiation in the developing pituitary becameapparent when a spontaneous mutation in micewas identi®ed which resulted in the failure of threepituitary-speci®c cell lines to differentiate. Thismutation was found to be localised to the Pit-1gene, con®rming that Pit-1 was crucial for develop-ment of the pituitary gland (Li et al., 1990).Mutations in the human form of Pit-1, POU1F1,have been identi®ed (reviewed by Pfaf¯e et al.,1996) and although the effect seems to be lessdramatic than in mouse, the result is that individ-uals who carry the mutations are de®cient inprolactin and human growth hormone (McEvilly &Rosenfeld, 2000).

Recognition of DNA through dimerisation

The Pit-1 POU domain binds an assortment oftargets, and the variation makes it dif®cult toachieve anything more then a weak consensussequence for the majority of Pit regulatoryelements. Pit-1 has accommodated this variationthrough the high degree of conformational ¯exi-bility conferred by the POU domain, whichendows it not only with an ability to re-orientatethe sub-domains to recognise different sites, butalso to co-operatively oligomerise (Figure 2)(Holloway et al., 1995; Ingraham et al., 1990). ThePit-1 POU domain was crystallised on a DNAsequence (Jacobson et al., 1997) based on the palin-dromic prolactin promoter, PrlP (Figures 1(c) and2). The PrlP promoter is much longer than the octa-mer element, which is intriguing, as the Pit POUdomain happens to have the shortest linker pep-

tide in the POU family, which might be thought torestrict the size of the element that it could cover.This apparent conundrum is solved by the crystalstructure, which shows that Pit-1 does not bind asa highly extended monomer, but instead dimerisesto span the 12 base-pairs of the promoter (Figure 3).

The crystal structure of the Pit-1/PrlP responseelement complex shows a very different sub-domain arrangement on the DNA compared tothat seen in the Oct-1/octamer complex (Figures 2and 3). The dimer binds on one face of the DNA.Both POU sub-domains form a concave surfacecontinuous with the major groove of the promoter(Figure 3(b)). As the structure has been determinedto an intermediate resolution of 2.3 AÊ , a fewordered water molecules can be located in the


structure. Five water molecules are found in theprotein/DNA interface. Only one water moleculeappears to be involved in mediating a sequence-speci®c interaction to a base which is outside ofthe PrlP consensus site (POUH Arg46 to Tÿ2,Figure 2), while the remaining four molecules med-iate phosphate backbone contacts.

The Pit-1 homeodomains dock in the majorgroove in the same manner as the Oct-1 homeodo-main, binding to the AT site at the 50 end of thepalindrome (Figure 1(c)). Again, the three mainplayers in sequence-speci®c recognition of the AThalf-site are Val47, Cys50 and Asn51. However,this structure was the ®rst to clearly reveal theinteraction between Gln54 and an adenine base(A1). This residue is one of the most highlyconserved in the POU family, and as suchdistinguishes POUH from conventional homeodo-mains. The N-terminal Pit-1 POU homeodomainarm contacts the minor groove, but again, this isthe only region of the linker which is clearly visiblein the electron density map, as the preceding 16residues are disordered.

Comparing the Pit-1 and Oct-1 POU structuresshows that the sub-domains have undergone adramatic rearrangement. Not only has Pit-1 POUS

¯ipped its orientation relative to the Oct-1 POUS

(Figure 2) but also it binds a completely differentface of the DNA, and the spacing between the twosub-domains changes. The POUS domain of Pit-1recognises an ATAC half-site (A7-C10 inFigure 1(c)), as opposed to the ATGC site (A1-C4 inFigure l(c)) recognised by Oct-1. It is the POUS`sspeci®city for the ATA

GC pattern that causes thesub-domain to switch its orientation. The speci®cinteractions, which are conserved by this reorienta-tion, are hydrogen bonds between Gln44 and A7,and between Thr45 and T8 and T90, which are onopposing strands. Arg49 interacts with G100 on thecomplementary strand of the ATAC half-sitethrough bidentate hydrogen bonds.

As in the case of the Oct-1 POU linker, there wasno continuous electron density linking the twosub-domains. This left two possible combinationsof linkages, one of which placed the sub-domainson opposing strands, while the other situated bothon a single strand. The latter arrangement appearsto permit the more relaxed linker conformations.The problem was resolved experimentally byengineering Pit-1 mutants with shorter linkers sothat it could only bind the PrlP site if both sub-domains bound the same strand. Although the lin-ker was shortened by ®ve residues, the mutantPOU domain still bound PrlP, con®rming the top-ology shown in Figure 2 (Jacobson et al., 1997).

