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Originally published In Press as doi:10.1074/jbc.M603371200 on July 6, 2006

J. Biol. Chem., Vol. 281, Issue 35, 25781-25790, September 1, 2006
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Decapentaplegic-responsive Silencers Contain Overlapping Mad-binding Sites*

Sheng Gao and Allen Laughon1

From the Laboratory of Genetics, University of Wisconsin, Madison, Wisconsin 53706

Received for publication, April 10, 2006 , and in revised form, June 21, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Smad proteins regulate transcription in response to transforming growth factor-beta signaling pathways by binding to two distinct types of DNA sites. The sequence GTCT is recognized by all receptor-activated Smads and by Smad4. The subset of Smads that responds to bone morphogenetic protein signaling recognizes a distinct class of GC-rich sites in addition to GTCT. Recent work has shown that Drosophila Mad protein, the homologue of bone morphogenetic protein rSmads, binds to GRCGNC sites through the same MH1 domain beta-hairpin interface used to contact GTCT sites. However, binding to GRCGNC requires base-specific contact by two Mad proteins, and here we provide evidence that this is achieved by contact of the two Mad subunits that overlap across the two central base pairs of the site. This topology is supported by results indicating that His-93, which is located at the tip of the Mad beta-hairpin, is in close proximity to base pairs 2 and 5. Also consistent with the model is disruption of binding by mutation of Glu-39 and Glu-40, which are predicted to lie at the interface of the two overlapping Mad MH1 domains. As predicted from the overlapping model, binding is disrupted by insertion of 1 bp in the middle of the site, whereas insertion of 2 bp creates abutting sites that can be bound by the Mad-Medea heterotrimer without requiring Glu-39 and Glu-40. Overlapping Mad sites predominate in decapentaplegic response elements, consistent with a high degree of specificity in response to signaling.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
TGF2-beta signaling contributes to numerous developmental processes, including axis formation, organogenesis, and limb development (1-5), and is a factor in cancer (6, 7). A TGF-beta ligand initiates signaling by binding type I and type II receptor serine/threonine kinases at the cell surface (8). Consequently, activated type I receptors phosphorylate Smad proteins at C-terminal serines, and these receptor-activated Smads (rSmads) then form a complex with Smad4 (9, 10). The activated Smad complexes translocate into the nucleus where they regulate transcription of target genes, through physical interaction and functional cooperation with co-activators and co-repressors (9, 10).

Decapentaplegic (Dpp) is a well characterized TGF-beta ligand in Drosophila that regulates transcription through the Smad proteins, Mothers Against Dpp (Mad), and Medea (2, 11). Mad is homologous to the BMP-specific rSmads (Smad1, Smad5, and Smad8), and Medea is the homologue of Smad4. Discrete patterns of Dpp expression during many phases of development (12, 13) result in graded patterns of phosphorylated Mad (pMad) across developing tissues (14, 15), which are interpreted by individual cells as positional information that instructs cell-type specification (2, 11). pMad levels are read out differentially by target genes with various thresholds for activation or repression by pMad, in combination with Medea and tissue-specific cofactors (16-20). Considerable progress has been made in understanding how Smad proteins interact with cofactors to activate or repress transcription, and it is expected that response thresholds will be determined by their combined affinities for particular cis elements. In Drosophila this is complicated by a double-negative mechanism, in which Dpp represses the brinker (brk) gene, and the Brk protein then represses other Dpp targets, in some instances by competing for Mad-binding sites (21-29). Repression of brk by Dpp is through a small silencer element (brkS) that contains a high affinity Mad-Medea-binding site (30, 31). A nearly identical silencer controls repression of bag of marbles (bam) in germ line stem cells in response to Dpp secreted from an adjacent somatic layer of the ovary or testis (32, 33). Alignment of these sites and mutational analysis revealed that these silencers are bipartite, with a GRCGNC Mad-binding site spaced 5 bp from a GTCT Medea-binding site (30-33). Once bound, Mad and Med together recruit Schnurri, a large zinc finger transcription factor with a repression domain responsible for silencing of brk and bam (31). Smad proteins contact DNA with a beta-hairpin contained in the MH1 domain. beta-Hairpin side chains make specific major groove contact with bases at the 1st, 3rd, and 4th positions of GTCT (34), and each of these amino acids is required in both Mad and Medea for silencer binding (35). However, an analysis of stoichiometry revealed that the GRCGNC site is contacted by two Mad MH1 domains and that all three base-contact residues are required in both subunits (35). This then leads to the question of how two Mad MH1 domains can squeeze onto a 6-bp site using the same contacts involved in binding of a single MH1 to a 4-bp site. The silencer GRCGNC Mad-binding site resembles the previously identified GCCGNCG Mad consensus deduced from alignment of Dpp-activated enhancers (16, 36), and which also occurs in vertebrate BMP-response elements (37-44). Here we provide evidence that two Mad MH1 domains bind to GRCGNC by overlapping across the two central base pairs of the site.


Figure 1
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  		FIGURE 1.
Mutational analysis of the brkS Mad-binding site supports an overlapping model. A, base-specific contacts by Smad3 beta-hairpin. Labeled vertical arrows indicated base contacts. Horizontal arrow indicates convention used for designating a Smad-binding site in B and Fig. 3A. B, models for binding of two MH1 domains to the brkS Mad-binding site. Positions 1-6 above the sequences correspond to the previously defined Mad-binding site. Positions -1 and 7 are flanking bases. Arrows pointing rightward indicate putative binding sites oriented as shown in A (e.g. the Medea sites in each model). Arrows pointing leftward are sites oriented in the opposite direction. C, competitive gel shift assay comparing the effects of flanking base substitutions on binding of a heterotrimeric complex of FLAGMad and HAMedea. Molar excess of unlabeled double-stranded oligo relative to the 32P-labeled DNA probe is indicated above each lane, with the identity of the unlabeled oligos given for groups of three lanes each. For all reactions the labeled DNA probe was wild-type brkS, and the overexpressed proteins were wild-type Mad and wild-type Medea. Arrow indicates position of the Mad-Medea-brkS complex; asterisk marks the nonspecific band. Prior to electrophoresis, radioactive DNA probes were incubated with lysates of human 293T cell transiently expressing FLAGMad, HAMedea, and constitutively active type I Dpp receptor (TkvQD).

