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9574–9591 Nucleic Acids Research, 2011, Vol. 39, No. 22 Published online 2 September 2011 doi:10.1093/nar/gkr672 Role of sequence encoded jB DNA geometry in gene regulation by Dorsal 1,2, 1 1, Nirotpal Mrinal *, Archana Tomar and Javaregowda Nagaraju * Laboratory of Molecular Genetics, Centre for DNA Fingerprinting and Diagnostics, Nampally, Hyderabad 500001 and Molecular Biology & Genetics Laboratory, Faculty of Life Sciences and Biotechnology, South Asian University, JNU Campus, New Delhi 110067, India Received October 26, 2010; Revised July 28, 2011; Accepted July 29, 2011 ABSTRACT to regulate gene expression. They are capable of homing on the correct binding site out of a vast number of poten- Many proteins of the Rel family can act as both tran- tial sites scattered in the genome, by virtue of having a scriptional activators and repressors. However, surface that is chemically complementary to that of the mechanism that discerns the ‘activator/repressor’ DNA motif. The current model of DNA motif recognition functions of Rel-proteins such as Dorsal by a TF is based on the sequence dependent read out of (Drosophila homologue of mammalian NFiB) is not H-bond donors and acceptors in the major groove (1). understood. Using genomic, biophysical and bio- Although efforts have been made to understand the char- acteristic chemical signature of nucleotides in the major chemical approaches, we demonstrate that the groove, there is as yet no protein–DNA code for base underlying principle of this functional specificity recognition (2,3). lies in the ‘sequence-encoded structure’ of the jB- There are different ways in which a protein surface can DNA. We show that Dorsal-binding motifs exist in take a structure that is ‘chemically’ complementary to distinct activator and repressor conformations. DNA of a particular sequence e.g. through different types Molecular dynamics of DNA-Dorsal complexes of DNA-contacting protein folds, flexibility of protein revealed that repressor jB-motifs typically have side chains, TF-induced structural changes in DNA, etc. A-tract and flexible conformation that facilitates Despite the lack of a well-defined set of rules governing interaction with co-repressors. Deformable struc- sequence recognition, some principles and common themes ture of repressor motifs, is due to changes in the in DNA–protein interactions have emerged (4,5). hydrogen bonding in A:T pair in the ‘A-tract’ core. The current paradigm is that sequence specificity in The sixth nucleotide in the nonameric jB-motif, ‘A’ DNA–protein interaction comes from precise near collin- ear apposition of donor and acceptor groups leading to (A ) in the repressor motifs and ‘T’ (T ) in the activa- 6 6 formation of hydrogen bonds between the protein and the tor motifs, is critical to confer this functional speci- DNA (3). ficity as A ! T mutation transformed flexible 6 6 The grooves of DNA are rich in hydrogen bond- repressor conformation into a rigid activator con- forming functional groups because of which substantial formation. These results highlight that ‘sequence number of intermolecular hydrogen bonds are observed encoded jB DNA-geometry’ regulates gene expres- in protein–DNA complexes (6,7). Traditionally, contacts sion by exerting allosteric effect on binding of Rel between DNA and protein have been explained in terms of proteins which in turn regulates interaction with direct hydrogen bonds, water-mediated hydrogen bonds, co-regulators. Further, we identified and char- van der Waal interactions, electrostatic and, hydrophobic acterized putative repressor motifs in Dl-target contacts (8–10). The role of water-mediated hydrogen bond genes, which can potentially aid in functional anno- was revealed in the crystal structure of the trp repressor– tation of Dorsal gene regulatory network. operator-specific complex in which several water-mediated contacts between protein and DNA were observed but no hydrogen bond to the bases (11,12). Furthermore, INTRODUCTION structure of phage 434 repressor–operator complexes re- Transcription factors (TFs) are DNA-binding proteins vealed role of non-contacted bases in binding, implying that bind to cognate DNA motifs with sequence specificity a role for base sequence-induced DNA structure (13). *To whom correspondence should be addressed. Tel: +91 11 2674 1471; Fax: +91 11 2674 1741; Email: nmrinal@sau.ac.in Correspondence may also be addressed to Javaregowda Nagaraju. Tel: +91 40 2474 9342; Fax: + 91 40 24749448; Email: jnagaraju@cdfd.org.in The Author(s) 2011. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Nucleic Acids Research, 2011, Vol. 39, No. 22 9575 Later, crystal structures of DNA with catabolite gene ac- patterning in Drosophila (36,37). It is not known that tivator protein, IHF and TATA-binding protein (14–19) how Dl recognizes ‘the addresses’ of the target genes as illustrated the role of sequence-dependent structural adap- activators or repressors. In other words, how does Dl tation in DNA. These studies laid the foundations of a decide which genes to activate and which to repress? structural code for DNA recognition (20). Dl, like other proteins of the Rel family, binds to a The role of DNA structure and its sequence-dependent ‘loosely’ conserved DNA sequence (GGGRRYYCCC) structural adaptation to facilitate protein binding has only called as B-motif (37). Predicting a functional B-motif recently begun to be understood (21,22). Apart from is particularly difficult because of the sequence heterogen- sequence specificity, other factors that determine the inter- eity. Furthermore, it has not been ascertained whether the action between a TF and its cognate binding motif are B-motif sequence plays any role in activation or repres- poorly understood, e.g. interactions with phosphate back- sion of target gene transcription by Dl. It has been shown bones in DNA–protein complexes are also common but that a single base change in the B-motif can determine they are relatively less studied (5). It is now accepted that the co-factor specificity of NFkB dimers thus highlighting TFs recognize a sequence-based DNA shape which is ap- the role of B sequence in Rel-mediated gene regulation propriately called as shape readout. Shape readout is an (38). Recently, we demonstrated ‘sequence specificity’ in important concept as it can explain specificity in DNA– Dl interaction with its co-regulator AP1, as we found that protein interactions at higher resolution (20–22). It the Dl–AP1 complex was recruited on an atypical AGAAA includes both global (e.g. DNA bending) as well as local AACA motif but not on canonical GGGAATTCC motif in shape (e.g. major and minor grooves) recognitions. These the same promoter (39). This raised the question as to how local and global structural readouts are sequence depend- Dl-binding motif sequence decides co-regulator specificity. ent, e.g. A-T rich sequence takes typical B-DNA form In the study reported here, we investigated ‘nucleotide while GC-rich motif assumes A-form (23–26). The struc- signatures’ in Dl-binding motifs with respect to their acti- ture assumed by A-tract DNA (stretch of >4 A residues) is vator/repressor functions. We show that the ability of Dl bent with a narrow minor groove, and is sometimes called to activate or repress target genes depends on the geometry B -DNA (27–29). of B–DNA which in turn depends on its sequence. The The critical differences in structural changes in different genome-wide analysis of different B-motifs in Drosophila DNA forms is best revealed in the electrostatic potential revealed distinct bias in the sequence of B-motifs present differences as it has been shown that minor grooves locally in genes repressed by Dl as compared to those activated by enhance negative electrostatic potential (30,31). Hence any it. We have also investigated the effect of sequence change change in minor groove shape is likely to change the elec- on DNA structure by molecular dynamics studies, and trostatic potential which in turn will affect DNA–protein show that different activator motifs, in spite of sequence interactions. In such a scenario, can the logic of transcrip- differences, have comparable major groove geometries. tional regulation be reliably understood merely by the Our findings indicate that the structure of the B-DNA matching of consensus TF sites from the genomic se- backbone is an important factor that determines not only quences? For example, a study of SRY and SOX family the ability of Dl to bind the B-motif but also its ability to proteins in mouse revealed that each of these TFs has the interact with cofactors. ability to identify two distinct binding sites: one primary and the other secondary (32). Thus, a major limitation in understanding transcriptional regulation is the rarity of MATERIALS AND METHODS determined specificities for a majority of the encoded Homology modelling TFs. Recent studies highlighting the role