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EXD homeodomain proteins
REVIEWS
Extraspecificity from O v e r the past two years, a fruitful intersection of two fields, cancer biology a n d developmental biology, has helped to explain h o w a special class o f transcription factors e n c o d e d by the g e n e s o f the homeotic complex ( H O X ) achieve their functional specfficities in vivo. These genes, which are structurally a n d functionally conserved throughout the animal kingdom (Fig. 1), are critical for animal development because they control the choice between parallel developmental pathwaysJ. For example, in the fruittly Drosophila melanogasterthe choice between executing a pathway that results in a n antenna or, alternatively, a p a t h w a y that results in a leg is governed b y the HOX gene Antennapedia tangO) 2,3. The execution of these alternative pathways almost certainly- requires tlLat ANTP protein, which contains a homeodomain, binds to DNA a n d controls the transcription of a unique set of "leg-promoting' a n d 'antennalsuppressing" target genes 4-6. ANTP binds with high affinity to the sequence, 5' [C/TI[C/A!ATFA (Refs 7-9). (For reviews on homeodomain structure a n d DNA recognition, see Refs 10-12.) O n average, this sequence Ls present approximately once per kilobase of DNA in most eukaryotic genomes. Thus, it is highly unlikely that all of these binding sites, which w o u l d be present in most genes in multiple copies, are functional ANTP-binding sites. Therefore, there must b e mechanisms for ANTP to select the correct subset of binding sites that mediate its in vit,o functions. For example, chromatin stmcture might limit the accessibility of AN'I'P to some of these binding sites. Alternatively, ANTP probably needs to bind in conjunction with other transcription factors to control transcription. Another alternative might b e that through the cooperative DNA binding of ANTP with cofactors, some binding sites might be selected in preference to others. In addition to having only a 6 b p consensus binding site, ANTP must overcome an additional problem to achieve specificity. This problem is due to the fact that many h o m e o d o m a i n proteins, including other HOX proteins, also bind this consensus sequence ~.l~,qs. O n e example is the HOX protein Uhrabithorax (UBX) which. in vitro, has an indistinguishable sequence preference to that of ANTP (Ref. 8}. Yet, although u b x also differentiates between antenna a n d leg, it makes a leg that is morphologically different from the one made by Antp (Refs 5, 16. 17). In addition. Ubxis required for choosing haltere development (a small balancing organ) instead of wing development, whereas Antp is required for proper wing development s,ts. Thus, DI vie0, there must be a mechanism to distinguish the target genes of UBX from those of ANTP a n d other HOX proteins. Recent studies on HOX cofactors e n c o d e d by the Drosophila extradenticle (exd) a n d the mammalian PBX geues have provided some intriguing answers to this problem. Cooperative DNA b i n d i n g o f HOX a n d PBC proteins The genetic dtaracterization of exd, which was identified in a screen for mutations that altered embryonic patterning in Drosophila t'y, suggested that it was important for the HOXgenes to execute their specific functions and, therefore, that it could encode a HOX cofactor20, Most importantly, exd mutations altered developmental fates without altering the expression patterns of the
extradenticle
the partnershipbetween H0X and PBX/EXD homeodomain proteins R I ~
S. MANN AND SIU-KWONGCHAN
For maay DNA-bi~U~ l r a ~ r i p t i o u f a a o r s it is often diff~ult to reco~ile their higb~ specific i~ vivo funaioms with their less specific i~ vitro DNA.bt~i~g properlie~
Cooperative DNA Mmflmgwith cofaaors often provides part o f the answer to this paradox a~ld r e c e g studies have demosstrated this to be the case f o r the bomeotic
com~e= (nox) fam~y of t r a ~ p i o J faaom Howe~er, the uaiqueproblem posed by these lagbly related and deveiopmestally importa~ transcriptiou factors requires addfliomd twists to the standard solulio~ which are beg~gmg to become apparemt f r o m the charaeterizatioa o f the HOX cofactors encoded by the extrademtide and PBX geue~
teleran: HOX genes z0. Thus, in an e,r d mutant embryo at least some HOX g e n e products are present in the correct place a n d time, yet they execute incorrect pathways. Also consistent with the cofactor hypothesis. exd regulates some o f the same target genes that are regulated by the H O X g e n e s -'x a n d encodes a homeodomain protein-'-'.-'-*. The exd g e n e is highly related to the C. d e g a m gene ceh-20 and to the mammalian PBX genes and, together, this g e n e family is referred to as the PBC family2~ 2,, {Fig. 1). The PBX genes were independently identified because a h u m a n chromosome translocation that fuses the transcriptional activation domain of E2A with the h o m e o d o m a i n of PBX1 has the potential to cause leukemia r-a'~. The connection between the I'BC gene family a n d leukemia underscores their role in controlling developmental decisions. Consistent with t h e m indirect arguments, EXD binds cooperatively to DNA with H O X gene products -~-.¢2. Similarly. PBX proteins also bind to DNA cooperatively with mammalian HOX proteins3.*--3". PBC proteins can also interact with HOX proteins in cultured cells~.3s. The oncogenic fusion, E2A-PBX1. but not PBX1, can activate the transcription of reporter genes containing PBX consensus binding sites, suggesting that PBX1 does not contain a transcriptional activation doutain -'8,39. Curiously, activation by E2A-PBX1 was suppressed b y HOXB8, but not b y HOXC,q (Ref. 34). Although these results were consistent with the cofactor hypothesis several points remained unclear. Many of these studies, in partkadar those that used binding sites derived In vitro, demonstrated that PBC proteins could cooperatively bind with a wide spectrum of HOX proteins-~z--~-¢.Therefore, these studies could not address whether or not the PBC proteins contribute to HOX specificity, as suggested by the genetic characterization
TIG JuLY 1996 VOL. 12 No. 7 Cnpytight ~'1 1'~et¢,EL~viet .~lence Lid. All rights r L ~ - e d PlI: SR)ltxq+952~t)lOO2t~5
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REVIEWS
of exd. In contrast, two investigations that used in vivo binding sites showed that, given the correct DNA sequence, EXD could bind cooperatively and selectively with different HOX proteins~°.3L More recent studies, which are summarized below, support the idea that PBC proteins contribute to the specificity of DNA recognition by HOX proteins. There are two important conclusions from these studies: first, subtle differences in the DNAbinding site determine which HOX protein is preferred in the ternary complex; and second, PBC proteins might alter the conformation of the HOX homecuJomain, thus causing a change in DNA recognition.
Protein requirementsfor the PBC-HOX interaction Before describing the experiments that pertain to specificity, the features of HOX and PBC proteins that are important for heterodimer formation are first discussed.