 

   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmids—Mad, Med, TkvQD, ShnCT, and 2XGFP-Mad/-Med effector plasmids for expression in human 293T cells have been described previously. Site-directed mutagenesis of Mad and Med effector plasmids was carried out using a method described previously (35, 45). Plasmid constructs and products of site-directed mutagenesis were verified by DNA sequencing. For reporter assays, the brkS sequence or derivatives were inserted between the EcoRI and NotI sites of 3xSu(H)-HSC (35). pPacTkvQD, pPacSu(H), and pPacNAct have been described previously (28).

Gel Shift and Reporter Assays—Gel shift assays and preparation of DNA probes and lysates from transfected human 293T cells were performed as described previously (35). Gel images were collected with a Storm 870 PhosphorImager (Amersham Biosciences), and the amount of probe present in bands was determined using ImageQuant software. Reporter assays were performed by transfection of Drosophila S2 cells as described previously (35) with pPac-luc as an internal control. S2 cells were harvested 2 days after transfection, and chemiluminescent beta-galactosidase and luciferase assays were performed on cell extracts using the GalactoStar assay system (Tropix, Inc.) according to the supplier's instructions. Reporter assays were performed in triplicate with results normalized to the internal control. Western blotting was performed as described previously (35).

Molecular Modeling—The Swiss-Model feature of Deep View (46) was used to thread amino acids 26-146 of the Mad MH1 domain sequence onto the Smad3 MH1-DNA co-crystal structure of Chai et al. (47). Compared with Smad3, Mad has a 3-residue gap in loop L1 between helices H1 and H2, as do the homologous BMP rSmads. By using the alignment feature, this gap was manually positioned between Lys-35 and Gln-36, allowing the Mad sequence to thread along the complete lengths of helices H2 and H3. Manual alignment was also used to position a single residue gap between Smad3 Lys-44 and Thr-45 in the turn between helices H2 and H3. The threaded alignment was submitted as a modeling request to the Swiss Model server. The resulting improved Mad MH1 domain model was manually docked to a B-DNA model of the brkS element generated using the model.it program of the DNAtools website (48). The Mutate feature of DeepView/Swiss-Pdb-Viewer (46) was used to explore possible hydrogen bonding between side chains at the interface between the two Mad MH1 domains. The same homology modeling and docking procedures were used to generate the Medea MH1 domain model shown in Fig. 5A. The images shown in Fig. 5 were generated by POV-Ray version 3.5 from screens created using DeepView.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Binding of Two Mad MH1 Domains to a 6-bp Site Suggests an Overlapping Model for DNA Contact—Alignment and mutational analysis of the bam and brk silencer elements identified a Mad-binding site spanning 6 bp but in which only 4 positions are rigidly specified (31, 35). These Mad sites are contacted by two Mad MH1 domains, each involving all three canonical beta-hairpin contact residues. In Mad these base-contact amino acids are Arg-88, Gln-90, and Lys-95 (Fig. 1A). Fig. 1B shows three models for how two Mad MH1 domains might contact GRCGNC. In each model, Arg-88 and Lys-95 contact guanines spaced 2 bases apart on opposite strands, as they do in the Smad3MH1-DNA structure. In the first model, the two MH1 domains abut facing outward so that 1 additional base on each end of the site (positions -1 and 7) would be contacted by Gln-90, a side chain that has been shown to be critical for binding of Mad to brkS (35). Pyrowolakis et al. (31) showed that substitution of G at -1 or C at +7 had little effect on binding of the Mad-Medea complex, inconsistent with critical base-specific contact at either position. However, contact by Gln-90 might be compatible with more than 1 base, which appears to be the case for the corresponding Gln-111 of Medea, because either T or G is tolerated at the 4th position of its binding site in brkS (i.e. GTCT or GTCG) (31, 35). To explore this further, we measured the effects of all possible single base substitutions of the flanking base pairs using a competitive gel shift assay (Fig. 1C). In this experiment excess unlabeled wild-type and mutant brkS DNAs were added to gel shift binding reactions containing labeled wild-type brkS probe. The observed effects on binding to brkS were minor, as judged by comparison of 3-fold steps in the concentration of cold competitor DNAs. Furthermore, according to model 1, -1A and 7T would be expected to improve binding because they match the Smad box consensus, but instead diminished binding affinity. Thus, these results do not lend support for model 1. For the same reasons, the results also weigh against a second model in which the MH1 domains bind pointed in the same direction with 1 bp of overlap, such that the predictions would apply to one flanking side or the other. The results do not rule out a third model in which the two Mad MH1 domains point toward each other with their beta-hairpins overlapping across the two central base pairs in the major groove (Fig. 1B, model 3). According to this model, all base contact by beta-hairpin side chains occurs within the 6-bp site, and the observed minor effects of substitutions at flanking positions would be indirect (e.g. perhaps reflecting constraints on DNA conformation). Consistent with this model, we note that the symmetry of -1T and 7A matches the conserved T adjacent to the Medea-binding site of brkS or bamS (TGTCT).


Figure 2
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  		FIGURE 2.
Silencer binding is sensitive to mutations affecting the tip of the Mad beta-hairpin. A, proposed arrangement of base contacts in overlapping Mad-binding site model. Solid arrows and non-italic lettering indicate base contacts by first Mad subunit; open arrows and italic lettering show second subunit contacts. Solid circle and non-italicized lettering indicates position of His-93 in first subunit; open circle and italic lettering show position in second subunit. B, gel shift assay showing that MadH93W strongly reduces binding of the Mad-Medea heterotrimer to brkS and that MedeaG114W has a much weaker effect. Bottom panel shows that expression levels of FLAGMad and FLAGMadH93W were indistinguishable when detected by probing of a Western blot with anti-FLAG antibody. C, gel shift assay showing approximately equal amounts of heterotrimers containing two wild-type Mad subunits or one wild-type and one H93W subunit. Compositions of Mad-Med-DNA complexes are diagrammed alongside the gel image with arrows pointing to the corresponding bands. Gray ovals indicate Mad subunits; white ovals indicate Medea subunits, and black circles indicate 2xGFP fusions to Mad. D, competitive gel shift assay comparing the effects of base substitutions at positions 2 and 5 (as numbered in Fig. 1B) on binding of the heterotrimeric Mad-Medea complex. The labeled probe is wild-type brkS. E, gel shift assay showing that MadH93A restores binding to brkS DNA probes with internal mutations in the Mad-binding site. Mutant probes are brkS with the indicated base pair substitutions according to numbering shown in Fig. 1B. MadH93W reduced binding to all four probes. At the bottom left is a Western blot showing that expression levels of FLAG-tagged MadH93A and H93W were indistinguishable from wild type when detected by probing with anti-FLAG antibody. B-E, arrow indicates position of the heterotrimer-DNA complex; asterisk indicates background band. Lysates used in all binding reactions were from 293T cells transfected to express TkvQD, Mad, and Medea. Use of mutant or GFP-fused versions or mutant proteins in place of wild type (WT) was as indicated for individual gel lanes.