of minor The protein–DNA system was modelled using default par- groove shapes in imparting specificity of TFs binding to ameters of Modeller 9v2 (40). Since the crystal structure of their target genes indicate the existence of more intricate Rel proteins of Drosophila or other insects is not known, programming of gene regulation than previously thought we used chicken c-Rel bound to B-motif of CD28 (31–33). promoter (PDB code-1GJI) as template for modelling Binding of TFs to cis-elements leads to activation/re- pression of gene transcription, and this has led to classifi- RHD of Dl bound to DNA (41). The RHD of chicken cation of TFs as repressors or activators e.g. KRAB c-Rel was closest to the RHD of the Dl as they share family of Zinc-finger TFs are known transcriptional re- 48% homology and their DNA-binding residues are com- pressors (34). An example of transcriptional activator is pletely conserved (Supplementary Figures S1 and S2). CF2 (Chorion Factor 2), which possesses a transcriptional Furthermore, the sequence of CD28 responsive element activation domain, but, its role as transcriptional repres- AGAAATTCC, is close to that of the dlB motif (AGA sor has also been documented (35). These studies suggest AAAACA) and hence it was chosen as the appropriate that gene activation/repression by a TF is context depend- DNA template for homology modelling of the dlB ent and thus they highlight the complexity in assigning a motif. TF as an activator or as a repressor. There are certain To generate the structure of the dlB DNA, the sixth TFs, called bifunctional transcriptional factors, which and seventh ‘T’and the nineth ‘C’ in the crystal structure can activate as well as repress gene transcription. Many of AGAAATTCC was replaced with ‘A’. While replacing developmentally important TFs fall under this category, the original base, nitrogen atom bonded to the sugar and e.g. Dorsal (Dl), a morphogen, activates as well as re- the two base carbon atoms bonded to this nitrogen were presses target genes during embryonic dorso-ventral aligned with the corresponding atoms of the new base so 9576 Nucleic Acids Research, 2011, Vol. 39, No. 22 that the sugar base bond and phosphate backbone remain Transfection, luciferase reporter assay unchanged. In other words, the base substitution was Protocols for cell transfection and luciferase assay were as done without affecting the backbone of DNA. described by Mrinal and Nagaraju (39). To model the DNA in the protein–DNA complex, the DNA atoms were defined as ‘HETATM’ and the resi- Electrophoretic Mobility Shift Assay (EMSA) dues as ‘BLK’ (Modeller 9v2) in the template. These residues are restrained more or less as rigid bodies to The nuclear extracts were prepared as mentioned previ- ously (39). A total of 100 ng of double-stranded retain the conformation of the equivalent residues in the oligo was labelled with 3 mlof[g- P] ATP and 1 mlof template. polynucleotide kinase in 1 ml PNK buffer (New England The mutations were introduced by keeping the orienta- BioLabs) for 1 h at 37 C. The labelled DNA was purified tion of the structure with reference to the first base pair by on a G50 column. DNA-binding reactions were done in using the frame_mol utility of X3DNA package (42). 5 mM Tris–HCl, pH 7.9; 12 mM HEPES; 50 mM KCl; Furthermore, using the rebuild facility the co-ordinates 3 mM EDTA; 1 mM DTT; 5 mg/ml BSA; 10% glycerol; of the atoms were regenerated and the nitrogenous base 0.1 mg/ml poly(dI-dC). Nuclear extracts prepared co-ordinates were manually replaced in the structure, from LPS+PGN-treated cells were pre-incubated in this keeping the phosphate backbone constant. DNA structure buffer in the presence/absence of 40-fold excess of un- generated after mutation was aligned with template DNA labelled oligonucleotide at room temperature for 15 min. and no significant distortion in the backbone was found Afterwards, 50–100 pg of the labelled oligonucleotide was (RMSD, 0.0008). added to the reaction mix, which was then incubated for another 15 min at 25 C. The binding reactions were Molecular dynamics analysed by electrophoresis on 6% native polyacrylamide All simulations were performed using the molecular gels. dynamics program NAMD2 (43) and CHARMM22 force field. The resulting structure was then subjected to 2000 steps of initial minimization to remove bad con- RESULTS tacts and reduce the strain in the system. The complex jB-motif has variable sequence but distinct organization was then immersed in the centre of a box of radius 10 A filled with TIP3P water. In order to obtain the proper Rel proteins are multidomain proteins. However, their geometry for each water molecule, all oxygens were held characteristic feature is the presence of b-sheet sandwich fixed and 1000 steps of energy minimization of the bond immunoglobulin fold. Such DNA-binding proteins typic- and angle energies was performed. All atoms were ally use loops for interaction with DNA (21,41). then relaxed, and the entire system was equilibrated at Interaction of different Rel proteins with cognate B- 300 K for 30 ps. Water molecules closer than 1.8 A to the motifs is also mediated through a series of loops. While, protein–DNA system were removed. The resulting system RHD, the DNA-binding domain of Rel proteins is highly was further subjected to 5 ps equilibration during which conserved, B-motifs, which interact with RHD of the Rel the protein–DNA backbone of the system was put into proteins, probably represent the most heterogeneous constraints. DNA-binding motifs. It is currently not known why To achieve electro-neutrality for the system, 30 Na and such heterogeneity may have evolved (Figure 1A). In 15 Cl were added by removing 45 water molecules, spite of sequence variations, the nucleotides in B-motifs ˚ ˚ located >9A apart from each other and 5 A apart from are arranged in a dyad fashion with two half-sites joined the protein or DNA atoms. The system was further eq- by a central hinge (41) (Figure 1A). Here, we explored the uilibrated for 15 ps without any constraints. This system functional significance of sequence variations in B- contained 1,180,85 atoms, and was finally subjected to motifs. molecular dynamics simulation for 10 ns. Constant tem- Terminal nucleotides G in the first half-site and C in the perature was maintained at 300 K using Langevin second half-site are critical for DNA–proteins interactions dynamics with a damping coefficient of 1 ps . Short- and hence are more conserved (Figure 1A and B). While range non-bonded terms were evaluated every step using nucleotides in the first and second half-sites are always pro- a10A cut-off for van der Waals interactions. An tein contacted the nucleotides 4–6, which form the core integrated time-step of 1fs (DCD freq 500) was used. of the B-motifs, usually, do not interact with the Rel The system was simulated with periodic boundary proteins directly (Figure 1B) and hence they would conditions and full electrostatics computed using the probably have experienced less selection pressure Particle Mesh Ewald (PME) method with a compared to the terminal nucleotides which form direct 120 125 108 point grid. The resulting trajectories interactions with the Rel proteins. In the absence of selec- were analysed by VMD (44). The conformational tion pressure, the probability of occurrence of any of the changes of the DNA during dynamics were evaluated 4 nucleotides would have been expected to be equal in the using the X3DNA program (42). Major groove and core region. However, A/T rich core region shows other DNA local structural parameters were also position-specific preference for T at the sixth and analysed using the same X3DNA package. All simulations seventh positions and A at the fourth and fifth positions were run on SunGrid Engine running the Red Hat (Figure 1B–D). An earlier SELEX study performed with Enterprise operating system for AMD architecture. the three Drosophila Rel proteins had also suggested Nucleic Acids Research, 2011, Vol. 39, No. 22 9577 Figure 1. Sequence bias in the composition of the activator and repressor B-motifs. (A) The second half-site of the kB-sequence dyad is more conserved than the first half-site. Hinge nucleotide (underlined) of the dyad is usually A or T. (B) Only the bases at the termini (shown in bold) form protein contacts while the core bases (underlined) usually do not form hydrogen bonds with proteins. (C and D) Repressor motifs have ‘A-tract’ which is lacking in the activator motifs as revealed by Weblogo consensus. The sixth base in the repressor B-motifs is always ‘A’. For the repressor motif consensus prediction, the B-motifs present in Dl repressor target genes dpp and zen were taken into consideration, however only one motif in each gene is functional. Thus all functional repressor motifs have A-tract and the sixth base is always ‘A’. (E) First half-site consensus is same for both activator and repressor motifs but not the second half site. First base of