The Pit-1/PrlP structure shows a surprisinglycompact arrangement of the individual sub-domains on the promoter compared to that seenfor the monomeric Oct-1/octamer structure(Figures 1(a) and 3(a)). The POUS of one monomercontacts the C-terminal end of the POUH DNAbinding helix of the adjacent monomer(Figure 3(a)). There are two interesting features of

this interaction. First, the C terminus of the shortPOUH recognition helix forms part of the dimerinterface. In conventional homeodomains, thishelix extends by gaining an extra helical turn onbinding DNA, but this does not happen with Pit-1.If the Pit-1 POUH helix were to gain the extra turnthe dimerisation interface would be disrupted, for-cing Pit-1 to bind as a monomer. Second, the dis-ruption of this interface is associated with pituitarydysfunction as a key residue (R271) that is situatedin the interface is often found mutated to trypto-phan in combined pituitary hormone de®ciency(CPHD). It is clear that the ability of Pit-1 to dimer-ise on certain promoter elements is an essentialaspect of its regulatory function (Jacobson et al.,1997).

Pit-1 is known to form a platform for the recruit-ment of co-activator/repressor complexes in theregulation of Pit-1 signal-transduction pathways,with different surfaces of the transcriptional com-plex mediating different responses. The histonedeacetylase protein N-Cor, which acts repressivelyon Pit-1 activity, interacts with both the POUH andPOUS sub-domains; however, the interaction withthe activator CREB binding protein is speci®callytargeted to a loop in the homeodomain (Xu et al.,1998). Other surfaces in the complex may alsorecruit components that are yet to be identi®ed,and it is conceivable that the concave surfaceformed by the POUS domains and the exposedmajor groove of the promoter (Figure 3(b)) mayform one of these surfaces.

A comparison of the Pit-1 and Oct-1 DNAstructures reveals some conserved features of thePOU/DNA interface (Figure 1(c)). Within themajor groove, many direct sequence-speci®c inter-actions are conserved, such as Arg49, Thr45 andGln44 from the POUS to the ATA

GC half-site, whileCys50 and Asn51 interact with an A and T base,respectively, in the other half-site. Cys50 is one ofthe most highly conserved residues in the POUfamily, yet it is rare to ®nd a cysteine residue inprotein/DNA interfaces (Nadassy et al., 1999). Wedo not believe that the cysteine is conservedbecause of redox control, as has been proposed forother transcription factors (Jayaraman et al., 1997;Xanthoudakis et al., 1996). Instead, the residue maybe required because of its hydrophobic character,which favours contacts to thymine methyl groups,and its hydrogen-bonding potential.

More recently, a novel structure of an Oct-1POU dimer bound to a palindromic binding site(ATGCATATGCAT) has been solved to a resol-ution of 1.9 AÊ (A. Remenyi et al., unpublishedresults). This structure is signi®cant for two princi-pal reasons, as it is the ®rst dimeric Oct-1 POUstructure, and secondly it is the ®rst time that aPOU domain has been determined at such highresolution. The overall conformation is very similarto that of the Pit-1 dimer, with RMS values of1.3 AÊ for one POU domain and 2.1 AÊ for thesecond. Despite the accuracy with which the struc-ture has been determined, the linker region is not


visible. However, the higher resolution hasallowed the clear observation of an extensivehydration network, with 17 well-de®ned watermolecules found in the protein/DNA interface.Five of these water molecules are found in similarpositions in the Pit-1/DNA structure, where fourof them are involved in mediating protein/phos-phate contacts and the ®fth mediates an Arg/Tÿ2

hydrogen bond. The structure reinforces the fantas-tic versatility of the POU domain which cannotonly adapt to bind in different conformations, butcan also bind promoter elements with differentstoichiometries.

Structural versatility in central nervoussystem development

The best-studied system for neuronal develop-ment is that of Unc-86 in the nematode C. elegans(reviewed by Sengupta & Bargmann, 1996). Unc-86was ®rst identi®ed as a protein involved in controlof cell lineage fate and neuronal terminal differen-tiation (Chal®e et al., 1981) and was later found tobe expressed exclusively in neuronal cells. Afterasymmetric cell division, Unc-86 is expressed inonly one of the daughter cells. The cell that lacksUnc-86 follows the differentiation pattern of themother cell, while daughter cells with functionalprotein are programmed to follow an entirelydifferent developmental pathway, suggesting thatUnc-86 regulates cell fate decisions (Finney &Ruvkun, 1990).