(a) Or~,oph~ta
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FIGtmEL The components of the PBC-HOXcomplex. Organizationof the HOXgenes (a) in Drosophila and (b) in mouse. (at In Drosophila a single cluster kssplit into two complexes,the BX'-CandAAr/'P-~,(b) in the mouse fllere are four clusters(Hoxa, -b, -c. -d) of linked Hox genes that each contains a subset of paralogous groups 1 to 13 (Ref. 51). labial(lab), prohoseipedia (pb), and Deformed (Did) are most similarto groups 1, 2 and 4 of the mouse, respectively;Sex combs reduced (Scr), Antennapedia (Antp), Ultrabithorax (Ubx), and abdominaI-A (alxl-A) are equally similarto groups 5 to 8; and AbdominaI-B (Abd-B) is most similarto group~ 9 to 13. (c) A typical HOX protein has a homeodomain (HD) near its C-terminusand a hexapeptide (HX) to the N-terminalside of its homeodomain. Althoughthe Abd-B group of genes do not have an obvious hexapeptide, they usually have a conmrved W-containingmotif to tile N-terminalside of their homeodomainss2 that also contributes to an interaction with PBC proteins~l. A typical (',0amino acid homecxiomainhas the followingstructural features: N-terminalarm (N-term); three ~-helices (HI. residues 10-22; H2. residues 28-37: and H3, residues 42-57): a loop .separatingHI and H2; and a turn separating H2 and H3. The arrowhead indicates the position of the three additional amino acids in the PBC homeodomains. (d) Shows three representative members of the PBC-encodinggene familyand the extem of amino acid identity between them. PBX1 is a human gene and ceb-2Ois from the nematode Caenorbabditis elegans2~
PBC requireme~tts The PBC homeodomain is sufficient to bind DNA cooperatively with HOX proteins ~°-~L4°.However. for some binding sites, the addition of approximately 15 amino acids to the C-terminal side of the PBXI homeodomain was important for forming a HOX-PBX complex3.~.~0. These additional residues also increased monomeric PBX binding~0. Therefore, the effect on heterodimer formation could be due, in part, to an increase in the stability of the PBX-DNA complex. Alternatively, a direct interaclion between the C-terminal tail of PBX and some HOX proteins might also exist*0.it The homeodomains of the PBC family are unusual because the loop between helices 1 and 2 contains an extra three amino acids 2s (Fig. 1). Based on mutagenesis studies, this loop appears to be important for monomeric PBX binding and cooperative binding with HOX proteins "l°. Interestingly, for some loop substitutions, ctyoperative binding was reduced more titan monomeric binding, suggesting that the loop might constitute patx of the HOX-imeraction surface. HOX requirements In addition to having similar homeodomains, most HOX proteins have a short conserved stretch of amino acids to the N-tenliinal side of their homeodom,zius, the hexapeptide (Fig. 1). These residues are also called the pentapeptide or 'YPWM' motif. The existence of several names reflects the fact that the length of similarity depends on the comparison: tIOX proteins from the same HOXcluster (e.g. UBX and ANTP) often share only the four amino acids, YPWM (Tyr-Pro-Trp-Met; Phe occasionally substitutes for Tyr). In contrast, HOX proterns from different species (e.g. Drosophila UBX and
mouse HOXB7) often share 10-12 amino acids in this region t2.2.. In other words, the hexapeptide appears to have co-evolved with its associated homeodomain. A role for the hexapeptide in the PBC-HOX interaction was first shown using the yeast two-hybrid assay42. In vitro, the HOX bexapeptide is required for cooperative DNA binding of PBX and IIOX proteins to consensus binding site#3.bs.3(,.-~3. Moreover, a 12 amino acid peptide containing the hexapeptide from HOXA5 can, on its own, stimulate the binding of PBX1 to its consensus binding site~0.~3. Although direct binding of the hexapeptide to PBC proteins has not yet been demonstrated, these studies strongly suggest that such an interaction exists (Fig. 2). Other experiments, however, demonstrate that the PBC-HOX interaction has additional complexity. (1) Using the yeast two-hybrid assay, EXD was shown to interact with fragments of UBX that did not contain a hexapeptide31.42. Although these interactions were weaker than with full-length UBX protein, they nevertheless suggest a hexapeptide-independent interaction between these two proteins. (2) Also using the yeast two-hybrid assay, EXD was unable to interact with hexapeptide-containing forms of UBX in which the UBX homeodomain was deleted or replaced with the ANTP homeodomain42. (3) When fused to the engrailed homeodomain, the HOXB8 hexapeptide was unable to form a complex with PBX proteins~. (4) HOX proteins in which the hexapeptide was deleted or mutated were still stimulated to bind DNA by EKD (Refs 31, 44). (5) A
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REVIEWS important for the h e x a p e p t i d e interaction, T a k e n together, these studies suggest that, in addition to the hexapeptide, other H O X a m i n o a d d s , p e d i a p s within3t.~0 o r C-terminal 3t to the HOX h o m e o d o m a i n , are also necessary for the interaction with PBC proteins. In addition to being a n important c o m p o n e n t o f the PBC-HOX interaction, the h e x a p e p t i d e probably has additional functions, For e x a m p l e , in the Drosophila HOX protein, labial, this motif a p p e a r s to inhibit DNA binding b e c a u s e labial proteins in w h i c h this motif w a s deleted o r mutated b o u n d DNA better than proteins that had thLs motif intact+.. Furthermore, a labial protein with point mutations in the h e x a p e p t i d e w a s m o r e effective at activating the transcription o f a labial-dependent reporter g e n e in fly e m b r y o s fllan wild-type labial (Ref. 44). Thus, at least for labial, END a p p e a r s to s e r v e two functions: it o v e r c o m e s a negative function o f the h e x a p e p tide and, together, the t w o proteins cooperatively bind to DNA witli high specificity. Although a n inhibitory role for the h e x a p e p t i d e has only b e e n d e m o n s t r a t e d for labial, it is possible that this motif is partially inhibitory in other H O X proteins. For example, deletion o f the h e x a p e p t i d e in HOXD4 resulted in an increase in the ability o f this H O X protein to activate the transcription o f a reporter g e n e in cultured cells 4~. An inhibitory m e c h a n i s m might help to limit which binding sites are productively b o u n d b y i IOX proteins m vivo. Alternatively, the h e x a p e p t i d e s of other HOX proteins might h a v e other functions. T h e fact that h e x a p e p t i d e s a p p e a r to h a v e co-evolved with their h o m e o d o m a i n s suggests that these t w o parts of HOX proteins might function together. Thus, while t h e labial h e x a p e p t i d e might h a v e e v o l v e d to inhibit the labial h o m e ( x t o m a i n from binding DNA, the h e x a p e p tides of other IIOX proteins might be optimized to modify their associated h o m e o d o m a i n s in other, perhaps m o r e subtle, ways.