the second half-site (bold) is A in the repressor motifs and T in the activator motifs. position-specific preference for A/T in the core region (45). position is present in all activator motifs whereas all re- Here, we studied the role and significance of position pressor motifs have ‘A’ at the corresponding position specific preference for nucleotide, in a B-motif sequence (Figure 1C–E). on DNA–Rel interactions in general and DNA-Dl inter- action in particular. A single base change can transform an activator jB-motif into a repressor motif Dl can discern activator and repressor jB-motifs Given the distinction of A or T at the sixth position in the Dl, a transcriptional regulator and morphogen, binds activator or the repressor motif respectively, we investi- GGGYYYYCCC consensus motif (46–48). We aligned gated whether this sequence difference could serve to dis- B-motifs of Dl-activated and repressed genes separately. tinguish between them. We selected two enhancer (dlB Only, the established Dl-target genes were chosen for the and phm) and two repressor B-motifs (dlB and zen) for I P analysis. dlB and dlB motifs are newly identified functional comparison by luciferase assay (Figure 2A). Dl-binding motifs present in dl gene (39). dlB is an atyp- phm (GGGATTACC) and zen (GGGAAATCC) regulate ical B-motif (AGAAAAACA) and lacks ‘T’ (Figure 1D). embryonic development and are known Dl-target genes. From this comparison, it is evident that the repres- While dlB is a typical B-motif (GGGAATTCC) and acts sor motifs have preference for A to T as a result of as an enhancer, atypical dlB (AGAAAAACA) acts as a which repressor motifs, but not the activator motifs, repressor (39). For the functional assay, these motifs were appear to have A-tract (Figure 1C and D). The sequence placed at the dlB position in the dorsal promoter con- differences between activator and repressor motifs are struct with a luciferase reporter (39). more obvious in their second half-site consensus, which For the functional analysis, we introduced an A ! T 6 6 is ‘ATCC’ for repressor and ‘TTCC’ for the activator mutation in the respective repressor motifs, and T ! A 6 6 motifs (Figure 1C–E and Supplementary Figure S3). mutation in the two activator motifs. We found that acti- Interestingly, the hinge base (fifth base) in the repressor vator function of phm and dlB motifs was switched to motifs is always ‘A’ whereas no such sequence preference that of repressor by T ! A mutation in the respective 6 6 is observed for the activator motifs (Figure 1C and D). Dl-binding motifs of phm and dlB (Figure 2B). However, the most noticeable distinction was observed at Reciprocally, the repressor zen and dlB motifs became the sixth base position. It is evident that ‘T’ at sixth inducible upon A ! T mutation (Figure 2C). 6 6 9578 Nucleic Acids Research, 2011, Vol. 39, No. 22 We obtained complete transformation of enhancer motifs into repressors by mutating sixth ‘T’ to ‘A’ and vice versa by mutating sixth ‘A’ to ‘T’. However, no change in gene expression was observed for any of the four B-motifs upon A ! T or T ! A mutations. In 7 7 7 7 fact, A ! T mutation in the phm B-motif led to further 7 7 enhancement of gene expression suggesting that the GGG ATTTCC motif is a strong activator compared to GGGATTACC motif (Supplementary Figure S4). These observations are consistent with those of Muroi et al. (49) which showed that nucleotide substitutions at the seventh position in a B-motif are tolerated but not those at the sixth position. These results also suggest that position-specific A or T bias at the sixth position in the enhancer or repressor motifs does carry functional implications. In other words distribution of A or T at in the B-motif is not a random feature. A is critical for Dl interaction with co-repressor Reversal of Dl-mediated gene expression (repressor to enhancer and vice versa) by single nucleotide change in the B-motifs was intriguing, and we set out to study how nucleotide A or T at the sixth position in the B- motif can specify activator or repressor function of Dl. Repression of gene expression by Dl requires interaction with co-regulator proteins like Groucho and dCTBP (50). Ventral repression of zen is dependent on interaction of Dl with an uncharacterized protein that binds to the neigh- bouring AT-rich motifs on the zen repressor element (51). These studies have led to the hypothesis that transcrip- tional repression by Dl is co-regulator dependent (51). Recently, we identified AP1 as co-repressor of Dl and showed that Dl-AP1 complex binds to dlB motif (which has A as the sixth nucleotide) but not to dlB motif (which has T as the sixth nucleotide), although both motifs are functional and present in the same dl gene. dlB motif and the AP1-binding cluster in the dl promoter Figure 2. Enhancer or repressor activity of B-motifs is encoded in its I are in close proximity. However, when (i) the dlB motif sequence. (A) Two activator motifs dlB and phm and two repressor was replaced with dlB motif or, (ii) the two motifs were motifs dlB and zen were used to decipher the sequence code of gene activation or repression. The sixth nucleotide is bold and underlined. swapped, there was no interaction between AP1 and Dl (B)T ! A mutation transforms enhancer motif into repressor. 6 6 bound to dlB motif (39). These findings highlighted the (C) Reciprocal mutation, A ! T in the repressor motif confers it 6 6 role of DNA sequence not only in Dl binding but also in enhancer activity. (D) Dorsal interaction with different B-motifs is the interaction of Dl with its co-regulator. sequence-dependent as seen in EMSA. Activator motifs (lanes 3, 8 and 9) form smaller DNA–protein complex while repressor motifs Thus, our data suggested that transcriptional repression (lanes 5, 7 and 10) form larger complexes indicating the presence of by Dl requires (i) A -B-motif, and (ii) a gene specific additional proteins. The zen–repressor complex (as shown by brackets) co-repressor. We hypothesized that possibly A in the re- (lane 5) was supershifted with anti-Dl antibody (lane 6) confirming the pressor B-motif is essential for the assembly of Dl presence of Dl in the complex (indicated by arrow head). A ! T 6 6 co-regulator complex, which in turn represses the target mutant of the zen motif retards smaller complex (lane 4) similar to control enhancer motif twi (lane 3) suggesting that Dl interaction gene (Figure 2). To decipher putative modulation of Dl– with the co-repressor is lost due to A ! T mutation resulting in a 6 6 co-regulator complex binding to a B-motif by its nucleo- smaller complex. Activator motifs phm (lane 8) and dlB (lane 9) tide sequence, we performed EMSA. Our assessment was retard small complexes. However, their T ! A mutant probes retard 6 6 that if sequence of the B-motif-regulated Dl interaction larger complexes (as shown in brackets) (lanes 7 and 10). The probe sequences are given at the top of the lane. The sixth nucleotide is with its co-regulator, then biochemically distinct Dl–DNA shown in bold while corresponding mutant nucleotides are underlined. complexes should be seen in EMSA. Role of A -B in Lanes: 1—cold competition with the specific oligo probe, 2—mutant Dl-mediated repression was tested for Dl-repressed gene oligo, 3—twi oligo, 4—T -mutant of zen,5—zen, 6—supershift with Dl zen. With zen motif as probe, a larger Dl–DNA complex antibody, 7—A -mutant of phm,8—phm,9—dlB probe, 10—A - 6 6 mutant of dlB . was indeed observed compared to that with a T -mutant of the same probe (Figure 2D, lanes 4–6), suggesting that the single nucleotide change (A ! T ) in the zen motif 6 6 Nucleic Acids Research, 2011, Vol. 39, No. 22 9579 results in loss of interaction of Dl with its co-regulator interaction with co-regulatory proteins, we undertook a although it does not affect binding of Dl per se. On the biophysical approach. We generated structures of differ- other hand, phm and dlB , both activator motifs, yielded ent B-motifs, taking CD28 Rel-binding motif (AGAAAT smaller complexes of similar size with Dl (Figure 2D, lanes TCC) as a template, by homology modelling followed by 7–10). Mutation of T ! A in these motifs resulted in molecular simulation (Figure 3A and Supplementary 6 6 larger DNA–protein complexes, indicating the presence Figure S5 and details therein) (41). of additional proteins in them (Figure 2D, lanes 7 and The model of Dl–DNA complex showed binding of the 10). Thus the EMSA results suggest that Dl interaction Dl monomers through the major groove and hence, we with co-regulator is possibly modulated by the sequence of compared geometry of major grooves of the enhancer I P the B-motif. These findings led us to postulate that the motif dlB and the repressor motif dlB (Figure function of Dl as an activator or repressor is probably 3B–E). It is evident that the repressor dlB motif has a encoded in the sequence of its binding motif. significantly reduced major groove at the fourth and the