In vertebrates, the homologue of C. elegansUnc86 is Brn3.0. The Brn group of POU domainproteins and Tst-1, which are found expressed inthe forebrain, also regulate cell fate decisions toestablish the development of neurons and theirintricate linkage patterns (reviewed by McEvilly &Rosenfeld, 2000). Type III Brn domains (Brn-2, Brn-4 and Tst-1) are expressed early on during centralnervous system (CNS) development, playing a rolein neural tube formation, and continue to beexpressed on into adulthood (Alvarez-Bolado et al.,1995; Xi et al., 1989). Deletion studies only lead tovery localised disruption of development, despitethe broad distribution of type III Brn proteins inthis tissue. This suggests that there is a largedegree of redundancy in development pathwaysassociated with this class of POU proteins. How-ever, this is not the case for type IV Brn POUdomains (Brn 3.x). This class of Brn proteins have amore distinct spatial distribution in the CNS andappear to regulate the development of certainspeci®c sensory functions (reviewed by McEvilly &Rosenfeld, 2000).

Like Pit-1, the Brn proteins are characterised byversatile DNA recognition, binding a selection ofpromoters in a variety of conformations by co-operative dimerisation as shown in Figure 2. Con-sensus promoter sites for Brn2, Brn3.x and Brn5domains have all been de®ned (Gruber et al., 1997;Li et al., 1993; Rhee et al., 1998; Xiang et al., 1996).Brn2 tolerates sites with a variety of spacers by co-

operative dimerisation (Figure 2). However, oligo-merisation turns out to play a repressive role inthis case, as a Brn2 site that was modi®ed to favourmonomer binding was shown to have an elevatedlevel of transcription (Rhee et al., 1998). The opti-mal Brn5 site and the Brn3.x class (Brn3.0, Brn3.1Brn3.2) consensus sites are very similar, lack spacerbases and bind the POU domain as a monomer(Figure 2). It is incredible that the site preferredby Brn-3.x proteins has been conserved fromC. elegans through to mammals, showing that con-servation of these factors is important in neuraldifferentiation and probably derives from the ear-liest development of nervous tissue in a commonancestor. Deviation from the Brn3.x consensus siteshows a concomitant reduction in transcriptionalactivation (Gruber et al., 1997). So, one group ofBrn transcription factors are naturally down-regu-lated, while the other has maximum transcriptionalactivity from sites that match the consensus mostclosely, resulting in ®ne tuning of regulation by thepromoter element itself.

Viruses and Oct

Viral transcriptional regulation has evolved sothat the virus itself has few of its own endogenoustranscription factors and, instead, appropriatesthose of the host cell. In the case of HSV, the virionreleases the viral protein, VP16 (aTIF) into the cell,which in turn associates with a host cellular pro-tein, called host cell factor (HCF) or C1 (Herr,1998). HCF is synthesised as a large polypeptide,of 300 kDa which becomes post-translationallymodi®ed by cleavage into fragments which remainnon-covalently associated (Kristie et al., 1995;Wilson et al., 1993, 1995). The N and C-terminalregions alone of HCF are essential for transcrip-tional activation, with the C-terminal domain ofHCF interacting in some way with the acidic acti-vation domain of VP16 to allow association of thecomplex (LaBoissiere et al., 1997). This cellularcomplex is recruited to modi®ed octamer sites byOct-1 through an interaction with the POUH sub-domain (Pomerantz et al., 1995). These sites areboth involved in regulation of expression of theICP0 and ICP4 immediate early (IE) genes duringthe viral life-cycle (OCTA�TAATGARAT site fromthe ICP0 promoter and OCTAÿTAATGARAT fromthe ICP4 promoter) and both recruit Oct-1 POU inseveral alternative conformations, as shown bycrosslinking experiments (Cleary et al., 1997)(Figure 2). However, both promoters are stillcapable of recruiting VP16 (Cleary & Herr, 1995;Cleary et al., 1997; Herr, 1998). Another transcrip-tional element found adjacent to the IE enhancer isthat of the cellular transcription factor GABP (GA-binding protein) (Jones & Tjian, 1985; Triezenberget al., 1988). Vogel & Kristie (2000) have shownthat HCF co-ordinates recruitment of GABP, Oct-1and VP16 at the enhancer site and that HCF is aco-activator of GABP mediated transcription.


Another POU domain, the central nervous sys-tem restricted Brn-3.0, has also been found to inter-act with sites in the HSV genome. Although thiscomplex has little intrinsic transcriptional activity(Turner et al., 1997), it is possible that Brn3.0 insome way reactivates HSV through interactionswith other viral-speci®c transcription factors.