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5" I C/T C/A
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Fmuat 2. [a) A model for the PBC-HOX-DNA complex. Sho~sn are PBC (dark blue) and HOX (pale blue) home(x.lomains docked on to DNA. This picture is based on known homeodomain-DNA ,structure# e except for the dashed line, which indicates a structurally unsnlved portion of the HOX protein. The homeodomains contain three ~t-helices. which are numbered (cylinders). loops and turns (thin black lines), and N-terminal arms (thick colored lines). Tbe t~vo A_~nSl-adenine contacxs are shown (A). In addition, a guanint- (G)+ predicted to Ix: comacted The role of the binding site in specificity by AreS.5of the PBC home(xlomain in the major gr(x)ve, is Most of the studies s h o w i n g cooperative DNA bindindicated. The PBC loop. HOX hexapcptlde (HX). HOX ing o f PBC and HOX proteins used c o n s e n s u s binding C-terminal tail (CI and individual amino acids (* ~are implicated sites~-'.~.-3".~" Initially. these binding sites i n d u d e d a in the PBC-HOX interaction: the HOX N-terminal arm is implicated HOX consensus site (5' [C/T][C/A]ATlrA) d o s e to a PBX in conferring specificity. (b) The ,~'quence of a PBC-HOX "consensus' binding site, "5"[C/TIIC/AIATNNATCA(this sequence consensus site (5" ATCAATCAA) but subsequently the correslxmds to the darker gray strand) showing the overlapping HOX-binding site was s h o w n to be unnecessary~.-~7. PBC and HOX half-sites The 5t-lntlM poMtit)ns (IC TI[C:AI)are Oligonucleotides containing a PBX c o n s e n s u s site largely determined by amino acids 50 and 54 of the HOX b o u n d PBX or EXD a n d s h o w e d little specificity for difhomeodomain, which are Gin and Met in n~,~arlyall HOX prtxeins. ferent HOX proteins w h e n forming a PBC-HOX c o m respecti~'eiv , lie choice ofHOX pn)tein in the heterodimer is plex: h o w e v e r , a h e x a p e p t i d e w a s required33.3~.~,.~3. largely due to the .sequence of two variable tx)sitions t NN) that are contacted by the HOX N-terminal amL In addition, we note O n e interpretation of this a p p a r e n t lack of specificity is that PBC hometxlomairts have a Gly at position 50 (ReL 2.5) and. that PBC proteins do not contribute to H O X specificity therefnre, thLsamim) acid (which contributes to sequence specifici~" but. instead, are general cofactors for all hexapeptidein other homeodomains) might not do so for these proteins. "Arm" containing HOX proteins. Alternatively. the lack of and "3"refer to the N-terminal arms and third a-helices of these specificity might indicate that a strong PBC-binding site homeodomains, and indicate their head-to-tail orientation. is sufficient to recruit most HOX proteins to the DNA via the h e x a p e p t i d e - P B C interaction. According to this view. the lack of HOX specificity, o b s e r v e d in these mutation at the beginning of helix 2 of the PBX1 h o m e r studies w a s d u e to using a binding site that w a s d o m a i n (residue 28) abolished the ability to form c o m selected to bind PBX instead of a PBX-HOX complex. plexes with HOXA5 without interfering with m o n o m e r i e T h e available data a r g u e that PBC proteins d o conDNA binding a°. H o w e v e r , the HOXA5 h e x a p e p t i d e w a s tribute to HOX specificity and that the DNA-binding site still able to stimulate this mutant PBK1 protein to bind plays a critical role. Systematic studies h a v e identified DNA (Ref, 40). Thus. residue 28 of the PBXl h o m e r particular basepairs within the PBC-HOX-binding site d o m a i n is critical for heterodimer formation, but is not that are important for specificity ~t.~0. T h e s e data are best T I G JuLY 1996 VOL. 12 No. 7 260
REVIEWS
understood in the context of a model for the PBC-HOX-DNA complex, which, from a combination of protein-DNA interaction, binding site selection (SELEX) and mutagenesis experiments, was independently proposed by three groupM°.41.~6 (Fig. 2). In this model, the PBC and HOX homeodomains are orientated as a head-to-tail heterodimer; the centers of their binding sites, defined by the interaction between Asn51 (conserved in all homeodomains) and an adenine, are only 4 bp apart. Furthermore, the N-terminal arm of the HOX homeodomain is in the middle of the complex, interacting with basepairs in the minor groove that also h::ve the potential to interact with the third helix of the PBC homeodomain in the major groove. It is these basepairs that contribute to HOX specificity in the PBC-HOX heterodimer~t.46. In one example, when the sequence was ~C'X']~C="AATCA(the HOX and PBC half sites are overlined and underlined, respectively), UBX and labial both formed complexes equally well with EXD; when it was C'~"A'I'~AAATCA complexes of EXD-UBX, but not EXD-labial, were formed; and when it was CCATCCATCA, complexes of EX'D-labial, but not EXD-UBX, were formed (Fig. 2) .6. The analysis of chimeric HOX proteins indicated that much of this specificity corcelates with the HOX N-terminal arm, a result that is consistent with the model d. Moreover, consistent with these in vitro studies, a 20bp oligonucleotide containing this third labial-specific binding site generated a labial- and Hoxbl-dependent expression pattern in Drosophila and mouse embryos, respectively~.44. If this model is correct, it suggesLs a new twist to homeodomain-DNA binding: the sequence specificity of a HOX m o n o m e r can be different from its specificity as a heterodimer with PBC proteins. On its own, UBX has a strong preference for the sequence CCAT'I'A over CCATAA (Ref. 7). As a heterodimer with EXD, CCATAA and CCA'I-I'A are both high affinity UBX-binding sites~l.4c,. The difference in these sequences again lies in basepairs that are predicted to interact with the UBX N-terminal arm (Fig. 2). One interpretation of this change in sequence preference is that the formation of the PBC-HOX complex alters the conformation of the HOX N-terminal arm, the DNA, or both, thus changing how HOX homeodomains contact DNA. Comparisons with the Matalp-Mata2p complex Apart from the PBC family, one of the most similar (65% identical) homeodomains to EXD in the sequence database is the Matalp homeodomain from yeastn-23. The PBC-HOX model described above suggests that the PBC proteins have much more in common with Matalp than just their sequence (Fig. 3). Matalp forms cooperative heterodimers with another yeast homeoprotein, Mat~x2p and the crystal structure of this complex has been solved~7. In the al--~2 heterodimer, the Matalp-binding site contains the sequence ATCA (Refs 47, 48). According to the model for the PBC-HOX heterodimer, the PBC-binding site also includes the sequence ATCA. Moreover, two important DNA contacts made by the Mat.alp homeodomain are conserved in the PBC-DNA model: the Asn51--adenine contact and the Arg55-guanine contact (Fig. 3). Furthermore, Matalp is :timulated to bind DNA by a small hydt~phobic stretch