fifth base positions with the maximum reduction for the I P Enhancer dljB and repressor dljB motifs have different latter, the hinge position (Figure 3B and D). On the other structures hand there were no significant structural differences DNA–protein interactions always occur through the between the major grooves of the two enhancer motifs major and/or minor groove of the cognate DNA-binding dlB and CD28-B (Supplementary Figure S5). motifs. To elucidate how single base change in a B-motif Major groove geometry of the CD28-B-motif, as in- completely reverses its transcriptional behaviour and its ferred from the molecular dynamics studies, was not Figure 3. AGAAATTCC and AGAAAAACA motifs have different structures. (A) Structure of dorsal homodimer bound to enhancer motif (AGAAATTCC) generated after homology modelling is shown. (B) Repressor dlB motif, AGAAAAACA shows kink conformation compared to enhancer AGAAATTCC motif of CD28RE (1GJI). (C) RMSD graph shows stable simulation of simulation dlB DNA backbone (AGAAAAACA) during 10 ns (20 000 frames) molecular dynamics. (D) Major groove of dlB motif shows reduced geometry at the fourth and the fifth base. Sharp dip at the fifth nucleotide position is striking. (E) The space filling model of the enhancer AGAAATTCC and repressor AGAAAAACA motifs shows change in the major groove conformation due to mutation of T T to A A as shown here. Sharp turn at the fifth nucleotide position and extremely 6 7 6 7 reduced major groove can be seen. Presence of ‘T’ or ‘A’ at the sixth and seventh positions are indicated. 9580 Nucleic Acids Research, 2011, Vol. 39, No. 22 different from that seen in its crystal structure, indicating motif compared to that of the activator CD28-B-motif that no structural changes were introduced during the simu- (Figure 4A). Next, we checked whether narrowing of the lation process (Supplementary Figure S5). Furthermore, major groove led to interaction of basic amino acids such while incorporating the mutations in the structure of the as arginine and lysine of Dl in the minor groove of the template DNA, the bases alone were replaced without dlB motif. Rel proteins bind the DNA through the altering the phosphate backbone. Taking these points major groove. Whether Dl, which is a Rel homologue, into consideration, therefore, we suggest that the unique binds only through the major groove or also involves P I geometry of the dlB DNA backbone generated follow- minor groove was checked for the activator motif dlB ing the same simulation protocol reflects its structural as well as for the repressor motif dlB . We performed property and that it is not a simulation-generated struc- competition experiments with the major and minor tural artefact (Figure 3). groove binding drugs and resolved the complexes by Recent studies have suggested that many major groove- EMSA. Dl binding was affected only when competition binding TFs also interact with the minor groove, e.g. was performed with the major groove binding molecule homeodomain proteins are major groove binders but but not with the minor groove-binding drug (Figure 4B they also extend their basic amino acids into the minor and C). These results provide biochemical evidence that groove to impart specificity (31). Arginine is the most minor groove is not involved in the binding of Dl–AP1 abundant residue that inserts into minor grooves, and ly- complex to the dlB motif (Figure 4C). These results also sines are also observed in such regions although less corroborate our structural modelling data (Figure 3A–E). favoured (33). Minor groove width is dependent on se- Usually reduction in major groove width is associated with quence composition (27–29). Hence, we compared the compensatory increase in the minor groove. However, in P I P minor groove of the dlB and dlB motifs. The minor case of dlB motif, narrowing of the major groove at the groove of the repressor dlB motif is straight, which is fifth position was not associated with increase in the minor typical of A-tract motifs (29). We did not find any sig- groove width (Figures 3D and 4A). Since, geometry of the nificant decrease in the minor groove width of the dlB minor groove remained unchanged at the fifth base P P Figure 4. Dl binding to the dlB motif does not involve minor groove interactions. (A) Minor groove of dlB motif (Dl) is uniform and straight. Minor groove width of dlB motif is bigger compared to that of cd28-B-motif (1GJI) at most of the nucleotide positions except the fifth nucleotide. (B) The Dl–DNA model showed the two Dl monomers bound to major groove of B-DNA (Figure 3A and Supplementary Figure S1). Whether or not Dl interacts with the major groove of B -motif was examined by performing competition experiments with major and minor grooves binding drugs and resolved by EMSA. Dorsal binding was lost upon competition with major groove binding drug, methyl green, but not upon competition with minor groove binding drug Hoechest. This suggests that there are no Dl interactions in the minor groove of the B-motif. (Lanes, 1—mutant probe, 2—cold competition, 3—B motif, 4—Methyl Green competition, —Hoechest competition, 6—non-specific competition, 7—free probe). (C) Binding of Dl–AP1 complex on the repressor dlB motif also does not involve interactions with the minor groove. (Lanes, 1—homologous cold competition, 2—mutant probe, 3—B motif probe, 4—Hoechest competition, 5—Methyl Green competition, 6—non-specific competition, 7—free probe). Nucleic Acids Research, 2011, Vol. 39, No. 22 9581 position which might have constrained the major groove Interestingly, all these motifs also act as activator motifs at this position, ultimately resulting in sharp narrowing of (Figure 2 and Supplementary Figure S7). However, the the major groove or the formation of the kink in the dlB major groove geometry of A containing B motifs 6– motif (Figure 3D and E). (Figure 5B and C) is distinct from that of the T motifs (Figure 5D–F). We found that these two motifs have constrained major grooves and form repressor type Role of jB major groove geometry in Dl–DNA interaction DNA-Dl complexes (Figure 5B, C and G, lanes 6 Though the DNA-binding RHD of Rel proteins is evolu- and 7) (39). tionarily highly conserved, their interaction with their The most significant change in the major groove geom- cognate B-motifs is very specific, partly because orienta- etry was observed for T ! A mutation (Figure 5C; 6 6 tion of the immunoglobulin folds along the DNA and base Supplementary Figures S6 and S7). Interestingly this is contacts are different for different Rel homo/ the same mutation that leads to reversal of activator hetero-dimers (21, 41). Specificity in Rel–B interaction function to that of repressor, thereby suggesting that A can be accounted for by the DNA contacting loops or T at the sixth position is the most important nucleotide which in contrast to helices and sheets are not rigid struc- in imparting shape to the B–DNA (Figure 2 and tures. The DNA contacting loops of Rel proteins are not Supplementary Figure S6). Residues R15, R17, E21 and constrained because the side chains of the C-terminal K174 of monomer I of Dl interact with G A of the first 2, 3 domain of the Rel monomers, which regulate dimer half-site whereas R15, R17, E21 and K174 of monomer II formation, lie along one face of the immunoglobulin form H-bonds with A ,C and A of the second half-site 7 8 9 sandwich (21). As a result, these loops are free to of the B-DNA (Supplementary Figure S8). The take different conformations. Furthermore, the role of DNA-binding residues of monomer I are in similar orien- B-DNA geometry cannot be overlooked as Rel proteins tation resulting in their normal interaction with G A in 2, 3 also interact with the DNA backbone. both the activator and repressor motifs (Supplementary Dl, like other Rel proteins, exhibits extensive elec- Figure S8). On the other hand, monomer II interaction trostatic interactions with the phosphate backbone of with the second half-site nucleotides is different between the core nucleotides apart from forming direct hydro- the activator and repressor motifs, especially interaction gen bonds with the nitrogenous bases (Supplementary of K174 (Figure 5B and C; Supplementary Figure S8). Figure S1). Furthermore, DNA backbone conformation Interestingly, K174 exhibits electrostatic interaction with is sequence dependent (21,23–29,31,33). In order to under- the phosphate backbone of upper strand of AGAAAAAC stand the role of ‘sequence-encoded backbone geometry’ A and lower strand of AGAAAATCC indicating that Dl in DNA-Dl interaction, we asked the following questions: interaction with different repressor motifs is probably (i) How do nucleotide changes affect the DNA backbone unique (Figure 5B and C). geometry? (ii) Do activator and repressor motifs have We have shown above that Dl-co-regulator interaction is distinct structures? sequence specific and co-regulator dependent (Figure 2D). Position specific requirement