Adenovirus has also adapted Oct-1 for its owndevices, recruiting it to contribute to DNA replica-tion. The adenovirus origin of replication is com-prised of two regions, the ®rst binds the precursorterminal protein/DNA polymerase heterodimer,and the second recruits nuclear factor 1 and Oct-1(Pruijn et al., 1986; Rosenfeld et al., 1987). Oct-1'srole in initiation is to recruit the heterodimer,which comprises the DNA polymerase, to the pre-initiation complex. The details of this interactionshow that the majority of the contacts are mediatedby the POUH, in the same general region that alsointeracts with VP16, although different residues areinvolved in specifying the interaction (Coenjaertset al., 1994). While the POUS domain is found notto participate at all in the interaction, DNA replica-tion is severely affected when the DNA-bindinghelix of the POUS domain is mutated and the af®-nity for octamer sites reduced. This indicates thateven though the preinitiation complex interactsdirectly with the POUH, the speci®c domain isrequired for correctly locating Oct-1 at the origin ofreplication (van Leeuwen et al., 1995). The Oct-1interaction also affects the conformation of pre-terminal protein and it is possible that this acti-vates the DNA polymerase and initiates viral DNAreplication (Bolting & Hay, 1999).

HSV and adenovirus are not the only viruses tohave co-opted the octamer. Mouse mammarytumour virus proximal regulatory region has sev-eral response elements, including an octamer-binding site and glucocorticoid response element.At this promoter, Oct-1 has no intrinsic transcrip-tional activity, and is only activated by glucocorti-coid, suggesting a direct interaction between theglucocorticoid receptor (GR) and Oct-1. The inter-action interface is located between the POUdomain and two residues in the GR DNA-bindingdomain which are situated in the helix of theC terminal zinc module. It seems likely that theassociation between Oct-1 and GR results in trans-criptional co-operativity (Prefontaine et al., 1998).

Octamer-regulated transcription

As Oct-1 and Oct-2 stimulate transcription byinteracting with transcription promoter sites, anobvious question is whether the proteins actuallyinteract directly with components of the preinitia-tion complex, such as TBP, TFIIB, TFIIA, or othercomponents found at the transcription start site.Both Oct-1 and Oct-2 have been shown to associatethrough their POU domains with the conservedcore domain of TBP in vitro. This interaction wascon®rmed in vivo by co-precipitation, and co-trans-fection experiments with Oct-2 and TBP showed

that the proteins can synergistically promote tran-scription at an octamer-containing promoter(Zwilling et al., 1994).

Oct-1 has also been shown to play a critical rolein preinitiation complex recruitment at some pro-moters that lack the TATA element. For instance,Oct-1 may be able to functionally replace the roleof TBP through its interaction with TFIIB at thelipoprotein lipase promoter to initiate transcriptionat the octamer-binding site (Nakshatri et al., 1995).So, in some contexts, Oct-1 appears to functionallysubstitute for TBP. Another TATA-less transcrip-tion initiator is human Ying-Yang protein (YY1)(Usheva & Shenk, 1994). The crystal structure ofthis protein with its initiator site shows that unlikeTBP, YY1 does not bend the DNA element(Houbaviy et al., 1996). Oct-1 POU is also knownto perturb DNA very little (Klemm et al., 1994).Therefore, Oct-1 and YY1 can both recruit TFIIB toinitiate transcription and are functionally equival-ent to TBP although neither appears to act bydeforming the DNA. Instead, they might functionby simply orienting TFIIB for presentation to thepolymerase.

Although all of the cases which we have pre-sented so far have been associated with the acti-vation of transcription, the other face of geneticregulation is transcriptional repression. The inter-action between the nuclear hormone receptor, reti-noic acid receptor (RXR), and Oct-1 appears tomediate transcriptional repression (Kakizawa et al.,1999). Both proteins interact through their DNA-binding domains: Oct-1 via the POUH and RXRthrough both the zinc-bearing domain and a hingeregion that follows it. This interaction disruptsthyroid hormone/RXR heterodimer assembly onthyroid hormone response elements. Therefore, itseems likely that Oct-1 down-regulates transcrip-tion by sequestering RXR and preventing it fromforming active dimers with other members of thenuclear hormone receptor family (Kakizawa et al.,1999). As mentioned in the previous section, theglucocorticoid receptor DBD can also interact withOct-1. Although the RXR and GR DBD arestructurally homologous it is not clear if they bindPOU in the same way.