HSG operator
..........L~I~ ~ tI~55 AGTACATTTTTAAATG
5"TCATG
~ a2Asn51
PBC-HOX site 5 ' -
.........CCAT~N~TCP~ ASh51 N-tonninatarm
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Fmta~ 3. A compari~sonof the M~talp Mat~t,;p,;nd proposed PBC-HOX complexes. The Matalp-Manx2p heterodimer binds m a haploid-specificgene operator (HSG)48and the Asn51-adenine contacts of the two homeodomains are indicated. In addition, Arg55of Matalp contacts a guanine in the majorgroove47.The Matalp- and PBC-bindiogsites both contain the sequence ATCA, and the AsnSl and Arg55contacts arc.pcteraially conserved. The conservation of the Arg55-guanine contact is likelyto be significant because Arg55is found in only about 70 of the known homeodomainszS. Two positions in the PBC-HOX-bindingsite iNN) are variable and are proposed to contribute to HOX specificity Ixcause of interactions with the HOX N-terminalarm. of amino acids C-terminal to tLe Matc~2p homeodomain, and PBX is stimulated to bind DNA by the HOX hexapeptide "j0.'~9. Finally, the residues in the Matalp homeodomain that interact with the ct2 tail are on the surface of helices 1 and 2, and in die loop separating these helices47; residues in the same region of PBX, in particular within the loop, have been implicated in the interaction with the HOX hexapeptide 40. The most apparent difference between these two heterodimers is the spacing: the two binding sites in the Matalp-Mat~t2p heterodimer are separated by 12bp whereas they are separated by only 4 b p in the PBC-HOX model. Furthermore, there appear to be no homeodomain-homeodomain contacts made in the al-cx2 heterodimer, whereas the close proximity of the two homeodomains in the PBC-HOX model, together with the interaction data cited above, suggest that there might be contacts hetween the PBC and HOX homeodomains. An additional difference between Matalp and EXD (less is known about the P/3X genes) is how they might contribute to specificity in ~v0. The expression of Matalp is differentially regulated in different yeast cell types, thus controlling the formation of the Matalp-Mam2p heterodimer. In contrast, EXD protein Js likely to be uniformly distributed during the first half" of Drosophila embryogenesis due to maternal expression 2t.z2. How might a uniformly distributed cofactor contribute to HOX specificity? One way is that the EXD-HOX heterodimer binding site (about 10bp) is larger than a HOX monomer binding site (about 6bp). Thus, additional specificity is achieved simply by specifying a more complex binding site. A second po~ibility is that, despite its uniform distribution, EXD activity might be regulated by post-translational modification~.Su. A third way for EXD to increase specificity, suggested by the model described above, is that EXT)-HOX-binding sites might be different for HOX proteins, that, on their own, bind to similar DNA sequences. For example, although as monomers ANTP and UBX bind to identical DNA sequences, the binding sites fbr EXD-ANrFP and EXD-UBX might be different. According to this view, EXD uncovers cryptic
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DNA-binding specificities that are built into H O X h o m e o d o m a i n s (especially their N-terminal a r m s ) b y altering h o w H O X proteins contact DNA. Conclmiom For a cofactor to contribute to H O X specificity it m u s t d o m o r e t h a n s i m p l y increase H O X - b i n d i n g affinity, it m u s t distinguish b e t w e e n different H O X proteins that, o n their o w n , bind to similar D N A s e q u e n c e s . As die n u m b e r o f partners for M a t a l p is likely to b e small, t h e differences b e t w e e n t h e a l - a 2 a n d P B C - H O X c o m p l e x e s might, in part, b e important for fulfilling this additional r e q u i r e m e n t . In s u m m a r y , w e s u g g e s t the foll o w i n g t h r e e a s p e c t s to the P B C - H O X interaction. First, in the a b s e n c e o f PBC proteins the H O X h e x a p e p t i d e directly o r indirectly interacts with, a n d modifies, t h e D N A - b i n d i n g properties o f its linked h o m e o d o m a i n . Second, all h e x a p e p t i d e s m i g h t s h a r e the ability to interact w i t h PBC proteins, but this interaction is not sufficient for c o o p e r a t i v e DNA binding; additional interactions are required. Third, the formation o f the P B C - H O X - D N A ternary c o m p l e x reveals t h e potential for H O X - D N A contacts that are not m a d e b y H O X m o n o m e r s . A m o n g the q u e s t i o n s to b e a n s w e r e d in t h e future is h o w m u c h specificity c a n result f r o m t h e P B C H O X interaction. Although differences in the P B C - H O X b i n d i n g site are sufficient to distinguish b e t w e e n relatively d i v e r g e n t H O X proteins "H.",, it is less clear if differences in the b i n d i n g site c a n distinguish b e t w e e n all H O X proteins or, alternatively, if additional proteins are required. Finally, a l t h o u g h PBC proteins c a n c h a n g e t h e w a y H O X proteins b i n d D N A in vitro, their ability, to d o s o in vivo is not as well established a n d will o n l y b e r e s o l v e d o n c e additional PBC- a n d H O X - d e p e n d e n t e n h a n c e r s e q u e n c e s h a v e b e e n dissected. Acknowledgements We thank M. Cleary. M. Featherstone. M Kamps. Q. Lu and C. Murre for communicating results t/el'ore publication, without which this review would not have been possible. We also thank A. AggarwaL M. Gottesman. G. Struhl and D. Thanos [or helpful di~'ussions and/or comments on this manusctipL
References I McGinnis, W. and Krumlauf, R. (loft)2) Cell68, 283-302 2 Struhl, G. (1981) Nantre 292, 635-638 3 Schneuwly, S., Klemenz. R. and Gehring, W.J. (1987) ?,,'tUure 325. 816-818 4 Garcia-BeUido, A. 11977) Ant Zool. 17, 613-630 5 Struhl, G. (1982) Pro¢. Nail Acad. SoL USA 79. 7380-7384 6 Botas, J. (1993) Cttrr. Opin. Cell Biol. 5. 1015-1022 7 Ekker. S.C. e t a l (1991) ~IIBOJ. 10. 1179-1186 8 Ekker, S.C. el al. (19941 EtIBOL 13, 3551-3560 9 Affolter, M. et ol. (1990) proc. Z~2ttlAcad. Sci. USA 87. 4O93-4O97 10 Gcl~ring. W.J. et al. ( 19#)4) Cell 78. 211-223 11 Laughon. A. (1991) Biocbemisto,30. 11357-11367 12 Mann. R.S. (1995) BioEssm's 17. 855-863 13 Desplan, C.. Theis, J. and O'Farrell. P.H. (1988) Cell54. 1081-1090 14 Hocy. T. and Levine. M (1988) Nature332. 858-861 / 5 Kalionis, B. and O'Farrell. P.H (1993) Mech. Dev. 43, 57-70 16 Mann. R.S. and Hogness, D.S, (1990) Cell60, 597-610 17 Morata. G. and Garcia-Bellido. A. f 19761 Wilbem Ronx~ Arab. Dev. Biol. 179. 125-143