for T in the activator and T ! A mutation in the CD28-binding element, which is 6 6 A in the repressor motifs was intriguing considering that an activator B-motif, reduces the major groove at the this base is not directly contacted by Dl or chicken c-Rel fifth position which probably facilitates Dl interaction proteins (Figures 1 and 3) (41). This suggests that Dl is with the co-repressors as seen in EMSA (Figures 2, 3, able to discriminate the geometry of an A–T base pair 5B, C and G, lanes 6 and 7, respectively). The T -activator from that of a T–A base pair, which is possible since the motifs have similar DNA geometry and also form Dl– carbonyl group of ‘T’ and amino group of ‘A’, if DNA complexes of similar sizes (Figure 5G, lanes 1–5 superimposed in the same plane, are separated by 1.1 A; and Supplementary Figure S6). Taken together, these hence, A–T and T–A are structurally not similar (52). data indicate that Dl interaction with the repressor Consequently, A–T and T–A phosphate backbones motifs is co-regulator dependent. Since different would also not be similar, as evident from the back- co-regulators would interact differently with Dl, binding bone geometry of these two base pairs (Figure 3). of Dl-repressor complexes on DNA would also be differ- This may explain the structural differences in the ent (Figures 2D and 5G). I P major groove of the dlB and dlB motifs, although both have equal number of A–T pairs (Figures 3D, E O –N hydrogen bond of sixth A in repressor jB-motifs 4 6 and 4A). imparts flexible major groove geometry To address the question how A–T or T–A distinctly affect the B-DNA geometry, we sequentially mutated As described above, molecular simulation studies demon- the nucleotides at the fifth, sixth, and seventh positions strated that repressor B-motifs have deformable major of the CD28-B activator motif AGAAA T T CC. The grooves. Next, we examined how the A-tract core imparts 5 6 7 effects of these mutations on the DNA geometry ranged flexibility to the repressor motifs. We compared DNA from mild to severe (Figure 5 and Supplementary local parameters, viz. roll, buckle etc of activator Figure S6). It is also evident that all the motifs that and repressor B-motifs. Buckle and opening showed have T as the sixth nucleotide exhibit similar major much larger differences compared to other parameters groove geometry and comparable DNA-Dl interactions (Supplementary Figure S9). Interestingly, buckling in- (Figure 5D–F) when compared to the crystal structure of creased with increase in number of A residues towards the c-Rel-cd28B, DNA–protein complex (Figure 5A). the 3 -end (least for 3A motif and maximum for 5A 9582 Nucleic Acids Research, 2011, Vol. 39, No. 22 Figure 5. Effect of mutations in the core region of the B-motif, on its geometry. (A–F) Effect of the base substitution on Dl interaction with the B- motif was analysed following 10 ns simulation. (A) Interaction of the chicken c-Rel with the cognate DNA is shown for comparison with other mutants. (B–F) The two repressor motifs (B and C) have reduced major groove (indicated by arrow) in comparison to the activator motifs (D–G). DNA-binding residues of Dl (R15, R17, E21 and K174) show similar interactions in all the activator motifs. However, interaction of the same four amino acid residues of Dl with the two repressor motifs (B and C) is different probably because their major grooves are more constrained. The AGAAAAACA repressor motif shows sharp bend at the fifth position whereas another repressor motif AGAAAATCC shows bending at the seventh position in the second half-site. (D–F) DNA–protein interactions are almost similar in these activator motifs implying that sequence change does not affect overall DNA–protein interactions with activator B-motifs. All structures were generated and visualized using PyMol. (G) EMSA was performed to validate if structural changes seen in DNA affect Dl binding or its interaction with co-regulators. The two repressor motifs (lanes 6 and 7) form bigger size complexes while the activator motifs form complex of almost same size though the affinity of Dl binding varies (lanes 1–5). Lanes 1—AGAAATTCC,2—AGAAATACC (T ! A ), 3—AGAATTTCC (A ! T ), 4—AGATATTCC (A ! T ), 5—AGATTTTCC (A A ! T T ), 7 7 5 5 4 4 4 5 4 5 6—AGAAAAACA (dlB ), 7—AGAAAATCC (T ! A ), 8—cold competition with AGAAATTCC probe. 6 6 motif) (Figure 6A). Although A of both repressor motifs the zen motif (A ). As a result, the major groove geometry 6 6 AGAAAA TCC and AGAAAA ACA displayed similar of dlB motif is more constrained compared to that of 6 6 opening values, it was significantly higher than that for the zen motif (Figures 5B, C and 6C). the activator motif AGAAAT TCC (Figure 6B). Opening Our analysis of different DNA local parameters angle is much larger for A , in addition to A , of the revealed dramatic changes in values of the opening angle 5 6 AGAAA A ACA motif (Figure 6B). Since, DNA contain- (Figure 6B). Because of a high opening angle, the A –T 5 6 5 5 ing 3A–T pairs in a row has displaced N of ‘A’ and O pair of the AGAAAAACA motif is so far stretched that 6 4 of ‘T’ towards the 3 -ends, we measured O –N hydrogen it does not form the O –N hydrogen bond, resulting in 4 6 4 6 bond in different B-motifs (27–29). Interestingly, the two loss of major groove at the fourth and fifth positions repressor motifs dlB (AGAAAA ACA) and zen (GGAA (Figures 3B, D, 6B and C). A–T pairs in an A-tract AA TCC), which have 4A, displayed increased O –N are weak and have the tendency to form hydrogen bond 6 4 6 hydrogen bond for A compared to activator motifs, diagonally (bifurcated hydrogen bond) to stabilize the which have T at the corresponding base position (only DNA, as seen in binding of 434 repressor to its operator motif (13). However, no bifurcated hydrogen bond was underlined bases are shown on the X-axis) (Figure 6C) We found that dlB motif has two abrogated O –N seen in the repressor motifs (Figure 5B and C). 4 6 hydrogen bonds (A and A ) in contrast to only one in Formation of bifurcated hydrogen bond in an A-tract 5 6 Nucleic Acids Research, 2011, Vol. 39, No. 22 9583 Figure 6. Repressor motifs have weak A-T pairing. (A and B) Longer A-tracts of repressor motifs (AAAT, AAAAA) show more buckling (5A > 4A > 3A-tract) and large opening in the second half-sites. Large opening angles of A and A correspond to reduced major groove width 5 6 at these positions (Supplementary Figure S6). Cartoon shows opening angle in nucleotide pair. (C)N –O hydrogen bond was measured for fourth 6 4 to seventh nucleotides. It is longer (>3.5 A) at sixth A in the two repressor motifs indicating weakening of the A –T bond in them. Due to 6 6 extremely large opening angle, A of AGAAAAACA does not form N –O hydrogen bond which results in acute narrowing of the major groove at 5 6 4 A (Figure 3D and Supplementary Figure S6). Also see Table 1 data which highlights stacking properties of A–T pairing in repressor and activator motifs. (D) This diagram shows that N –O hydrogen bond of the A–T pair faces the major groove. 6 4 motif leads to propeller twisting which in turn results in We surmise that presence of T in the activator motifs narrowing of the minor groove (27–29). breaks the ‘A-run’, resulting in normal O –N hydrogen 4 6 bond and stable DNA conformation (Figures 5D–F In our view, absence of bifurcated hydrogen bond in the and 6C). Thus, our data suggest that A-tract in a AGAAAAACA motif (which has A-tract) can explain why B-motif confers a structural deformability without there are no significant differences in the minor groove of involving bifurcated hydrogen bond or minor groove nar- the repressor and the activator motifs (Figure 4A and rowing. We propose that absence of bifurcated hydrogen Supplementary Figure S7). This further suggests that in bond or minor groove narrowing is compensated by inter- absence of any significant changes in the minor groove, action of Dl with the co-repressor protein. the unique structure of the repressor motifs is most probably on account of loss of O –N hydrogen bonds 4 6 Identification of co-regulator dependent expression in the major groove of the second half-site. Activator of Dl target genes motifs, in contrast, have all the hydrogen bonds intact (Figure 6C). This finding lends credence to our hypothesis Next, we attempted to understand if there is any evolu- that owing to the presence of A-tract in B-motifs with tionary pattern in the sequence composition of B-motifs. AAA A or AAA T sequence, structure of such motifs 6 7 In an earlier study, Copley et al. (57) reported evolution- is more deformable compared to that of AAT T type ary dynamics of B-motifs. There are 4028 B-motifs that motifs. are conserved in different Drosophila species. We identified Additionally, intramolecular hydrogen bonds are also 315/4028 motifs as A-tract B-motifs (minimum 4 ‘A’) known to impart specificity. For example, an intramo- (Supplementary Table S1). Chromosomal distribution