Transcriptional regulation also requires that oneof the three types of eukaryotic RNA polymerase isrecruited to the correct initiator site to transcribe aparticular class of genes. snRNA components ofthe spliceosome fall into two classes of gene: thosetranscribed by pol II and those that are transcribedby pol III. A conserved regulatory region appearsupstream of the promoter of both gene classes,called the distal sequence element (DSE) and thisturns out to contain another occurrence of theoctamer. Oct-1 binds at this site. The proximalsequence element (PSE) is conserved in the corepromoter region between the two gene classes.This element is bound by a transcription factorassembly, which regulates snRNA gene expression,and is collectively known as either the PTF(Murphy et al., 1992) or SNAP (snRNA activating


protein) complex (Henry et al., 1998; Sadowski et al.,1993). Unlike other Oct-1 associated transcriptionfactors, SNAP has an intrinsic DNA af®nity, andcan bind the PSE directly (Wong et al., 1998).

But what role does Oct-1 play in transcriptionalactivation if it is not the factor necessary forrecruitment to the PSE? It turns out that when Oct-1 binds the DSE, this enhances the af®nity ofSNAP for the proximal site via a direct protein/protein interaction (Ford et al., 1998). Glu7 at theN terminus of the POUS domain was identi®ed tobe the crucial residue in Oct-1 which interacts withthe SNAP assembly (Mittal et al., 1996). This resi-due is speci®c to Oct-1, and so other cellular POUdomain proteins will not substitute.

High-mobility group proteins (HMG) have alsobeen found to enhance Oct-1-regulated transcrip-tion; however, there is no evidence that this actionis through direct recruitment to the activator com-plex. Oct-1 and HMG2 interact through the POUand HMG domains, respectively, before recruit-ment into the regulatory complex (Zwilling et al.,1995), and it is conceivable that HMG may func-tion to increase POU/DNA association by pre-ordering the DNA-binding domain to enhance itspromoter af®nity.

Regulation of the immune response: Bob1 andits interaction with Oct-1/2

How gene transcription becomes tissue-speci®chas been an intriguing question. When the ®rstoctamer-binding factors were isolated, Oct-2 wasfound to be expressed exclusively in B cells andwas clearly very important in immunologicaldevelopment, as mice de®cient in Oct-2 die at birth(Corcoran et al., 1993). This led to the suggestionthat this transcription factor alone was responsiblefor B-cell limited expression of immunoglobulingenes. However, two groups also identi®edanother component of human B-cell nuclearextract, referred to as OCA-B, that was responsiblefor high levels of IgH expression in the presence ofeither Oct-1 or Oct-2. This suggested that sometissue-speci®c factor, other than Oct-1 or Oct-2,was essential for immunoglobulin gene control(Annweiler et al., 1992; Luo et al., 1992). The activecomponent from the B-cell extract was cloned inde-pendently by three groups and named Bob1, OCA-B and OBF-1, respectively (Gstaiger et al., 1995;Luo & Roeder, 1995; Strubin et al., 1995). It wasdemonstrated that Bob1 interacts speci®cally withOct-1 and Oct-2 to regulate transcription of the Igkgenes and is expressed solely in immunological tis-sue. Therefore, Bob1 directly regulates the tissue-speci®c expression of immunoglobulin genes.

Bob1 has the classical modular structure com-mon to transcription factors, with the DNA-binding domain located in the N-terminal regionand an acidic activation region at the C terminus.Another striking property of the peptide is its unu-sually high proline content (16 %), which mayimpart distinctive structural characteristics. As

Bob1 interacts with both Oct-1 and Oct-2, but notwith other octamer proteins with lower sequenceidentity, Gstaiger et al. (1995) reasoned that Bob1was probably recognising the highly conservedPOU regions (94 % sequence identity betweenOct-1 POU and Oct-2 POU).

Solution studies have shown that the Bob1 inter-action with Oct-1 POU/DNA is novel, as Bob1interacts with both POU sub-domains, even thoughthese are known to be spatially distinct whenbound to the H2B promoter (Gstaiger et al., 1996;Klemm et al., 1994; Sauter & Matthias, 1998). Infact, Bob1 can functionally replace the deletedPOU linker peptide to bring the independent POUS

and POUH domains together on the octamer site(Sauter & Matthias, 1998). In contrast, VP16 whichalso interacts with the Oct-1 POU domain, interactsonly through the POUH domain, and it would, inprinciple, be possible for both co-activators to berecruited simultaneously to a single octamerelement (Babb et al., 1998), although it is not clearif this happens in vivo.