18 Lewis, E_B. (1978) Nature276, 565-570 19 J0rgens, G. et aL (1984) Wilhem Roux's Arcb. Dev. Biol.
193, 283-295 2 0 Peffer, M. and Wic'schaus. E. (1990) Genes Dev. 4. 1209-1223 21 Rauskolb, C. and Wieschaus, E. (1994) EMBOJ. 13, 3561-3569 2 2 Rauskolb, C., Peifer, M_ and Wieschaus, E. (1993) Cell74, 1-20 •?3 Flegel, W.A. el al. (1993) Mech. Dev. 41,155-161 24 Burglin, T.R. and Ruvkun, G. (1992) Nat. Genet. 1,319-320 2 5 Burglin, T. (1994) in Gttidebooll to the Homeobox Genes (Duboule. D., ed.), pp. 26-71, Oxford University Press 2 6 Monica, K. et al. (1991) Mol. Cell. Biol. 11, 6149-6157 2 7 Cleary. M.L. (1992) CancerSttrv. 15, 89-104 2 8 Kamps, M.P. et aL (1990) Cell60, 547-555 2 9 Nourse, J. et al. (1990) Cell60, 535-545 3 0 POpped, H. et aL (1995) Cell81, 1031-1042 31 Chan, S-K. etal.(1994) Ce1178, 603-615 3,2 van Di]k. M. and Murre. C. (1994) Cell78, 617-624 33 Chang. C-P. et al. (1995) Genes Dev 9. 663-674 34 Lu, Q. et aL (1995) Mol. Cell. BioL 15, 3786-3795 3 5 Phelan, M.L, Rambaldi, 1. and Featherstone. M. (1995) Mot. Cell. Biol. 15, 3989-3997 3 6 Neuteboom, S. et al. (1995) Proc. Natl Acad. $ci. USA 92, 9166-9170 3 7 van Dijk, M.. Peltenburg. L. and Murre. C, (1995) Mech. Dev. 52. 99-108 38 Lu, Q., Wright. D.D. and Kamps, M.P. (1994) Mol. Cell. Biol. 14, 3938-3948 3 9 van Dijk. M.A.. Voorh0eve, P.M. and Murre. C. (19~3) Proc. Natl Acad. Sci. USA 90, 6061-6065 40 Lu, Q. and Kamps, M. (1996) Mol. Cell. Biol. 16, 1632-1640 41 Chang, C-P. et al. (1996) Mol. Cell. BioL 16. 1734-1745 42 Johnson. F.B.. Parker, E. and Krasnow. M. (1995) Proc. Natl Acad. ScL USA 92, 739-743 43 Knbx:pfler, P. and Kamps, M. (1995) Mot. Cell. BIoL 15, 5811-5819 44 Chan. 8-K. et al. (1996) PJffBOJ. 15. 2477-2488 45 Raml,aldi, 1., Kovacs. E. and Featherstone. M. (1994) Nttclelc Acids Res. 22,376-382 46 Chan. S-K. and Mann. R.S. (1996) Proc. NatlAcad. 5ci, USA 93, 5223-5228 47 Li. T. etal. (1995) 5~cience270, 262-269 48 Gourte, C. and Johnson, A.D. (1994) EIIBOJ. 13. 1434-1442 49 Stark. M.R. and Johnson. A.D. (1994) ?,2tlttre371, 429-432 5 0 Sun, B. et aL (1995) F~IIBOJ. 14, 520-53 =, 51 Scott. M.P. (1993) ,Vu¢leic Acids Res. 21. 1687-1688 52 IzpisOa-Belmonte. J.C. el at. (1991) F~tlBO.L 10. 2279-2289
I~S~ MA:CX ( r s m l O Q c o l u m b l a ~ e d u ) ~ D SIC. C ~ v (scSSQcolumbia.edu) ARE IN r i l e DEPAWOfeNr OF BIOCHEMISTRY AND MOLECULAR BIOPHYSIC~ COLUMBL4 U~'rvF~srn,, 6 3 0 W. 1 6 8 Z ~ Sr~L~;, N Y 1 0 0 3 2 . USA.
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T I G JULY 1996 VOL. 12 N o . 7 262
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Extraspecificity from O v e r the past two years, a fruitful intersection of two fields, cancer biology a n d developmental biology, has helped to explain h o w a special class o f transcription factors e n c o d e d by the g e n e s o f the homeotic complex ( H O X ) achieve their functional specfficities in vivo. These genes, which are structurally a n d functionally conserved throughout the animal kingdom (Fig. 1), are critical for animal development because they control the choice between parallel developmental pathwaysJ. For example, in the fruittly Drosophila melanogasterthe choice between executing a pathway that results in a n antenna or, alternatively, a p a t h w a y that results in a leg is governed b y the HOX gene Antennapedia tangO) 2,3. The execution of these alternative pathways almost certainly- requires tlLat ANTP protein, which contains a homeodomain, binds to DNA a n d controls the transcription of a unique set of "leg-promoting' a n d 'antennalsuppressing" target genes 4-6. ANTP binds with high affinity to the sequence, 5' [C/TI[C/A!ATFA (Refs 7-9). (For reviews on homeodomain structure a n d DNA recognition, see Refs 10-12.) O n average, this sequence Ls present approximately once per kilobase of DNA in most eukaryotic genomes. Thus, it is highly unlikely that all of these binding sites, which w o u l d be present in most genes in multiple copies, are functional ANTP-binding sites. Therefore, there must b e mechanisms for ANTP to select the correct subset of binding sites that mediate its in vit,o functions. For example, chromatin stmcture might limit the accessibility of AN'I'P to some of these binding sites. Alternatively, ANTP probably needs to bind in conjunction with other transcription factors to control transcription. Another alternative might b e that through the cooperative DNA binding of ANTP with cofactors, some binding sites might be selected in preference to others. In addition to having only a 6 b p consensus binding site, ANTP must overcome an additional problem to achieve specificity. This problem is due to the fact that many h o m e o d o m a i n proteins, including other HOX proteins, also bind this consensus sequence ~.l~,qs. O n e example is the HOX protein Uhrabithorax (UBX) which. in vitro, has an indistinguishable sequence preference to that of ANTP (Ref. 8}. Yet, although u b x also differentiates between antenna a n d leg, it makes a leg that is morphologically different from the one made by Antp (Refs 5, 16. 17). In addition. Ubxis required for choosing haltere development (a small balancing organ) instead of wing development, whereas Antp is required for proper wing development s,ts. Thus, DI vie0, there must be a mechanism to distinguish the target genes of UBX from those of ANTP a n d other HOX proteins. Recent studies on HOX cofactors e n c o d e d by the Drosophila extradenticle (exd) a n d the mammalian PBX geues have provided some intriguing answers to this problem. Cooperative DNA b i n d i n g o f HOX a n d PBC proteins The genetic dtaracterization of exd, which was identified in a screen for mutations that altered embryonic patterning in Drosophila t'y, suggested that it was important for the HOXgenes to execute their specific functions and, therefore, that it could encode a HOX cofactor20, Most importantly, exd mutations altered developmental fates without altering the expression patterns of the
extradenticle
the partnershipbetween H0X and PBX/EXD homeodomain proteins R I ~
S. MANN AND SIU-KWONGCHAN
For maay DNA-bi~U~ l r a ~ r i p t i o u f a a o r s it is often diff~ult to reco~ile their higb~ specific i~ vivo funaioms with their less specific i~ vitro DNA.bt~i~g properlie~
Cooperative DNA Mmflmgwith cofaaors often provides part o f the answer to this paradox a~ld r e c e g studies have demosstrated this to be the case f o r the bomeotic
com~e= (nox) fam~y of t r a ~ p i o J faaom Howe~er, the uaiqueproblem posed by these lagbly related and deveiopmestally importa~ transcriptiou factors requires addfliomd twists to the standard solulio~ which are beg~gmg to become apparemt f r o m the charaeterizatioa o f the HOX cofactors encoded by the extrademtide and PBX geue~
teleran: HOX genes z0. Thus, in an e,r d mutant embryo at least some HOX g e n e products are present in the correct place a n d time, yet they execute incorrect pathways. Also consistent with the cofactor hypothesis. exd regulates some o f the same target genes that are regulated by the H O X g e n e s -'x a n d encodes a homeodomain protein-'-'.-'-*. The exd g e n e is highly related to the C. d e g a m gene ceh-20 and to the mammalian PBX genes and, together, this g e n e family is referred to as the PBC family2~ 2,, {Fig. 1). The PBX genes were independently identified because a h u m a n chromosome translocation that fuses the transcriptional activation domain of E2A with the h o m e o d o m a i n of PBX1 has the potential to cause leukemia r-a'~. The connection between the I'BC gene family a n d leukemia underscores their role in controlling developmental decisions. Consistent with t h e m indirect arguments, EXD binds cooperatively to DNA with H O X gene products -~-.¢2. Similarly. PBX proteins also bind to DNA cooperatively with mammalian HOX proteins3.*--3". PBC proteins can also interact with HOX proteins in cultured cells~.3s. The oncogenic fusion, E2A-PBX1. but not PBX1, can activate the transcription of reporter genes containing PBX consensus binding sites, suggesting that PBX1 does not contain a transcriptional activation doutain -'8,39. Curiously, activation by E2A-PBX1 was suppressed b y HOXB8, but not b y HOXC,q (Ref. 34). Although these results were consistent with the cofactor hypothesis several points remained unclear. Many of these studies, in partkadar those that used binding sites derived In vitro, demonstrated that PBC proteins could cooperatively bind with a wide spectrum of HOX proteins-~z--~-¢.Therefore, these studies could not address whether or not the PBC proteins contribute to HOX specificity, as suggested by the genetic characterization
TIG JuLY 1996 VOL. 12 No. 7 Cnpytight ~'1 1'~et¢,EL~viet .~lence Lid. All rights r L ~ - e d PlI: SR)ltxq+952~t)lOO2t~5
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REVIEWS
of exd. In contrast, two investigations that used in vivo binding sites showed that, given the correct DNA sequence, EXD could bind cooperatively and selectively with different HOX proteins~°.3L More recent studies, which are summarized below, support the idea that PBC proteins contribute to the specificity of DNA recognition by HOX proteins. There are two important conclusions from these studies: first, subtle differences in the DNAbinding site determine which HOX protein is preferred in the ternary complex; and second, PBC proteins might alter the conformation of the HOX homecuJomain, thus causing a change in DNA recognition.