lecular hydrogen bond (cytosine N to a neighbouring of A-tract B-motifs with respect to 4028 conserved B- phosphate) has been shown to be critical for yeast phenyl- motifs did not suggest any chromosomal bias in the dis- alanine tRNA function (53,54). Also, A–T and T–A base tribution of A-tract B-motifs (Figure 7A). Our analysis reversals are more sensitive to major groove changes revealed that 126 of the 315 A-rich B-motifs were highly (55–56). Since O –N hydrogen bond is subject to conserved, and these were carried forward for further ana- 4 6 precise stereochemical constraints in an A–T or T–A lysis (cut-off 0.8) (Figure 7A). A majority of these A-tract base pair and hence it is the probable read out of the B-motifs (86/126) were repressor type (A-tract motifs structural difference that can discern deformable (repres- where A is the sixth nucleotide) while 36/126 were of sor) and non-deformable (activator) B-motifs. activator-type (A-tract motifs where sixth nucleotide 9584 Nucleic Acids Research, 2011, Vol. 39, No. 22 Figure 7. Annotation of repressor B-motifs in Drosophila genome. (A) Out of 4029 conserved B-motifs, 315 were found to have A-tract (minimum 4A). Their distribution did not suggest any chromosomal bias. (B) EMSA was performed with novel A-tract B-motifs, identified in this study, which retarded complexes of different sizes. (Lane 1—zen2 probe, lane 2—dll probe, lane 3—pnr probe, lane 4—gsb probe, lane 5—mutant probe, lane 6— free probe). Smaller size complexes retarded in lanes 3 and 4 correspond to activator motif dlB (GGGAATTCC). The protocol for the competitive EMSA with two different probes (lanes 4 and 5) is explained in reference 39. (C) Different B-motifs present in gsb, dll, pnr and zen2 are indicated along with their position with respect to TATA element. Motifs which retarded Dl-DNA complexes in EMSA in Figure 7B are putatively functional and are shown in bold and are underlined. (D) Loss/reduction of luciferase expression upon Dl activation consequent to peptidoglycan (PGN) treatment confirms repressor function of the functional B-motifs of gsb, dll, pnr and zen2 genes. was T) (Supplementary Table S2). The remaining four second half-site in their respective B-motifs motifs in this list are probably non-functional as they (Supplementary Table S2). lack the most crucial second position G, which is typically Next, we examined physical interaction of Dl with the present in all B-motifs characterized till now. In fact, B-motifs of four representative genes namely gsb, pnr, dll and zen2. These genes have multiple Dl-binding sites, and these four motifs completely lack G at any position (Supplementary Table S2). Presence of G in the first here we identified the functional B-motifs in them by half-site and C in the second half-site is a special feature EMSA (Figure 7B and C). We found that the functional of functional B-motifs (Figure 1). B-motifs of the four genes retarded bigger DNA-Dl The list of 86 repressor-type B-motifs included those complexes compared to the activator motif GGGAATTC present in known Dl-repressed genes like dpp, zen etc, C, which was used as a control, indicating the pres- which suggests that other genes featuring in this list ence of additional proteins in the Dl–DNA complexes might be repressor targets of Dl as well (Supplementary (Figure 7C). The functional B-motifs of all the four Table S1). Dl-activated and -repressed genes can also be genes have ‘A-tract’ with A as the sixth nucleotide loosely identified by their spatial expression along the (Figure 7C). Furthermore, luciferase assay also proves dorso-ventral axis. In early embryogenesis, the that Dl-mediated repression of these B-motifs indicating Dl-repressed genes are expressed in the dorsal ectoderm role of co-regulator-mediated repression of target genes by while Dl-activated genes are expressed on the ventral side. Dl (Figure 7D). Using a whole-genome approach Stathopoulos et al. (46) According to the currently accepted model of Dl regu- identified novel Dl target genes that are expressed in the lation, the co-regulator-dependent gene regulation by Dl ectoderm. According to our findings, ectodermal genes may lead to repression of the target gene expression. that are repressed by Dl should have A -type B-motifs, Whether gene activation by Dl requires a co-regulator or and indeed we found that all these genes have A ACC as not, needs to be investigated further but such a possibility 6 Nucleic Acids Research, 2011, Vol. 39, No. 22 9585 cannot be ruled out. It will be interesting to see if on the same strand. This is the distinguishing feature of co-regulator dependent gene activation by Dl would also the two motifs. require A-tract in the B-motif for the binding of Dl– co-activator protein complex as is the case with the Role of jB-DNA sequence in determining Dl-co-factor binding of the Dl–co-repressor complexes. specificity A tract of four to six consecutive (dA): (dT) residues DISCUSSION imparts typical narrow and straight minor groove geometry to DNA (28,29). However A-tract B-motifs The mechanisms that control the precisely regulated switch have reduced major groove at certain points which, in from gene repression to gene activation represent a central our opinion, is due to partial structural collapse triggered question in transcriptional regulation. One feature of this by loss of O –N hydrogen bond. The A-tract has an transcriptional reorganization is the cross-talk with co- 4 6 inherent property to bend but a single nucleotide insertion factor. It is understood that activating stimuli induce re- that interrupts the run of A also breaks this bending (60). cruitment of TFs along with their co-activators to target Thus an AAAA/AAAT motif is more deformable than gene promoters while a repressor signal leads to the assem- AATT or AATA motif. Furthermore, co-repressor bly of co-repressors at the gene promoter. In general, binding is favoured by DNA sequences for which con- co-repressors and co-activators most often act in a gene formational constraints have low energy requirements. specific manner. Although it is known that gene activation Accordingly co-repressor binding is more favoured on or suppression by bi-functional TFs is context dependent, AAA motif than it is on ATA, as seen in the case of inter- it is not clear what factors determine that such TFs action between bacteriophage 434 repressor with its interact with a co-repressor at one gene and with a co-activator at another. operator (61). It is for this reason that repressor motifs that have AAAA/AAAT core can accommodate Co-regulator specificity in Dl binding more structural distortions in order to facilitate binding of Dl in complex with different co-regulators. A similar The current paradigm is that interaction of a TF with its mechanism has been proposed for differential binding co-factor is dependent only on protein–protein interaction affinity of 434 operator in complex with different (58). Here we report that Dl interaction with its co- repressor and Cro proteins (62). While the typical regulator is also decided by the sequence of the geometry of the 434 operator is stabilized by a Dl-binding DNA motif. An analysis of activator and re- bifurcated hydrogen bond, such a bond is not seen in pressor Dl motifs suggested that activator or repressor the AGAAAAACA motif. Interestingly the unique function of Dl might be encoded in the sequence of the geometry of AGAAAAACA motif is due to kink at B-DNA motif. Interestingly, Dl–co-regulator interaction the hinge base (A ), and based on our biophysical and was seen with the repressor motifs but not with the acti- biochemical data we propose that this geometry is import- vator motifs, implying that repressor function of Dl ant for binding of Dl–AP1 complex to this motif is probably co-regulator dependent (Figures 3 and 8). (Figures 5G and 6A–D). Previous reports have also suggested that transcriptional Our study provides structural insights into the nucleo- repression by Dl is co-regulator dependent (51,39). Our tide preferences in co-regulator-dependent and -independ- study suggests that nucleotide at the sixth position is an ent transcriptional regulation by Dl. This serves to important determinant of not only Dl binding but also of highlight the unique ways by which DNA can exert speci- its function as a transcriptional activator or repressor ficity not only on DNA–protein interactions but also on (Figures 2 and 3). Importance of sixth T of a B-motif interactions between regulator and co-regulator. There is in binding with Rel proteins has been implicated in at least evidence also that alterations in the overall structure of two previous studies involving 17 mammalian B-motifs DNA-binding domains can influence the DNA sequence using biochemical approaches (49,59). The only exception preferences in numerous ways (63,64). Previous work has to this rule is the B-binding motif of the IL2 receptor, shown that co-operative binding of ATF-2/c-Jun