 
Silencer Binding Is Sensitive to Mutations Affecting the Tip of the Mad beta-Hairpin—To further distinguish between the Mad-binding site models, we sought a way of determining how the beta-hairpins of the two Mad subunits are positioned with relation to specific base pairs within the brkS-binding site. At the tip of the beta-hairpin, where it makes the turn between beta-sheets B2 and B3, rSmads contain a histidine residue (His-93 in Mad) whose imidazole side chain is pointed toward the neighboring MH1 domain in the Smad3-DNA structure (34, 47). The overlapping model for binding of Mad MH1 domains to brkS positions His-93 near the DNA-contacting Arg-88 side chain of the opposite Mad subunit (Fig. 2A). The predicted close proximity of these residues suggests that binding might be affected by substitution of a bulky residue in place of His-93. To test this a H93W mutation (MadH93W) was made and found to cause substantial reduction in binding affinity for brkS (Fig. 2B). By co-expressing MadH93W together with 2XGFP-Mad, it was found that complexes in which just one Mad subunit contains the H93W mutation were relatively unaffected compared with wild type (Fig. 2C). The corresponding position in Medea and Smad4 is a glycine instead of histidine (Gly-114 in Medea), and it was found that a MedeaG114W mutation had a weaker effect on silencer binding (Fig. 2B), consistent with the binding of Medea to the unpaired GTCT and indicating that a Trp substitution at this position in the beta-hairpin does not drastically interfere with DNA contact if the site is nonoverlapping. In separate experiments it was noticed that substitutions at position 2 or position 5 of the overlapping site caused reductions in binding affinity (Fig. 2D). Position 2 shows a bias for G and position 5 shows the same bias on the opposite strand, consistent with a completely symmetrical Mad-binding site of optimal sequence GGCGCC. In the overlapping model, bases at positions 2 and 5 are not predicted to make direct contact with the MH1 domains but are near the His-93 side chain. To test whether an unfavorable interaction with His-93 might be responsible for the reduced binding affinity, MadH93A was tested for binding to brkS, whose Mad site sequence is GGCGAC, and to the position 2 and 5 mutations, GCCGAC and GGCGGC (Fig. 2E, numbering as in Fig. 1B). The MadH93A mutation had little or no effect on binding to the GGCGAC site or to the optimal GGCGCC but enhanced binding to the 2C and 5G probes, consistent with proximity of these 2 bases to the corresponding His-93 side chains. Western blot analysis showed that neither H93A nor H93W detectably affected the level of Mad protein in the cell lysates used for gel shift experiments (Fig. 2, B and E, lower panels). These results support an overlapping model in which the tip of the beta-hairpin of one Mad subunit is close to position 2 and on the other subunit is close to position 5.


Figure 3
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  		FIGURE 3.
The brkS Mad site is disrupted by interruption of symmetry in the proposed overlap. A, sequences of DNA probes used in gel shift assays to probe Mad-binding site substructure. In the left column of sequences the boldface lettering indicates alterations from the brkS site shown at the top. The right column indicates alterations from the 2SB_RL site shown at the top. In both columns the arrows indicate the putative position of MH1-binding sites as in Fig. 1B. B, gel shift assay showing that the brkS binding by the Mad-Medea heterotrimer was disrupted by 1-bp insertions into the middle of the Mad site. Open arrow indicates position of complexes with slightly slower mobility than brkS complex (solid arrow); asterisk indicates background band; same gel annotation applies to C-F and H. C, gel shift assay showing that binding was not disrupted by 2-bp insertions, with enhanced binding when the Mad site was replaced by symmetrical SBEs (2SB_RL). Insertions of 3 or 4 bp reduced binding but not as severely as the 1-bp insertions. D, competitive gel shift assay showing effects of insertions of varying length at the midpoint of the Mad-binding site. Labeled probe is wild-type brkS. Molar excesses of unlabeled competitor DNAs are indicated above each lane. E, gel shift assay showing that other arrangements of nonoverlapping GTCT were poor substrates for Mad-Medea heterotrimer binding in comparison to 2SB_RL. F, mutation of the Medea-binding site reduced binding affinity for a probe with abutting Mad sites (compare 3(GC)4 with 3(GC)4GTCTm), although the effect was not as severe as for brkS (compare brkS with brkSGTCTm). A stronger abutting site was also less dependent on a Medea site (compare SB_RL with 2SB_RLGTCTm). The effect of mutating the Medea site shows that Medea binding still contributes to overall affinity when the Mad sites abut rather than overlap. G, gel shift using GFP fusion strategy showed that a 2SB_RL probe with nonoverlapping Mad sites was bound by a 2:1 Mad:Med heterotrimer. Positions of complexes containing 2Mad + Medea or 2(2xGFP-Mad) + Medea are indicated by arrows on the left; position of an intermediate complex containing Mad + 2xGFP-Mad + Medea is indicated by the upper arrow of the two middle arrows; position of complex containing 2Mad + Medea or 2Mad + 2xGFP-Medea are indicated by arrows flanking the right-hand set of lanes. The observation of an intermediate band with the 2xGFP-Mad but not with 2xGFP-Medea is consistent only with a 2:1 Mad-Medea stoichiometry. H, gel shift showing that GGCG can function as a nonoverlapping Mad-binding site (compare 2GC_RL with brkS and 2SB_RL) but is incompatible when paired with a GTCT (GC_SB). Right side of gel shows that recruitment of ShnCT was still strong when the Mad site was changed to a nonoverlapping GGCG pair (Mad-Medea-ShnCT complexes indicated by right-hand open arrow; compare brkS and 2GC_RL probes) but was weaker when the Mad site included a GTCT (2SB_RL and GC_SB). Pairing of GTCT with the alternative Smad1 site GCAT resulted in weak binding that was slightly enhanced by Shn (S1_SB). I, results of S2 reporter assays in which brkS and derivative sites were tested for the ability to repress transcription in response to TkvQD. Reporters also contained Su(H)-binding sites and were activated by co-expression of Su(H) and an activated form of Notch, as indicated. Replacement of the brkS Mad site with nonoverlapping GGCG sites still allowed repression (compare brkS with GC_SB), but substitution with a site containing GTCT almost eliminated repression (GC_SB) or brought about activation in response to TkvQD (2SB_RL). Other arrangements of nonoverlapping GTCT sites also failed to repress reporter expression (2SB_LL, 2SB_RR, and 2SB_LR).