High-af®nity binding by Bob1 requires the pre-assembled Oct-1/DNA complex. The direct inter-action between Bob1 and the promoter has a veryhigh Kd (Cepek et al., 1996) that is unmeasurable atmM concentration ranges when tested by isother-mal titration calorimetry (Chang et al., 1999). Bob1interacts directly with the octamer site, but withonly a distinct subset of those sites bound by Oct-1(Cepek et al., 1996; Gstaiger et al., 1996). It speci®-cally recognises the ®fth base-pair of the octamer(A-T) but only upregulates transcription from theIgk promoter, even though it interacts with the H2Bpromoter (Gstaiger et al., 1995), suggesting the octa-mer ¯anking regions affect the interaction in someway, perhaps by recruiting additional factors.

Biophysical characterisation has shown Bob1 tobe unstructured when free in solution (Chang et al.,1999). A logical conclusion from this observation isthat any interaction with Oct-1 would very likelybe associated with some structural rearrangementof Bob1 (Chang et al., 1999). Any such folding pro-cess can be monitored by the change in speci®cheat capacity upon complex formation (Spolar &Record, 1994). For Oct-1/Bob1/DNA the exper-imentally observed heat capacity change suggeststhat signi®cant regions of peptide, contributed byone or both proteins, become ordered or buriedupon association (Chang et al., 1999; LundbaÈcket al., 2000).

The transcriptional activation domain of Bob1 isunusual as it appears not to share the main featureof classical activation domains which can regulatetranscription autonomously. Instead, the Bob1 acti-vation domain has little intrinsic activationcapacity, and appears to require interactions withthe Oct-1 activation domain to function. Recruit-ment by homologous transcription factors to theoctamer site does not bring about upregulation(Krapp & Strubin, 1999). Bob1 also interacts withother components of the transcriptional machinery:TBP and TFIIB, but this transcriptional activity is

Figure 4. (a) View of one of the Oct-1Bob1/DNAcomplexes seen in the crystal structure (Chasman et al.,1999). The view is seen in the same orientation asFigure 1(a), looking into the major groove. The POUdomain is shown in green and the Bob1 peptide inpink ribbons. (b) View of the two Oct-1/Bob1/DNAstructures looking down the major groove showing thatOct-1 POU and Bob1 form a continuous ring around thepromoter, with the POU linker and Bob1, respectively,running along the minor and major grooves at the ®fthoctamer base-pair. (c) Sequence alignment of Oct-1 POUinteraction peptides found in SNAP and Bob1. Identicalresidues are in red. The residues of Bob1 that contactthe POUS domain are highlighted with blue boxes.These residues from a hydrophobic contact surface. Theregion of Bob1, which is helical in the crystal structure,is underlined in blue.


limited to certain promoter sites (Schubart et al.,1996). As the Bob1 activation domain has littleintrinsic structure, our conjecture is that it foldsupon binding to the activation domain of Oct-1 orto another co-activator protein, and that this com-plex acts as a scaffold for higher-order assembly.

Oct-1/Bob1/DNA crystal structure: how doesBob fit in?

What does a POU domain/co-activator complexlook like, and does the addition of the third partyaffect the other two components in any way? AsBob1 is known to have little intrinsic structure,what structural transitions accompany the assem-bly? The crystal structure of the Oct-1/Bob1/octa-mer element, with two copies of the complex in theasymmetric unit, provided the ®rst opportunity toaddress these questions (Figures 1(c) and 4)(Chasman et al., 1999).

The structure of the ternary complex showedthat Oct-1 POU changes its conformation littlecompared to the binary complex, and that only 20residues from the 44 residue Bob1 peptide are vis-ible in the electron density map. The peptide tracksfrom the POUH domain, across the major grooveperpendicular to the DNA axis, and ®nally foldsinto a short region of alpha helix running up theside of the POUS domain (Chasman et al., 1999).The overall fold of the complex shows thattogether, the two peptides completely encircle theDNA (Figure 4(b)), with Bob1 linking the two sub-domains on the opposite face of the DNA from thePOU linker, as had been predicted by Sauter &Matthias (1998).

Bob1 only interacts speci®cally with one base-pair in the major groove of the octamer. The pep-tide runs along the edge of the ®fth base-pair (A-T)that is also thought to mediate co-operativitybetween the POU sub-domains. The peptidebackbone of Val22 contributes two hydrogenbonds to the base edge of A5 (Figure 1(c)). Thepeptide is stabilised in this conformation by vander Waals contacts between Val22 and Val24 andhydrophobic groups in the major groove (T50 andT60). Leu55 from POUS, which is essential forrecruiting Bob1 to the ternary complex, alsocontributes to POU/octamer recognition, as it but-tresses Val24, and is responsible for speci®cally sta-bilising the peptide backbone conformation at A5.So, although there are few direct sequence-speci®cinteractions between Bob1 and the major groove,the de®ned contacts are reinforced by the Oct-1/DNA platform.