and which has C in place of T at the sixth position. Although IRF3 depend on inherent asymmetry of the ATF-2/ Muroi et al. (49) showed the importance of sequence com- c-Jun-binding site, so that mutation of the latter to a con- position in binding of Rel proteins, the mechanism by sensus AP1 recognition element resulted in loss of inter- which the B sequence imparts specificity has remained action (65). In another study, interaction of Fos-Jun unclear. heterodimer with NFAT on its binding site on the IL-2 Our study reveals that T motifs characterize co- enhancer was shown to be dependent on the co-operativity regulator-independent transcriptional regulation by Dl, ‘at some level of assembly’ between the three proteins and whereas ‘A’ at the sixth position in a B-motif signifies a the ‘DNA backbone’ (66). It is believed that assembly of co-regulator-dependent regulation by Dl. This specificity proteins on DNA is a result of co-operativity between is on account of Dl–co-repressor complex binding on an DNA and the binding proteins and that co-operative ‘A-tract’B-motif (A-tract is due to presence of ‘A’ at the binding can arise through nucleotide sequence-guided sixth position, as a result there are four continuous ‘As’ in the repressor B-motifs) (Figure 2D). On the other hand, structural changes in the DNA which allow formation of activator motifs (T -B-motif) have ‘T’ at the sixth complementary DNA conformations for the adjacently position which breaks the run of continuous ‘A’ residues bound TFs (67–69). 9586 Nucleic Acids Research, 2011, Vol. 39, No. 22 Figure 8. Putative model of transcriptional activation and repression by Dl. (A) Different activator B-motifs (sixth T is common) retard similar size Dl-complexes suggesting that gene activation by Dl is probably independent of co-regulators. (B) Gene repression by Dl is context-based and co-repressor dependent as different repressor motifs (sixth A is common) retard Dl-complexes of varying sizes indicating presence of proteins of different sizes (also see Figures 2 and 5). A-tract core in the repressor motifs has intrinsic property to bend. This intrinsic deformability in repressor motifs is sequence specific which allows specific recruitment of Dl–co-repressors complexes in gene specific manner as shown in the schematic. Dl interaction with the co-regulator could be either cis (B1, upper panel) or trans (B2, lower panel). (C and D) For successful binding, the DNA–protein interface should match molecular surfaces which includes protein contacts to not only base pairs in the major groove but also to sugar–phosphate backbone. We have shown that activator and repressor motifs have different major groove conformations. As a result, Dl interaction with the two motifs is different (e.g. interaction of K173 and K174 of Dl with the two activator motifs is very similar). On the other hand, binding of the same two residues with the two repressor motifs is very different. Hence, we opine that ‘A-tract B’ motifs have deformable structure to facilitate context-based interaction of the same Dl with different co-repressors in gene-specific manner as revealed by differences in their DNA geometry. indicate core nucleotides) (Figure 1A). Surprisingly, Mammalian jB-motifs have comparable DNA shape DNA geometry of these terminal nucleotides is not so Our analysis of major groove geometries of B-motifs for conserved (Supplementary Figure S10). On the contrary which crystal structures are available indicated a rather major groove structures of the core nucleotides (positions similar DNA geometry. All these B-motifs have their 4–6), where sequence conservation is the least, is relatively major groove maxima at second and eighth positions high (Figure 1 and Supplementary Figure S10). Maximum and minima at the fifth position that gives the stretched conservation of major groove width was observed for the eagle geometry (Supplementary Figure S10). However, hinge base (Supplementary Figure S10). This suggests that none of these motifs can be classified as A-tract containing these B-motifs, in spite of sequence differences, have and thus a crystal structure of Rel monomers bound common structural paradigm (Supplementary Figure to an ‘A-tract’ kB-motif is still not known. In a typical S10). Conserved geometry of major grooves at positions B-motif, nucleotides at the termini are involved in 4–6 was unexpected (Supplementary Figure S6). DNA–protein interactions and are more conserved than Nitrogenous bases at these positions do not form core nucleotides GGRNWTTCC (underlined bases hydrogen bonds with Rel proteins (direct contact) Nucleic Acids Research, 2011, Vol. 39, No. 22 9587 although they are involved in electrostatic interactions Transcriptional property of Dl is encoded in its binding with different Rel monomers and hence conserved motif sequence geometry phosphate backbone at these positions was Dl, like its mammalian homologues p65, c-Rel and RelB, intriguing. In our view, conserved geometry of core nu- possesses transcriptional activation domain and hence is cleotides is essential as it provides unique framework to classified as transcriptional activator (36,37,75). However, B-DNA. We have shown that mutations that affect DNA Dl has been shown to activate as well as repress transcrip- geometry in this region are associated with strong pheno- tion of different genes along the dorso-ventral axis. There types (Figure 2 and Supplementary Figure S6). If electro- are at least two models to explain the ability of Dl to static interactions involving phosphate backbone of activate or repress target genes. According to one model, protein non-contacted bases were not important then Dl by default is an activator and works as a repressor only T ! A mutation should not have affected repressor/ 6 6 in specific promoter contexts (51,76,77). However, another activator function of Dl or its interaction with model suggests that Dl recognizes two classes of sites co-repressor (Figures 2–5 and Supplementary Figure S6). which have different allosteric effects on the protein and These results suggest that phosphate backbone geometry that can result in either transcriptional activation or re- is sequence dependent and guides Rel–DNA binding. pression, e.g. Dl-binding sites in twi (Dl as activator) is less Thus, we have revealed distinct roles played by protein symmetrical compared to that in zen (Dl as repressor) (78). contacted and protein non-contacted nucleotides in a Even so, it has not been established whether specific B-motif. sequence variations in the Dl-binding motifs lead to dif- ferential regulation of Dl target genes. The present study reveals the sequence-specific allosteric jB-DNA geometry explains permissiveness in Rel binding changes in B-motifs which regulate binding of Dl to DNA-binding RHD is highly conserved, yet different Rel precise B-motifs with complementary structure and proteins bind B-motifs of their respective target genes thus confer specificity not only to Dl–DNA interaction without overlap (70). This raises a question that how dif- but also to Dl–co-regulator interaction. This might have ferent Rel proteins identify their binding motifs specifical- led to the evolution of distinct sequence heterogeneity in ly. We suggest that specificity in Rel–DNA interaction is Dl-binding motifs viz. ‘A-rich’ B-motifs in genes re- regulated by protein non-contacted ‘core’ as well as pressed by Dl and ‘A or T-rich’ B-motifs in genes protein contacted ‘terminal’ nucleotides in respective Rel activated by it, i.e. Dl-binding motifs exist in two forms binding motifs. A similar example of DNA–protein inter- (i) activator, and (ii) repressor conformation (Figure 8). action is seen in the HPV E2 system in which the ‘A-tract’ This is also true for another Rel protein, human p65 which core, which is also not contacted by the protein, ensures is a known transcriptional activator but has been shown to specificity of interaction (71). The importance of the struc- bind to human P-sequence ‘GAAAATTTCC’ of IL-4 gene ture of protein non-contacted nucleotides in DNA– leading to transcriptional repression of IL-4 (A-tract is protein interaction is also highlighted by the overwound underlined). Interestingly, the P-sequence motif has an configuration of central nucleotides of the phage 434 A-tract as well. This suggests that A-tract in a B-motif operator (13). is required for transcriptional repression by activator Although core nucleotides of B-DNA are not con- human Rel protein p65 as well. Whether co-regulator is tacted by Rel proteins, they play a critical role by impart- involved in transcriptional repression of IL-4, by p65 ing the correct conformation to B-DNA, and hence they homodimer or not, is currently not known and needs display a conserved geometry. However, it is the major further investigation. groove variations at the termini that make each B- Further, we have used this structural information to motif structurally different