Protein requirementsfor the PBC-HOX interaction Before describing the experiments that pertain to specificity, the features of HOX and PBC proteins that are important for heterodimer formation are first discussed.
(a) Or~,oph~ta
BX-C
ANTP-C
Abd-B abd-A Ubx
(b) Mouse 13
HOxb HOXC Hoxd
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FIGtmEL The components of the PBC-HOXcomplex. Organizationof the HOXgenes (a) in Drosophila and (b) in mouse. (at In Drosophila a single cluster kssplit into two complexes,the BX'-CandAAr/'P-~,(b) in the mouse fllere are four clusters(Hoxa, -b, -c. -d) of linked Hox genes that each contains a subset of paralogous groups 1 to 13 (Ref. 51). labial(lab), prohoseipedia (pb), and Deformed (Did) are most similarto groups 1, 2 and 4 of the mouse, respectively;Sex combs reduced (Scr), Antennapedia (Antp), Ultrabithorax (Ubx), and abdominaI-A (alxl-A) are equally similarto groups 5 to 8; and AbdominaI-B (Abd-B) is most similarto group~ 9 to 13. (c) A typical HOX protein has a homeodomain (HD) near its C-terminusand a hexapeptide (HX) to the N-terminalside of its homeodomain. Althoughthe Abd-B group of genes do not have an obvious hexapeptide, they usually have a conmrved W-containingmotif to tile N-terminalside of their homeodomainss2 that also contributes to an interaction with PBC proteins~l. A typical (',0amino acid homecxiomainhas the followingstructural features: N-terminalarm (N-term); three ~-helices (HI. residues 10-22; H2. residues 28-37: and H3, residues 42-57): a loop .separatingHI and H2; and a turn separating H2 and H3. The arrowhead indicates the position of the three additional amino acids in the PBC homeodomains. (d) Shows three representative members of the PBC-encodinggene familyand the extem of amino acid identity between them. PBX1 is a human gene and ceb-2Ois from the nematode Caenorbabditis elegans2~
PBC requireme~tts The PBC homeodomain is sufficient to bind DNA cooperatively with HOX proteins ~°-~L4°.However. for some binding sites, the addition of approximately 15 amino acids to the C-terminal side of the PBXI homeodomain was important for forming a HOX-PBX complex3.~.~0. These additional residues also increased monomeric PBX binding~0. Therefore, the effect on heterodimer formation could be due, in part, to an increase in the stability of the PBX-DNA complex. Alternatively, a direct interaclion between the C-terminal tail of PBX and some HOX proteins might also exist*0.it The homeodomains of the PBC family are unusual because the loop between helices 1 and 2 contains an extra three amino acids 2s (Fig. 1). Based on mutagenesis studies, this loop appears to be important for monomeric PBX binding and cooperative binding with HOX proteins "l°. Interestingly, for some loop substitutions, ctyoperative binding was reduced more titan monomeric binding, suggesting that the loop might constitute patx of the HOX-imeraction surface. HOX requirements In addition to having similar homeodomains, most HOX proteins have a short conserved stretch of amino acids to the N-tenliinal side of their homeodom,zius, the hexapeptide (Fig. 1). These residues are also called the pentapeptide or 'YPWM' motif. The existence of several names reflects the fact that the length of similarity depends on the comparison: tIOX proteins from the same HOXcluster (e.g. UBX and ANTP) often share only the four amino acids, YPWM (Tyr-Pro-Trp-Met; Phe occasionally substitutes for Tyr). In contrast, HOX proterns from different species (e.g. Drosophila UBX and
mouse HOXB7) often share 10-12 amino acids in this region t2.2.. In other words, the hexapeptide appears to have co-evolved with its associated homeodomain. A role for the hexapeptide in the PBC-HOX interaction was first shown using the yeast two-hybrid assay42. In vitro, the HOX bexapeptide is required for cooperative DNA binding of PBX and IIOX proteins to consensus binding site#3.bs.3(,.-~3. Moreover, a 12 amino acid peptide containing the hexapeptide from HOXA5 can, on its own, stimulate the binding of PBX1 to its consensus binding site~0.~3. Although direct binding of the hexapeptide to PBC proteins has not yet been demonstrated, these studies strongly suggest that such an interaction exists (Fig. 2). Other experiments, however, demonstrate that the PBC-HOX interaction has additional complexity. (1) Using the yeast two-hybrid assay, EXD was shown to interact with fragments of UBX that did not contain a hexapeptide31.42. Although these interactions were weaker than with full-length UBX protein, they nevertheless suggest a hexapeptide-independent interaction between these two proteins. (2) Also using the yeast two-hybrid assay, EXD was unable to interact with hexapeptide-containing forms of UBX in which the UBX homeodomain was deleted or replaced with the ANTP homeodomain42. (3) When fused to the engrailed homeodomain, the HOXB8 hexapeptide was unable to form a complex with PBX proteins~. (4) HOX proteins in which the hexapeptide was deleted or mutated were still stimulated to bind DNA by EKD (Refs 31, 44). (5) A
TIG JULY 1996 VOL, 12 No. 7
259
REVIEWS important for the h e x a p e p t i d e interaction, T a k e n together, these studies suggest that, in addition to the hexapeptide, other H O X a m i n o a d d s , p e d i a p s within3t.~0 o r C-terminal 3t to the HOX h o m e o d o m a i n , are also necessary for the interaction with PBC proteins. In addition to being a n important c o m p o n e n t o f the PBC-HOX interaction, the h e x a p e p t i d e probably has additional functions, For e x a m p l e , in the Drosophila HOX protein, labial, this motif a p p e a r s to inhibit DNA binding b e c a u s e labial proteins in w h i c h this motif w a s deleted o r mutated b o u n d DNA better than proteins that had thLs motif intact+.. Furthermore, a labial protein with point mutations in the h e x a p e p t i d e w a s m o r e effective at activating the transcription o f a labial-dependent reporter g e n e in fly e m b r y o s fllan wild-type labial (Ref. 44). Thus, at least for labial, END a p p e a r s to s e r v e two functions: it o v e r c o m e s a negative function o f the h e x a p e p tide and, together, the t w o proteins cooperatively bind to DNA witli high specificity. Although a n inhibitory role for the h e x a p e p t i d e has only b e e n d e m o n s t r a t e d for labial, it is possible that this motif is partially inhibitory in other H O X proteins. For example, deletion o f the h e x a p e p t i d e in HOXD4 resulted in an increase in the ability o f this H O X protein to activate the transcription o f a reporter g e n e in cultured cells 4~. An inhibitory m e c h a n i s m might help to limit which binding sites are productively b o u n d b y i IOX proteins m vivo. Alternatively, the h e x a p e p t i d e s of other HOX proteins might h a v e other functions. T h e fact that h e x a p e p t i d e s a p p e a r to h a v e co-evolved with their h o m e o d o m a i n s suggests that these t w o parts of HOX proteins might function together. Thus, while t h e labial h e x a p e p t i d e might h a v e e v o l v e d to inhibit the labial h o m e ( x t o m a i n from binding DNA, the h e x a p e p tides of other IIOX proteins might be optimized to modify their associated h o m e o d o m a i n s in other, perhaps m o r e subtle, ways.