The majority of the protein/protein interactionsare mediated through complementary hydrophobicpatches on the helical region of Bob1 and the POUS

domain, which contributes about half of the inter-face buried in complex formation. POUS residuescontributed from the ®rst and fourth helices andone from the loop linking helices 3 and 4 comprisea pocket in which Val28 rests. Leu31 and Leu32,which also contribute to the Bob1 hydrophobic

patch, pack against the surface of POUS. The inter-face is augmented by a series of complementaryelectrostatic interactions between the two peptides,

Figure 5. Perpendicular views of the Oct-1/H2B andOct-1/Bob1/octamer crystal structures superimposed onthe DNA. The POUH domains of POU in the presenceof Bob1 (pink) and without Bob1 (green) are shown. ThePOUS domains in the presence of Bob1 and the Bob1peptide have been omitted for clarity.


but these differ between the two copies. Whetherthis is due to alternative crystal packing or limitsof the crystallographic re®nement procedure is notclear. Interactions between the N-terminal regionof the Bob1 peptide and the POUH domain are lessextensive, limited to one hydrogen bond and threevan der Waals contacts. Side-chains from Bob1 alsointeract with deoxyribose rings and phosphategroups from the DNA phosphate backbone. Theturn in the Bob1 peptide, which directs it out ofthe major groove and up the side of the POUH

domain, is stabilised by Tyr19, which sits snugly ina hydrophobic pocket formed by Bob1 and theDNA.

Solution studies of Bob1 peptides containing theDNA-binding region had con®rmed structure pre-dictions that suggested that free Bob1 was largelyunstructured, with some residual helical structure(Chang et al., 1999). It is possible that this peptideis structured when Bob1 is free in solution, andthat it somehow nucleates the assembly beforefurther ordering of the peptide through contactswith the protein/DNA platform. The short helixfound between residues 28 and 36 in the crystalstructure happens to be the region which hassequence homology with the POU-binding regionof SNAP (Figure 4(c)). Although conventionalsequence alignment techniques had not found anyregions of homology between Bob1 and SNAP,direct visual comparison of the two sequencesrevealed a short region of strong homologybetween the DNA-binding region of Bob1 and resi-dues 891-903 of SNAP190 (Ford et al., 1998). It ispossible that SNAP and Bob1 interact with POU invery similar fashions, with a short, vestigial helixnucleating the protein/protein interaction.

Does Bob1 docking in the major groove perturbthe Oct-1/DNA interaction at all? To test whetherthe POU sub-domains had undergone anyrearrangement as a result of Bob1 recruitment, wesuperimposed both copies of the Oct-1/Bob1/DNA complex on the Oct-1/DNA complex usingthe phosphate atoms of all the Watson-Crick base-paired bases (RMS of 1.3 AÊ for one copy of theBob1 complex onto the Klemm structure and 1.2 AÊ

for the second copy) (Figure 5). This results inhelix 3 of the POUS domain superimposing almostperfectly (maximum Ca deviation of 0.8 AÊ and aminimum of 0.13 AÊ ) with the Ca atoms in theDNA-binding helix showing virtually no deviationbetween the POUS domains even though they havenot been included in the superposition. However,this is not the case for the homeodomain, whichrotates away from its position in the major groove,down towards the Bob1 peptide. The N-terminalend of the POUH helix 3 superimposes well (Ca

deviation of 1 AÊ ) but at the C-terminal end, thehelix has rotated, such that the Ca positions ofequivalent residues are now shifted by up to 6 AÊ .

This is quite remarkable, as there are far fewerinteractions between Bob1 and the POUH domainthen with the POUS. Associated with this shift inposition of the POUH, there is an increase of the

order of one residue in the POUH/DNA interface(Figure 1(c)). Gln54 is disordered in the absence ofBob1, and was not assigned in the original Oct-1/DNA complex. However, in both copies of theBob1 ternary complex this residue has becomeordered, but appears to take alternate confor-mations in the two complexes. Lys57 towards theC terminus of the recognition helix is also broughtcloser to the phosphate backbone, so that it too canmake a further hydrogen bond to the A8' phos-phate group. The interaction of Bob1 with the Cterminus of the POUH domain may also contributeto stabilising the extra turn induced in the recog-nition helix when POUH binds DNA.