from others (Supplementary identify and characterize the functional Dl-binding Figure S10). These unique structures of B-motifs are motifs in the Drosophila genome and classify them as ac- compatible with one particular homo/hetero-dimer of tivator or repressor motifs. Conventionally whole genome Rel proteins and not others, which can explain the speci- and tiling arrays studies have been employed to identify Dl ficity with which different Rel proteins identify and bind target genes. A major limitation of these approaches is their cognate B-motifs. e.g. base-specific contacts within that they do not give information whether the target the p50 and p65 homodimers and p50–p65 hetero-dimer gene is activated or repressed by Dl. Our molecular suggest that the two RHDs contained in each dimer relate dynamics approach circumvents this limitation by predict- to each other differently in different structures ing the structure of the B-motif as activator/repressor (Supplementary Figure S10) (72–75). Hence, only Rel conformations. This can potentially aid in ‘functional an- isoforms which have the chemically complementary notation’ of Dorsal gene regulatory network. DNA geometry will bind that specific B-motif and not Our study, importantly, suggests that specificity in Dl– others. We propose that structural variations at the DNA binding may also derive from interactions involving terminal positions may explain how different Rel double helical backbone. Backbone contacts impart speci- proteins selectively interact with target gene promoters. ficity in DNA–protein interactions through the position- Thus, it is the structural compatibility which is more im- ing of protein recognition elements in orientations portant than the sequence of nucleotides in a B-motif that allow them to make other more specific contacts. which in turn can explain the evolution of sequence vari- This is particularly important as sequence dependent ation in a B-motif (sequence permissiveness). deformability of DNA has been observed in 9588 Nucleic Acids Research, 2011, Vol. 39, No. 22 DNA–protein complexes (79,80). Hence, while a typical while dlB motif (T = 38.68 C) is a repressor (Figures B-motif is recognized by Dl alone the A-tract B- 2, 3 and Supplementary Figure S6). This T difference is motifs, due to their unique shapes, are not. As a result significant and can potentially explain the kink in the such motifs are recognized by Dl only when bound to its major groove of the dlB motif (Figures 3D, E, 5B, 6 co-regulator. It is also possible that sequence-dependent and 8). It is interesting to note that although both activa- DNA structure may also contribute to co-operative tor and repressor motifs have equal number of A or T nucleotides still T -motif has higher T compared to A binding of Dl with its co-regulators. This suggests that 6 m 6 motif which points to the role of stacking interactions. The sequence-dependent DNA structure may be critical for binding of individual factors (e.g. co-operative binding same pattern is also seen in the repressor zen motif (GGGA of AP1 and Dl complex on the Dl promoter), but also AAACC, T = 49.29 C) and its T mutant (GGGAATAC m 6 in the assembly of Dl–AP1 multi-protein complex (39). C, T = 50.11 C), which acts as activator. In all these cases the difference in T of the respective activator–re- Further structural studies would be needed to evaluate pressor pair is <1 C (Table 1 and Figure 2A–D). It is not the proposals made here. The major limiting factor at clear whether or not small changes in T will have signifi- this stage is the lack of crystal structures of Drosophila cant impact on DNA–protein interactions, but the present Rel proteins. Another limitation is the fundamental diffi- culty in accommodating small movements of amino acid analysis suggests that motifs with low T will be more amenable to structural perturbations as compared side-chains, which are likely to occur in Dl interaction to motifs with high T . We have shown that with its binding motif. Additionally, role of hydrophobic AGAAAATCC motif, where sixth nucleotide is A, facili- stacking interactions in the recognition process needs to be tates binding of the co-regulator, but interaction of Dl understood. Stacking interactions are somewhat sequence with its co-regulator is lost upon A ! T mutation dependent, and it is not clear at present how the intercal- 6 6 (Figures 2, 5 and Supplementary Figure S6). These ation of planar amino-acid side chains can be used in a analyses suggest that B-motifs with lower T may facili- DNA–protein recognition system. m tate Dl–co-regulator interaction probably because there Significance of stacking interactions can also be are less stacking interactions in such motifs which make envisaged from the melting temperatures of different B- them more amenable to structural perturbation. In other motifs as shown in Table 1. The T was calculated online words A-tract motifs are more deformable which might be using T Predictor software (http://www.scfbio-iitd.res.in/ required for Dl interaction with co-activator/co-repressor chemgenome/Tm_predictor.jsp). This programme expli- proteins (Figure 8). citly accounts for disruption in stacking interactions, In recent years, information on the role of nucleosome breakage of hydrogen bonds apart from other in gene transcription has begun to emerge. It has been physico-chemical parameters to predict T of a given shown that nucleosomes frequently assume specific pos- DNA sequence (81). It is evident that the repressor itions on DNA, which is called as nucleosome positioning motifs have lesser T compared to corresponding activa- (82,83). The signals on DNA for the nucleosome tor motif e.g. dlB motif (T = 49.29 C) is an activator packaging code reside in the structural properties of DNA base pair combinations, indicating the role of con- formation of particular DNA sequences in deciding the Table 1. Comparison of melting temperatures (T ) of activator re- code (84,85), e.g. positioning of nucleosomes was shown pressor combinations of different B-motifs to operate by excluding nucleosomes from ‘A-tract’ DNA. Motif Sequence T m ( C) A-tracts take a context-independent structure, distinct from canonical B-DNA, due to their ability to switch in dlkB GGGAAT TCC (Act) 49.29 P a cooperative manner (86–88). Because of this A-tracts dlkB AGAAAA ACA (Rep) 38.68 AGAAAAA TCC (Rep) 41.94 become a conformationally rigid DNA motifs that con- AGAAAT ACC (Act) 42.76 strain B-DNA regions bordering them (29). Furthermore, zen GGGAAA ACC (Rep) 49.29 short A-tracts stabilize the deformation required for GGGAAT ACC (Act) 50.11 histone facing nucleosomal DNA as a result of which phm GGGATT ACC (Act) 50.11 DNA bending, deformability and other shape readouts GGGAAA ACC (Rep) 49.29 become important in nucleosome positioning (89). (Act—Activator; Rep—Repressor). Currently it is not known whether Dl binding to its It is evident that activator motifs have higher T compared to corres- P cognate motif requires a nucleosome-free region or ponding repressor motifs. T difference is highest for dlB motif and nucleosome-bound DNA. To study the role of nucleo- its corresponding activator mutant and their backbone structures are drastically different (Supplementary Figure S6). This is indicative of the somes in transcriptional regulation by Dl would require correlation between T and structural flexibility of the DNA sequence. the knowledge of nucleosome positions in target genes. This might be an important determinant for interaction of TFs (in Such a study would also uncover the role of sequence combination with or without co-regulators) with cognate composition on nucleosome positioning in relation to Dl DNA-binding motifs. This is also to be noted that this comparison holds true for the repressor– binding, if any. We have recently shown that swapping of activator combination of the same B-motif but not for two different a repressor motif with an activator motif or vice versa motifs. e.g. T of wild-type zen motif can be compared with zen-mutant does not affect the transcriptional property of the respect- and similarly wild-type phm motif can be compared with phm mutant. ive motifs in autoregulation of dl gene by Dl (39). Thus Nucleotide at the sixth position is indicated in bold while, mutated nucleotide at this position is also underlined. one can speculate that Dl binding to its cognate activator Nucleic Acids Research, 2011, Vol. 39, No. 22 9589 6. Jones,S., van Heyningen,P., Berman,H.M. and Thornton,J.M. or repressor motifs in the dl gene, at least, might be inde- (1999) Protein-DNA interactions: a structural analysis. pendent of nucleosome position. J. Mol. Biol., 287, 877–896. Current study suggests that the geometry of B-motif 7. Saenger,W. (1983) Water and nucleic acids. 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Nucleic Acids Research – Oxford University Press
Published: Dec 2, 2011
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