 
The brkS Mad Site Is Disrupted by Interrupting Symmetry in the Proposed Overlap—The overlapping model predicts that a 1-bp insertion at the center of the GGCGAC site will disrupt overlap of the two sites, creating an asymmetric 1-bp overlap or two tandem sites (Fig. 3A). A varying degree of disruption by such insertions was observed (Fig. 3B), the least damaging of which (the 3(A)4 construct) correlated with the creation of a GTCT site. In contrast, a series of 2-bp insertions left binding largely intact (3(GC)4, 3(TA)4, and 2SB_RL probes in Fig. 3C), whereas further separation of the sites reduced binding affinity (Fig. 3C, right 4 lanes) suggesting that proximity of the Mad MH1 domains is required for stable binding. These effects were confirmed in a competitive gel shift assay (Fig. 3D). Binding was also disrupted when the GGCGAC Mad-binding site was replaced with other arrangements of two SBEs (2SB_LR, 2SB_LL, and 2SB_RR probes in Fig. 3E), indicating that stability of the bound Mad-Medea complex depends upon a specific topology of the Mad MH1 domains.

The failure to disrupt binding by 2-bp insertions might be due to the creation of two abutting sites in the same configuration as the Smad3/Smad4 consensus binding site (49), (indicated by the arrows marking each sequence in Fig. 3A). In support of this idea, binding was not disrupted by substitution of GTCTAGAC (the Smad3/4 consensus) in place of GGCGAC; rather, slight enhancement of binding was observed (Fig. 3, C and D, 2SB_RL). The resulting gel shift complex had a slightly slower mobility than the brkS complex and was still dependent on the Medea site for high affinity binding (Fig. 3F, compare brkS, 3(TA)4, and 2SB_RL to corresponding GTCTm probes). Stoichiometry of the complex was assessed using 2xGFP fusions to Mad or Medea (Fig. 3G). The substitution of 2xGFP-Mad for Mad caused a supershift consistent with the presence of two Mad subunits (35), and an intermediate band was visible when Mad and 2xGFP-Mad were both present in the complex. In contrast, the supershifted 2xGFP-Medea aligns with the immediate Mad+2xGFPMad band and caused no obvious intermediate band in combination with Medea, consistent with the presence of just a single Medea subunit. Together, these results indicate a 2:1 Mad:Medea heterotrimer complex, as was found for the brkS Mad-Medea complex (35).

Reporter assays were carried out in Drosophila S2 cells to determine whether an abutting site still allows silencer function. In this assay the brkS and bamSE silencers can over-ride reporter activation by Notch and Suppressor Hairless (30, 31, 35). By using this assay, we found that when abutting Mad-binding sites matched the GC-rich Mad consensus, i.e. GGCGCGCC, then ShnCT still bound (2GC_RL probe in Fig. 3H) and the element repressed reporter expression in response to Dpp signaling (2GC_RL reporter in Fig. 3I). If the abutting site was changed to GTCTAGAC, then ShnCT bound was reduced (2SB_RL probe in Fig. 3H), and repression was lost (Fig. 3I), perhaps reflecting a situation where, in vivo, the binding of co-activators wins out when Shn binding affinity drops below a critical threshold, despite its ability to bind weakly in vitro. Repression was also disrupted when the overlapping Mad sites were replaced with other arrangements of two SBEs (Fig. 3I).


Figure 4
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  		FIGURE 4.
GGCG and GTCT are both contacted by the beta-hairpin but with different contributions by helix 2 side chains. A, positions of Mad helix 2 and beta-hairpin mutations that were tested for effects on binding to 2SB_RL and 2GC_RL. Indicated positions of structural elements are taken from the Smad3 structure. Bracket indicates location of the Smad3 beta-hairpin. B, gel shift assays showing the effects of alanine substitutions in the Mad helix 2 and beta-hairpin on binding to two brkS derivatives containing nonoverlapping Mad-binding sites. The ratios given below each lane are binding affinities of mutant Mad proteins (complexed with Medea) relative to that of wild-type Mad, as calculated by dividing the fraction of bound versus free probe DNA for each mutant complex by the same fraction from the control lane containing wild-type Mad (an approximation that is valid because Mad-Medea complexes are in >10-fold excess over probe, as shown by oligo competition assays in Figs. 1D and 2E). beta-Hairpin base contact residues were required for binding regardless of whether the nonoverlapping Mad site contained GTCT or GGCG half-sites (2SB_RL versus 2GC_RL). In contrast, helix 2 mutations affected binding to the two probes differently, particularly K46A and K54A (lanes marked with filled circles above labels). For the 2SB_RL probe, loss of binding by the Mad-Medea complex (solid arrow) coincided with the appearance of a slower mobility band (open arrow) identified in separate experiments as a homomeric Medea complex (see Fig. 6C). Panels at bottom left are Western blots showing the FLAGMad (or mutant FLAGMad) and HAMedea levels in each assay.