~b)
5" I C/T C/A
3
A
HOX
T
N"I3 Arm NI
PBC
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Q
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5"
Fmuat 2. [a) A model for the PBC-HOX-DNA complex. Sho~sn are PBC (dark blue) and HOX (pale blue) home(x.lomains docked on to DNA. This picture is based on known homeodomain-DNA ,structure# e except for the dashed line, which indicates a structurally unsnlved portion of the HOX protein. The homeodomains contain three ~t-helices. which are numbered (cylinders). loops and turns (thin black lines), and N-terminal arms (thick colored lines). Tbe t~vo A_~nSl-adenine contacxs are shown (A). In addition, a guanint- (G)+ predicted to Ix: comacted The role of the binding site in specificity by AreS.5of the PBC home(xlomain in the major gr(x)ve, is Most of the studies s h o w i n g cooperative DNA bindindicated. The PBC loop. HOX hexapcptlde (HX). HOX ing o f PBC and HOX proteins used c o n s e n s u s binding C-terminal tail (CI and individual amino acids (* ~are implicated sites~-'.~.-3".~" Initially. these binding sites i n d u d e d a in the PBC-HOX interaction: the HOX N-terminal arm is implicated HOX consensus site (5' [C/T][C/A]ATlrA) d o s e to a PBX in conferring specificity. (b) The ,~'quence of a PBC-HOX "consensus' binding site, "5"[C/TIIC/AIATNNATCA(this sequence consensus site (5" ATCAATCAA) but subsequently the correslxmds to the darker gray strand) showing the overlapping HOX-binding site was s h o w n to be unnecessary~.-~7. PBC and HOX half-sites The 5t-lntlM poMtit)ns (IC TI[C:AI)are Oligonucleotides containing a PBX c o n s e n s u s site largely determined by amino acids 50 and 54 of the HOX b o u n d PBX or EXD a n d s h o w e d little specificity for difhomeodomain, which are Gin and Met in n~,~arlyall HOX prtxeins. ferent HOX proteins w h e n forming a PBC-HOX c o m respecti~'eiv , lie choice ofHOX pn)tein in the heterodimer is plex: h o w e v e r , a h e x a p e p t i d e w a s required33.3~.~,.~3. largely due to the .sequence of two variable tx)sitions t NN) that are contacted by the HOX N-terminal amL In addition, we note O n e interpretation of this a p p a r e n t lack of specificity is that PBC hometxlomairts have a Gly at position 50 (ReL 2.5) and. that PBC proteins do not contribute to H O X specificity therefnre, thLsamim) acid (which contributes to sequence specifici~" but. instead, are general cofactors for all hexapeptidein other homeodomains) might not do so for these proteins. "Arm" containing HOX proteins. Alternatively. the lack of and "3"refer to the N-terminal arms and third a-helices of these specificity might indicate that a strong PBC-binding site homeodomains, and indicate their head-to-tail orientation. is sufficient to recruit most HOX proteins to the DNA via the h e x a p e p t i d e - P B C interaction. According to this view. the lack of HOX specificity, o b s e r v e d in these mutation at the beginning of helix 2 of the PBX1 h o m e r studies w a s d u e to using a binding site that w a s d o m a i n (residue 28) abolished the ability to form c o m selected to bind PBX instead of a PBX-HOX complex. plexes with HOXA5 without interfering with m o n o m e r i e T h e available data a r g u e that PBC proteins d o conDNA binding a°. H o w e v e r , the HOXA5 h e x a p e p t i d e w a s tribute to HOX specificity and that the DNA-binding site still able to stimulate this mutant PBK1 protein to bind plays a critical role. Systematic studies h a v e identified DNA (Ref, 40). Thus. residue 28 of the PBXl h o m e r particular basepairs within the PBC-HOX-binding site d o m a i n is critical for heterodimer formation, but is not that are important for specificity ~t.~0. T h e s e data are best T I G JuLY 1996 VOL. 12 No. 7 260
REVIEWS
understood in the context of a model for the PBC-HOX-DNA complex, which, from a combination of protein-DNA interaction, binding site selection (SELEX) and mutagenesis experiments, was independently proposed by three groupM°.41.~6 (Fig. 2). In this model, the PBC and HOX homeodomains are orientated as a head-to-tail heterodimer; the centers of their binding sites, defined by the interaction between Asn51 (conserved in all homeodomains) and an adenine, are only 4 bp apart. Furthermore, the N-terminal arm of the HOX homeodomain is in the middle of the complex, interacting with basepairs in the minor groove that also h::ve the potential to interact with the third helix of the PBC homeodomain in the major groove. It is these basepairs that contribute to HOX specificity in the PBC-HOX heterodimer~t.46. In one example, when the sequence was ~C'X']~C="AATCA(the HOX and PBC half sites are overlined and underlined, respectively), UBX and labial both formed complexes equally well with EXD; when it was C'~"A'I'~AAATCA complexes of EXD-UBX, but not EXD-labial, were formed; and when it was CCATCCATCA, complexes of EX'D-labial, but not EXD-UBX, were formed (Fig. 2) .6. The analysis of chimeric HOX proteins indicated that much of this specificity corcelates with the HOX N-terminal arm, a result that is consistent with the model d. Moreover, consistent with these in vitro studies, a 20bp oligonucleotide containing this third labial-specific binding site generated a labial- and Hoxbl-dependent expression pattern in Drosophila and mouse embryos, respectively~.44. If this model is correct, it suggesLs a new twist to homeodomain-DNA binding: the sequence specificity of a HOX m o n o m e r can be different from its specificity as a heterodimer with PBC proteins. On its own, UBX has a strong preference for the sequence CCAT'I'A over CCATAA (Ref. 7). As a heterodimer with EXD, CCATAA and CCA'I-I'A are both high affinity UBX-binding sites~l.4c,. The difference in these sequences again lies in basepairs that are predicted to interact with the UBX N-terminal arm (Fig. 2). One interpretation of this change in sequence preference is that the formation of the PBC-HOX complex alters the conformation of the HOX N-terminal arm, the DNA, or both, thus changing how HOX homeodomains contact DNA. Comparisons with the Matalp-Mata2p complex Apart from the PBC family, one of the most similar (65% identical) homeodomains to EXD in the sequence database is the Matalp homeodomain from yeastn-23. The PBC-HOX model described above suggests that the PBC proteins have much more in common with Matalp than just their sequence (Fig. 3). Matalp forms cooperative heterodimers with another yeast homeoprotein, Mat~x2p and the crystal structure of this complex has been solved~7. In the al--~2 heterodimer, the Matalp-binding site contains the sequence ATCA (Refs 47, 48). According to the model for the PBC-HOX heterodimer, the PBC-binding site also includes the sequence ATCA. Moreover, two important DNA contacts made by the Mat.alp homeodomain are conserved in the PBC-DNA model: the Asn51--adenine contact and the Arg55-guanine contact (Fig. 3). Furthermore, Matalp is :timulated to bind DNA by a small hydt~phobic stretch