The Bob1 peptide seems to bring about thisrearrangement by behaving as a rigid extensionfrom the POUS domain. First, it is tightly pinned inthe complex through its interactions with the octa-mer ®fth base-pair. This is followed by a tight turn,


just after a glycine residue at position 21, whichforms a pocket that accepts Tyr19. The confor-mation assumed by Bob1 is dictated by contactswith the POUS and octamer which in turn force thePOUH to reorientate to maximise its interface withBob1. It seems that the POUH domain is tetheredby this interaction with the N terminus of Bob1.

Although it had been thought that assemblingthe ternary complex would lead to a signi®cantordering of the Bob1 peptide (Chang et al., 1999),the crystal structure has shown that it is not asextensive as expected. However, there is evidencethat both Oct-1 and Bob1 are sensitive to the octa-mer ¯anking regions (Cepek et al., 1996; Changet al., 1999). So, it is possible that there is still somesubtle rearrangement that may occur in the pre-sence of the full site that would lead to furtherordering of the peptide. It is clear that assembly ofcomplexes with more components of the activationcomplex will be necessary to understand furtherthe complex and intricate interactions which mustbe satis®ed before transcription is initiated.

Not only is the POU domain incredibly versatilein its ability to recognise a myriad of differentsites, but it is also suf®ciently plastic to be subtlymodi®ed by interactions with co-activators,increasing its ability to interact with promotersthat are co-activator speci®c. Bob1 causes a tiny,but measurable change in the relative orientationsof the POUS and POUH domains in such a waythat the POUH is able to make two more hydrogenbonds with the promoter in the presence of Bob1than when free. It is not possible at this resolutionto say whether these new interactions are speci®cand it is not clear whether they need to be, as theaddition of two hydrogen bonds, whether speci®cor not, would contribute favourably to the stabilityof the entire complex.

Summary and perspectives

Lefstin & Yamamoto (1998) have suggested thatsome transcription factors have the potential toassume a variety of conformations, and the choiceis governed by the bound DNA element itself.Therefore, the target can be thought of as an allo-steric modulator, serving to present different inter-faces for the recruitment of element speci®c co-factors to regulate a wide variety of transcriptionalevents (Lefstin & Yamamoto, 1998). In the case ofthe POU domain, it is the linker which permits thedomain to assume widely differing conformationsand oligomersiation states depending on the pro-moter context (Figure 2). It is this facility whichpermits a single transcription factor, such as Oct-1,to regulate widely differing transcription eventsthroughout the lifetime of an organism.

The crystal structure of the DNA-bindingdomain of Bob1 complexed with its partners intranscription adds another dimension to the vari-able life-style of the POU domain. The POU/pro-moter platform may become a paradigm forstructure-speci®c recruitment, allowing unstruc-

tured cofactors, such as Bob1, to nucleate and foldon the concave surface offered by the POU clampand the promoter major groove. Not only can theindividual POUH and POUS sub-domains adapt tobind a wide range of variations on the octamertheme, but they can pick and mix promoters,ignoring spacers and modulations to interact withfar more than conventional octamers. The Pit-1complex showed how POU can bind larger sites byforming homodimers. So, as well as behaving as aplatform to recruit complex regulatory assemblies,POU can also undergo some minor rearrangementsto optimise its interactions. This domain's chame-leon nature allows it to remould itself to ®t thecontext, whether it's the promoter it recognises, theco-activator or the class of polymerase it activates.

The crystal structure of this fragment of theimmunoglobulin transcriptional machinery alsoshows how it will become more of a challenge inthe future to successfully predict the structure andfold of many important regulatory proteins. As thequantity of sequence data expands enormously, itis anticipated that more and more intrinsicallyunstructured proteins will be identi®ed and thatthese will often be components of fundamentalregulatory assemblies. The stability of such assem-blies requires ``induced ®t'' type interactions,where these peptides will fold onto, and maybeeven rearrange, the structural platform providedby its partners (Wright & Dyson, 1999). Formationof the ternary complex between Oct-1/Bob1 andthe octamer site is one example of such an inter-action. We must await more data on such large-scale induced-®t interactions to see whether thisadditional level of protein folding can help in ourfundamental understanding of the mechanisms ofmolecular recognition.

Acknowledgements

We are very grateful to Matthias Wilmanns for shar-ing results with us before publication, and the refereesfor all their extremely helpful and constructivecomments on the manuscript. We also thank MartynSymmons, Larissa Lee, Elliott Stollar and all members ofthe Luisi/Blundell groups (past and present) for helpfuldiscussions. K.P. and B.L are supported by the WellcomeTrust.

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Edited by P. Wright

(Received 26 May 2000; received in revised form 11 August 2000; accepted 14 August 2000)

the virtuoso of versatility: pou proteins that flex to fit

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