 
The results described above show that Mad binds to GTCT or GGCG with similar affinity despite a difference in the base apparently contacted by Mad Gln-90. In the overlapping site the sequence is constrained to GNCGNC, but in abutting sites it seemed possible that GTCT and GGCG might be interchangeable. However, although the 2SB_RL and 2GC_RL probes were bound with similar affinity, a composite of the two, GTCTCGCC, was bound only weakly (GC_SB probe in Fig. 3H). In addition, we tested the alternative Smad1-binding site, GCAT (50), and we observed that it too was a poor partner for GTCT, although binding was enhanced by the addition of ShnCT (S1_SB probe + ShnCT in Fig. 3H).

GGCG Is Contacted by the beta-Hairpin but with Different Helix 2 Contribution—Incompatibility of abutting GTCT and GGCG suggested a possible difference in docking geometry of the Mad MH1 domain. To test this, we compared the effects of a series of alanine substitutions in the Mad helix 2 and beta-hairpin (Fig. 4A). The binding activities of these mutants were compared for the 2SB_RL and 2GC_RL probes (Fig. 4B). All of the beta-hairpin substitutions disrupted binding to both probes, but helix 2 substitutions had differential effects. For example, K46A and K54A (Fig. 4B, filled circles) caused a similar reduction of binding to the 2GC_RL probe, whereas binding affinity for 2SB_RL was unaffected by K46A but reduced more than 3-fold by K54A (ratio of mutant versus wild-type binding affinities is given below each lane in Fig. 4B). Both of these conserved lysines have side chains positioned near the DNA phosphate backbone in the Smad3-DNA structure. Thus the observed differential effects of these substitutions on binding affinity are indicative of a different docking geometry for GTCT versus GGCG, although docking to GGCG must still allow for contacts involving all three base-specific beta-hairpin residues. Overall, the results showed that Lys-42, Lys-46, and Asp-49 were more important for contact with GGCG than for GTCT, although the reverse was true for Lys-53-57 and Lys-59. One additional difference with 2SB_RL was that disrupted binding of Mad mutants was accompanied by the appearance of a slower mobility band, seen most clearly for the R58A mutant. This band appears to have been due to binding by a Medea complex that had moderate affinity for 2SB_RL but not for 2GC_RL or for the natural brkS element (see Fig. 6, B and C). The slow mobility of this Medea complex suggested that it was a homotrimer.


Figure 5
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  		FIGURE 5.
brkS binding is affected by mutation of Mad residues predicted to lie at an interface between overlapping MH1 domains. A, ribbon model showing binding of Mad MH1 domains to overlapping sites in brkS. Right-hand panel shows view along the DNA axis with the Medea MH1 domain omitted for clarity. Derivation of the model is described under "Experimental Procedures." B, oblique close-up view of putative hydrogen bonding between overlapping MH1 subunits. For clarity the ribbon backbone outline is shown only for the helix 2 and beta-hairpin regions of each Mad subunit, with side chains shown only for a subset of interface residues. Arg-94 and Glu-40 side chains are from the blue upper subunit; Glu-39 and the Glu-40 backbone amide are from the purple lower subunit; hydrogen bonds calculated by DeepView are shown as dotted green lines. According to this model Glu-39 positioned at the tip of helix 2 makes inter-subunit hydrogen bonds with hairpin residue Arg-94 and helix 2 residue Glu-40, with Glu-40 also sharing a hydrogen bond with the Glu-40 backbone amide of the opposite subunit. Not shown is the reciprocal set of inter-subunit H-bonds. C, gel shift assay showing that brkS binding was substantially weakened by alanine substitutions for Glu-39, Glu-40, and Arg-94 (left panel), with little or no effect on binding to a 2SB_RL probe containing nonoverlapping Mad sites (right panel). Cell lysates used for gel shifts contained FLAGMad (wild-type or the indicated mutant), HAMed, and TkvQD. Anti-FLAG Western blot at bottom shows that levels mutant Mad proteins were as high as for wild-type Mad. Arrow indicates Mad-Medea heterotrimer complex; asterisk marks the background band. Mutant versus wild-type binding affinity ratios shown below each lane were derived as in Fig. 4B.

 
A Structural Model Predicts Contacts between Overlapping Mad MH1 Domains—To explore the idea that Mad MH1 domains might contact each other when bound to the overlapping site, a molecular model of the Mad MH1 domain was generated by homology modeling. The Mad MH1 amino acid sequence was threaded onto the Smad3 MH1 structure using the Swiss-Model feature of DeepView/Swiss-PdbViewer (described under "Experimental Procedures") (46). The resulting MadMH1 structural model was then used to model docking to brkS DNA using DeepView (Fig. 5A). The use of a uniform B-DNA model provided only an approximation of how Mad MH1 domains might contact the silencer because the Smad3MH1-DNA structure exhibits substantial base-to-base variation in twist and major groove width (34). With this caveat, it was possible to generate a model in which Gln-90 and Lys-95 of two MadMH1 domains make overlapping contact with the central 2 bp of GGCGAC (underlined). This model also allowed for most of the same phosphate backbone contacts present in the Smad3-DNA structure.


Figure 6
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  		FIGURE 6.
Silencer binding requires MH1 domains from both Mad and Medea. A, diagram showing EA and AE chimeric proteins containing swapped Mad and Medea MH2 domains. Lengths are proportional to actual lengths. B, gel shift assay testing the indicated combinations of Mad, Med, and chimeric proteins for binding to a brkS probe. Only Mad + Med and EA + AE bound strongly (arrows). Vertical lines mark weak homomeric Mad and homomeric Medea complexes. C, gel shift assay as in B but with the 2SB_RL probe containing a nonoverlapping Mad-binding site. AE failed to bind this probe, but all other combinations resulted in readily detectable gel shift complexes. Arrows mark the complexes containing EA + Med, which bound with all Med MH1 domains, and AE + Mad, which bound with all Mad MH1 domains.