HSG operator
..........L~I~ ~ tI~55 AGTACATTTTTAAATG
5"TCATG
~ a2Asn51
PBC-HOX site 5 ' -
.........CCAT~N~TCP~ ASh51 N-tonninatarm
PBC ASh51
Fmta~ 3. A compari~sonof the M~talp Mat~t,;p,;nd proposed PBC-HOX complexes. The Matalp-Manx2p heterodimer binds m a haploid-specificgene operator (HSG)48and the Asn51-adenine contacts of the two homeodomains are indicated. In addition, Arg55of Matalp contacts a guanine in the majorgroove47.The Matalp- and PBC-bindiogsites both contain the sequence ATCA, and the AsnSl and Arg55contacts arc.pcteraially conserved. The conservation of the Arg55-guanine contact is likelyto be significant because Arg55is found in only about 70 of the known homeodomainszS. Two positions in the PBC-HOX-bindingsite iNN) are variable and are proposed to contribute to HOX specificity Ixcause of interactions with the HOX N-terminalarm. of amino acids C-terminal to tLe Matc~2p homeodomain, and PBX is stimulated to bind DNA by the HOX hexapeptide "j0.'~9. Finally, the residues in the Matalp homeodomain that interact with the ct2 tail are on the surface of helices 1 and 2, and in die loop separating these helices47; residues in the same region of PBX, in particular within the loop, have been implicated in the interaction with the HOX hexapeptide 40. The most apparent difference between these two heterodimers is the spacing: the two binding sites in the Matalp-Mat~t2p heterodimer are separated by 12bp whereas they are separated by only 4 b p in the PBC-HOX model. Furthermore, there appear to be no homeodomain-homeodomain contacts made in the al-cx2 heterodimer, whereas the close proximity of the two homeodomains in the PBC-HOX model, together with the interaction data cited above, suggest that there might be contacts hetween the PBC and HOX homeodomains. An additional difference between Matalp and EXD (less is known about the P/3X genes) is how they might contribute to specificity in ~v0. The expression of Matalp is differentially regulated in different yeast cell types, thus controlling the formation of the Matalp-Mam2p heterodimer. In contrast, EXD protein Js likely to be uniformly distributed during the first half" of Drosophila embryogenesis due to maternal expression 2t.z2. How might a uniformly distributed cofactor contribute to HOX specificity? One way is that the EXD-HOX heterodimer binding site (about 10bp) is larger than a HOX monomer binding site (about 6bp). Thus, additional specificity is achieved simply by specifying a more complex binding site. A second po~ibility is that, despite its uniform distribution, EXD activity might be regulated by post-translational modification~.Su. A third way for EXD to increase specificity, suggested by the model described above, is that EXT)-HOX-binding sites might be different for HOX proteins, that, on their own, bind to similar DNA sequences. For example, although as monomers ANTP and UBX bind to identical DNA sequences, the binding sites fbr EXD-ANrFP and EXD-UBX might be different. According to this view, EXD uncovers cryptic
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DNA-binding specificities that are built into H O X h o m e o d o m a i n s (especially their N-terminal a r m s ) b y altering h o w H O X proteins contact DNA. Conclmiom For a cofactor to contribute to H O X specificity it m u s t d o m o r e t h a n s i m p l y increase H O X - b i n d i n g affinity, it m u s t distinguish b e t w e e n different H O X proteins that, o n their o w n , bind to similar D N A s e q u e n c e s . As die n u m b e r o f partners for M a t a l p is likely to b e small, t h e differences b e t w e e n t h e a l - a 2 a n d P B C - H O X c o m p l e x e s might, in part, b e important for fulfilling this additional r e q u i r e m e n t . In s u m m a r y , w e s u g g e s t the foll o w i n g t h r e e a s p e c t s to the P B C - H O X interaction. First, in the a b s e n c e o f PBC proteins the H O X h e x a p e p t i d e directly o r indirectly interacts with, a n d modifies, t h e D N A - b i n d i n g properties o f its linked h o m e o d o m a i n . Second, all h e x a p e p t i d e s m i g h t s h a r e the ability to interact w i t h PBC proteins, but this interaction is not sufficient for c o o p e r a t i v e DNA binding; additional interactions are required. Third, the formation o f the P B C - H O X - D N A ternary c o m p l e x reveals t h e potential for H O X - D N A contacts that are not m a d e b y H O X m o n o m e r s . A m o n g the q u e s t i o n s to b e a n s w e r e d in t h e future is h o w m u c h specificity c a n result f r o m t h e P B C H O X interaction. Although differences in the P B C - H O X b i n d i n g site are sufficient to distinguish b e t w e e n relatively d i v e r g e n t H O X proteins "H.",, it is less clear if differences in the b i n d i n g site c a n distinguish b e t w e e n all H O X proteins or, alternatively, if additional proteins are required. Finally, a l t h o u g h PBC proteins c a n c h a n g e t h e w a y H O X proteins b i n d D N A in vitro, their ability, to d o s o in vivo is not as well established a n d will o n l y b e r e s o l v e d o n c e additional PBC- a n d H O X - d e p e n d e n t e n h a n c e r s e q u e n c e s h a v e b e e n dissected. Acknowledgements We thank M. Cleary. M. Featherstone. M Kamps. Q. Lu and C. Murre for communicating results t/el'ore publication, without which this review would not have been possible. We also thank A. AggarwaL M. Gottesman. G. Struhl and D. Thanos [or helpful di~'ussions and/or comments on this manusctipL
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