 
The model indicated that overlapping binding would bring into proximity the N-terminal ends of helix 2 from each Mad MH1 such that several hydrogen bonds might form between subunits and stabilize the complex (Fig. 5B). Specifically, it appeared that the Glu-39 side chain of one subunit might be able to form hydrogen bonds with the Glu-40 and Arg-94 side chains of the overlapping subunit. In addition, the Glu-40 side chain of one subunit may be able to hydrogen-bond with the Glu-40 backbone amide of the opposite subunit. The results of gel shift assays using alanine substitutions for Glu-39, Glu-40, and Arg-94 showed 3-10-fold reductions in binding to brkS (Fig. 5C, ratio of mutant versus wild-type binding affinities given below each lane). In contrast, E39A and E40A had little effect on binding to the 2SB_RL site. These results indicate that E39A and E40A specifically disrupt binding of Mad to brkS without affecting the intrinsic affinity of Mad for nonoverlapping sites. R94A resulted in reduced binding to both probes but had less of an effect on the nonoverlapping site than on brkS, again consistent with a specific effect on brkS. Western blot analysis showed that mutant protein expression levels were indistinguishable from that of wild-type Mad (lower panel of Fig. 5C).

Silencer Binding Requires MH1 Domains from Both Mad and Medea—Experiments to determine the stoichiometry of silencer-bound Mad-Medea complexes demonstrated a 2:1 Mad:Medea ratio (35). This ratio could be strictly dictated by MH2 trimerization (51, 52) or it might also be influenced by interactions between overlapping Mad MH1 domains, as suggested by the effects of mutating putative MH1-MH1 interface residues. To address this question, Mad-Medea chimeric proteins were generated in which the MH2 domains were swapped to create a heterotrimer with one MH1 from Mad and two from Medea. These chimeras are designated "AE" for MadMH1 + linker plus Medea MH2 and "EA" for Medea MH1 + linker plus Mad MH2 (Fig. 6A). Binding of brkS required a heteromeric combination of MH2 domains because the combinations of AE + Medea and EA + Mad failed to bind. The combination of AE and EA was able to bind brkS with only a moderate reduction in affinity in comparison to the Mad-Medea complex (Fig. 6B). Western blot analysis showed that EA and AE were both expressed at levels indistinguishable from those of Mad and Medea (data not shown). The results indicate that a Medea MH1 domain is able to substitute for one Mad MH1 in contacting GGCGAC. In contrast, EA was unable to bind in combination with Medea (Fig. 6B), demonstrating that at least one Mad MH1 is necessary. Similarly, AE was unable to bind in combination with Mad (Fig. 6B), demonstrating that at least one Med MH1 is also necessary. Two of the residues involved in the interface between overlapping Mad MH1 domains, Glu-39 and Arg-94, are conserved in Medea, possibly accounting for the ability of one Medea MH1 to substitute for that of Mad. In contrast all three combinations bound with fairly high affinity to a probe containing GTCTAGAC in place of GGCGAC (Fig. 6C), demonstrating that there is little difference in the affinity of Mad and Medea MH1 domains for GTCT sites. These results indicate that binding to brkS requires at least one Mad and one Medea and that this is exclusively a property of the overlapping site. The nonoverlapping arrangement is less specific in that it can be contacted entirely by Mad or Medea MH1 domains. In fact, it was observed that the nonoverlapping arrangement allowed Mad to bind weakly without Medea and vice versa (Fig. 6C). Thus the overlapping Mad sites impose a requirement for both signaling and co-Smad participation, a requirement that is less stringent for abutting sites.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
DNA contact by Smad proteins has been shown to play an important role in many instances of target regulation by TGFbeta pathways. For the consensus Smad3/Smad4-binding site, GTCT can also be bound by Mad- and BMP-specific Smad1 (35, 53), but Smad3 does not bind to GC-rich Mad/Smad1-binding sites (42, 54), leaving open the question of whether such sites are contacted by a different mechanism. Recent work had shown that Mad-binding sites within the brk and bam silencers are bound by two Mad subunits and that in each case two Mad MH1 domains simultaneously contact a single 6-bp site using the same three beta-hairpin residues that are responsible for base-specific contact by Smad3 (35). Here, by using mutational analysis and directly measuring binding, we provide evidence that two Mad MH1 domains bind to the 6-bp site by overlapping across the two central base pairs. Smad1-binding sites match this 6-bp motif, a likely indication that overlap is also a feature of BMP-response elements.

The overlapping structure of Mad sites explains the seeming discrepancy between the Mad consensus and that of Smad3/Smad4. Smad3 differs from Mad at two positions that influence binding to brkS. Arg-58 at the C terminus of helix 2 is absolutely essential for binding to brkS (35); in Smad3 this position is a threonine, whereas the adjacent Lys-59 of Mad is absent in Smad3 (Fig. 4A). Glu-39 at the N terminus of helix 2 contributes substantially to binding affinity for brkS (Fig. 5), and Smad3 has instead a glutamine at this position. In addition, the loop between helices 1 and 2 is 3 residues shorter in Mad than in Smad3, a difference that modeling suggests will affect the structure of the {alpha}-carbon backbone and side chains near the N terminus of helix 2. Each of these differences is conserved between Smad3 and Smad2 and between Mad and the vertebrate BMP-specific rSmads.

Our mutational analysis indicated the optimal sequence for an overlapping Mad site is GGCGCC, meaning each Mad MH1 prefers GGCG in the context of overlap. However, we also show that even when the two Mad sites are spaced such that they do not overlap, GGCG is still bound by Mad with about the same affinity as GTCT. The structural basis for this compatibility with two distinct sites remains to be determined, but the differential effects of helix 2 alanine substitutions suggest distinct docking geometries. Individual GGCG motifs occur in Dpp and BMP-response elements (20, 37, 40, 41, 44, 55-60), and our results indicate that these are likely sites for contact by Mad and Smad1.

Although the natural brkS element was specific for the Mad-Medea heterotrimer, changing the Mad site to abutting SBEs allowed binding by Mad alone or by Medea alone. The ability of such a site to be bound by Medea oligomers (putatively homotrimers) without Dpp signaling seemingly would make it ill-suited to function as a Dpp-response element, although signaling-induced activation was observed by reporter analysis (perhaps an indication that Medea alone is a poor activator). However, the brkS derivative with abutting GGCG sites (i.e. GGCGCGCC) showed little or no Medea binding in the absence of active Mad (compare Fig. 4, B and C), was able to recruit Shn, and caused repression in response to signaling. Similar sites in the Dpp-response element of Race (GACGCGAC) (55), which does not respond to repression by Brk protein (61), and in a BMP-response element of Smad7 (GGCGCGCC) (57, 59, 60, 62) appear to be examples of functional nonoverlapping Mad/Smad1 sites. In Drosophila a potentially significant difference between overlapping and nonoverlapping Mad sites is that the overlapping motif allows for competitive binding by the Brinker protein (26-28) and thus dual control of Dpp targets, whereas the nonoverlapping motif does not. This may account for the predominance of overlapping Mad sites in Drosophila. The predominance of overlapping sites in BMP-response elements may reflect specificity for Smad1 but not Smad3.


   FOOTNOTES
 
* This work was supported by the University of Wisconsin Graduate School. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Laboratory of Genetics, University of Wisconsin, 425G Henry Mall, Madison, WI 53706. Tel.: 608-262-2456; Fax: 608-262-2976; E-mail: alaughon{at}wisc.edu.

2 The abbreviations used are: TGF, transforming growth factor; BMP, bone morphogenetic protein; MH, Mad homology; GFP, green fluorescent protein; oligos, oligonucleotides; Dpp, decapentaplegic; rSmad, receptor-activated Smad; SBE, Smad-binding element. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. De Robertis, E. M., and Kuroda, H. (2004) Annu. Rev. Cell Dev. Biol. 20, 285-308[CrossRef][Medline] [Order article via Infotrieve]
  2. Raftery, L. A., and Sutherland, D. J. (2003) Trends Genet. 19, 701-708[CrossRef][Medline] [Order article via Infotrieve]
  3. Rusten, T. E., Cantera, R., Kafatos, F. C., and Barrio, R. (2002) Development (Camb.) 129, 3575-3584[Abstract/Free Full Text]
  4. Niswander, L. (2003) Nat. Rev. Genet. 4, 133-143[CrossRef][Medline] [Order article via Infotrieve]
  5. Hogan, B. L., and Kolodziej, P. A. (2002) Nat. Rev. Genet. 3, 513-523[CrossRef][Medline] [Order article via Infotrieve]
  6. Kang, Y., He, W., Tulley, S., Gupta, G. P., Serganova, I., Chen, C. R., Manova-Todorova, K., Blasberg, R., Gerald, W. L., and Massague, J. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 13909-13914[Abstract/Free Full Text]
  7. Mishra, L., Derynck, R., and Mishra, B. (2005) Science 310, 68-71[Abstract/Free Full Text]
  8. Hart, P. J., Deep, S., Taylor, A. B., Shu, Z., Hinck, C. S., and Hinck, A. P. (2002) Nat. Struct. Biol. 9, 203-208[Medline] [Order article via Infotrieve]
  9. Massague, J., Seoane, J., and Wotton, D. (2005) Genes Dev. 19, 2783-2810[Abstract/Free Full Text]
  10. Feng, X. H., and Derynck, R. (2005) Annu. Rev. Cell Biol. 21, 659-693
  11. Affolter, M., Marty, T., Vigano, M. A., and Jazwinska, A. (2001) EMBO J. 20, 3298-3305[CrossRef][Medline] [Order article via Infotrieve]
  12. Posakony, L., Raftery, L., and Gelbart, W. (1991) Mech. Dev. 33, 69-82
  13. Masucci, J. D., Miltenberger, R. J., and Hoffmann, F. M. (1990) Genes Dev. 4, 2011-2023[Abstract/Free Full Text]
  14. Dorfman, R., and Shilo, B. Z. (2001) Development (Camb.) 128, 965-972[Abstract]
  15. Tanimoto, H., Itoh, S., Ten, D. P., and Tabata, T. (2000) Mol. Cell 5, 59-71[CrossRef][Medline] [Order article via Infotrieve]
  16. Xu, X., Yin, Z., Hudson, J. B., Ferguson, E. L., and Frasch, M. (1998) Genes Dev. 12, 2354-2370[Abstract/Free Full Text]
  17. Knirr, S., and Frasch, M. (2001) Dev. Biol. 238, 13-26[CrossRef][Medline] [Order article via Infotrieve]
  18. Halfon, M. S., Carmena, A., Gisselbrecht, S., Sackerson, C. M., Jimenez, F., Baylies, M. K., and Michelson, A. M. (2000) Cell 103, 63-74[CrossRef][Medline] [Order article via Infotrieve]
  19. Lee, H. H., and Frasch, M. (2005) Development (Camb.) 132, 1429-1442[Abstract/Free Full Text]
  20. Xu, M., Kirov, N., and Rushlow, C. (2005) Development (Camb.) 132, 1637-1647[Abstract/Free Full Text]
  21. Marty, T., Muller, B., Basler, K., and Affolter, M. (2000) Nat. Cell Biol. 2, 745-749[CrossRef][Medline] [Order article via Infotrieve]
  22. Sivasankaran, R., Vigano, M. A., Muller, B., Affolter, M., and Basler, K. (2000) EMBO J. 19, 6162-6172[CrossRef][Medline] [Order article via Infotrieve]
  23. Jazwinska, A., Kirov, N., Wieschaus, E., Roth, S., and Rushlow, C. (1999) Cell 96, 563-573[CrossRef][Medline] [Order article via Infotrieve]
  24. Campbell, G., and Tomlinson, A. (1999) Cell 96, 553-562[CrossRef][Medline] [Order article via Infotrieve]
  25. Minami, M., Kinoshita, N., Kamoshida, Y., Tanimoto, H., and Tabata, T. (1999) Nature 398, 242-246[CrossRef][Medline] [Order article via Infotrieve]
  26. Rushlow, C., Colosimo, P. F., Lin, M. C., Xu, M., and Kirov, N. (2001) Genes Dev. 15, 340-351[Abstract/Free Full Text]
  27. Saller, E., and Bienz, M. (2001) EMBO Rep. 2, 292-297[CrossRef][Medline] [Order article via Infotrieve]
  28. Kirkpatrick, H., Johnson, K., and Laughon, A. (2001) J. Biol. Chem. 276, 18216-18222[Abstract/Free Full Text]
  29. Zhang, H. L., Levine, M., and Ashe, H. L. (2001) Genes Dev. 15, 261-266[Abstract/Free Full Text]
  30. Muller, B., Hartmann, B., Pyrowolakis, G., Affolter, M., and Basler, K. (2003) Cell 113, 221-233[CrossRef][Medline] [Order article via Infotrieve]
  31. Pyrowolakis, G., Hartmann, B., Muller, B., Basler, K., and Affolter, M. (2004) Dev. Cell 7, 229-240[CrossRef][Medline] [Order article via Infotrieve]
  32. Chen, D. H., and McKearin, D. (2003) Curr. Biol. 13, 1786-1791[CrossRef][Medline] [Order article via Infotrieve]
  33. Chen, D. H., and McKearin, D. M. (2003) Development (Camb.) 130, 1159-1170[Abstract/Free Full Text]
  34. Shi, Y., Wang, Y., Jayaraman, L., Haijuan, Y., Massague, J., and Pavletich, N. (1998) Cell 94, 585-594[CrossRef][Medline] [Order article via Infotrieve]
  35. Gao, S., Steffen, J., and Laughon, A. (2005) J. Biol. Chem. 280, 36158-36164[Abstract/Free Full Text]
  36. Kim, J., Johnson, K., Chen, H. J., Carroll, S., and Laughon, A. (1997) Nature 388, 304-308[CrossRef][Medline] [Order article via Infotrieve]
  37. Benchabane, H., and Wrana, J. (2003) Mol. Cell. Biol. 23, 6646-6661[Abstract/Free Full Text]
  38. Ishida, W., Hamamoto, T., Kusanagi, K., Yagi, K., Kawabata, M., Takehara, K., Sampath, T. K., Kato, M., and Miyazono, K. (2000) J. Biol. Chem. 275, 6075-6079[Abstract/Free Full Text]
  39. Kang, Y., Chen, C. R., and Massague, J. (2003) Mol. Cell 11, 915-926[CrossRef][Medline] [Order article via Infotrieve]
  40. Karaulanov, E., Knochel, W., and Niehrs, C. (2004) EMBO J. 23, 844-856[CrossRef][Medline] [Order article via Infotrieve]
  41. Korchynskyi, O., and ten Dijke, P. (2002) J. Biol. Chem. 277, 4883-4891[Abstract/Free Full Text]
  42. Kusanagi, K., Inoue, H., Ishidou, Y., Mishima, H. K., Kawabata, M., and Miyazono, K. (2000) Mol. Biol. Cell 11, 555-565[Abstract/Free Full Text]
  43. Lopez-Rovira, T., Chalaux, E., Massague, J., Rosa, J. L., and Ventura, F. (2002) J. Biol. Chem. 277, 3176-3185[Abstract/Free Full Text]
  44. Seki, K., and Hata, A. (2004) J. Biol. Chem. 279, 18544-18549[Abstract/Free Full Text]
  45. Sawano, A., and Miyawaki, A. (2000) Nucleic Acids Res. 28, 78
  46. Guex, N., and Peitsch, M. C. (1997) Electrophoresis 18, 2714-2723[CrossRef][Medline] [Order article via Infotrieve]
  47. Chai, J., Wu, J. W., Yan, N., Massague, J., Pavletich, N. P., and Shi, Y. (2003) J. Biol. Chem. 278, 20327-20331[Abstract/Free Full Text]
  48. Vlahovicek, V., Kajan, L., and Pongor, S. (2003) Nucleic Acids Res. 31, 3686-3687[Abstract/Free Full Text]
  49. Zawel, L., Dai, J. L., Buckhaults, P., Zhou, S. B., Kinzler, K. W., Vogelstein, B., and Kern, S. E. (1998) Mol. Cell 1, 611-617[CrossRef][Medline] [Order article via Infotrieve]
  50. Henningfeld, K. A., Rastegar, S., Adler, G., and Knochel, W. (2000) J. Biol. Chem. 275, 21827-21835[Abstract/Free Full Text]
  51. Shi, Y., Hata, A., Lo, R. S., Massague, J., and Pavletich, N. P. (1997) Nature 388, 87-93[CrossRef][Medline] [Order article via Infotrieve]
  52. Chacko, B. M., Qin, B. Y., Tiwari, A., Shi, G., Lam, S., Hayward, L. J., de Caestecker, M., and Lin, K. (2004) Mol. Cell 15, 813-823[CrossRef][Medline] [Order article via Infotrieve]
  53. Johnson, K., Kirkpatrick, H., Comer, A., Hoffmann, F. M., and Laughon, A. (1999) J. Biol. Chem. 274, 20709-20716[Abstract/Free Full Text]
  54. Kusanagi, K., Kawabata, M., Mishima, H. K., and Miyazono, K. (2001) J. Biol. Chem. 276, 28155-28163[Abstract/Free Full Text]
  55. Wharton, S. J., Basu, S. P., and Ashe, H. L. (2004) Curr. Biol. 14, 1550-1558[CrossRef][Medline] [Order article via Infotrieve]
  56. Brugger, S. M., Merrill, A. E., Torres-Vasquez, J., Wu, N., Ting, M., Cho, J., Dobias, S. L., Yi, S. E., Lyons, K., Bell, J. R., Arora, K., Warrior, R., and Maxson, R. (2004) Development (Camb.) 131, 5153-5165[Abstract/Free Full Text]
  57. von Gersdoff, G., Susztak, K., Rezvani, F., Bitzer, M., Liang, D., and Bottinger, E. P. (2000) J. Biol. Chem. 275, 11320-11326[Abstract/Free Full Text]
  58. Brodin, G., Ahgren, A., Ten, D. P., Heldin, C. H., and Heuchel, R. (2000) J. Biol. Chem. 275, 29023-29030[Abstract/Free Full Text]
  59. Nagarajan, R. P., Zhang, J. M., Li, W., and Chen, Y. (1999) J. Biol. Chem. 274, 33412-33418[Abstract/Free Full Text]
  60. Denissova, N. G., Pouponnot, C., Long, J. Y., He, D. M., and Liu, F. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 6397-6402[Abstract/Free Full Text]
  61. Ashe, H. L., Mannervik, M., and Levine, M. (2000) Development (Camb.) 127, 3305-3312[Abstract]
  62. Dooley, S., Delvoux, B., Lahme, B., Mangasser-Stephan, K., and Gressner, A. M. (2000) Hepatology 31, 1094-1106[CrossRef][Medline] [Order article via Infotrieve]

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