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From the Hormone Research Center, School of Biological Sciences and Technology, Chonnam National University, Gwangju 500-757, South Korea and || Division of Diabetes, Endocrinology, and Metabolism, Department of Medicine, Baylor College of Medicine, Houston, Texas 77030
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Although yeast two-hybrid methods are suitable for the sensitive detection of protein/protein interactions in vivo, these interactions need to be tested in the relevant biological system. For such functional analyses, identification of missense mutations that specifically disrupt the interaction with a given partner (loss-of-interaction mutants) is very helpful for determining the functional significance and molecular basis of the interaction. Modified yeast two-hybrid systems, known as "reverse two-hybrid" or "split hybrid" systems, have been developed to rapidly isolate mutant prey proteins that are specifically defective in the interaction with a potential partner (4–7). The "reverse two-hybrid system" uses a URA3 reporter gene as a counterselective marker that, as with the yeast two-hybrid system, is activated by bait/prey interactions. In this system, expressed Ura3p inhibits growth of the cells on medium containing 5-fluoroorotic acid, which Ura3p converts into a toxic compound (5). In the "split hybrid system," the two-hybrid interaction of two proteins results in the expression of the TetR repressor. TetR subsequently binds to the tet operators, blocking expression of the HIS3 reporter gene and preventing yeast growth in medium lacking histidine (7). Thus, both systems are specifically designed for positive selection of interaction-defective mutants using specific counterselection markers, and they can be used to identify events that dissociate protein/protein interactions (8).
Despite the advantages of these modified two-hybrid methods in selecting non-interactors, they have two major technical obstacles, which prohibit a wider usage of these methods. First, they were not designed to positively select for informative missense mutations among interaction-defective alleles isolated by split hybrid or reverse two-hybrid screening. In the split hybrid system, for example, the prey protein is expressed as a triple fusion between the VP16 activation domain and ß-galactosidase to allow identification of uninformative mutations (truncation) as white colonies on 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (X-gal) plates, corresponding to a negative color selection for the missense mutants (7). In the reverse two-hybrid systems, several alternative strategies have also been used to isolate non-interacting full-length alleles by adding easily detectable C-terminal fusions such as green fluorescent protein (9–11) or an epitope tag (12) to prey proteins. In these systems, interruption of the prey by an uninformative mutation is indicated by green fluorescence or immunoblot analysis for the epitope. Accordingly these methods cannot positively select the missense mutants. Because greater than 97% of counterselected non-interacting alleles are expected to contain uninformative mutations (6, 7, 13), isolation of the small portion of full-length alleles from a large library of mutant alleles remains a major technical hurdle. Second, it has been reported that a high background of false positives (more than 65%) is generally produced during the first counterselection step of reverse two-hybrid screening (12, 13). This might be due to the spontaneous inactivation of the counterselection system itself, including the loss of marker gene function or bait plasmid. All these observations strongly suggest that negative selection of full-length allele (detectable C-terminal fusion) after counterselection of non-interactor (reverse two-hybrid screening) is not an effective strategy to isolate specific missense alleles from a randomly generated mutant library.
Recently Gray et al. (13) reported an alternative method designed to generate a full-length, high coverage allele library based on in vitro recombinational cloning and positive selection of full-length clones in Escherichia coli. In this method, mutagenized prey proteins are expressed as a C-terminal fusion of the kanamycin resistance gene to confer antibiotic resistance in E. coli to full-length clones. Accordingly this system requires two in vitro recombinational cloning steps and generation of a full-length allele library in E. coli prior to isolating non-interacting mutants using the yeast reverse two-hybrid system. In addition to this technical complication, more than 60% of non-interactors recovered from reverse two-hybrid screenings have turned out to be false positives. All these technical problems explain why we still need a more rapid and efficient system for the positive selection of full-length alleles as well as a simple manipulation method for allele library generation.
Many nuclear receptors (NRs) function as ligand-regulated transcription factors. NRs control numerous critical biological events, including development, growth, differentiation, and homeostasis (14–16). In the absence of ligand, the apo form of the NR recruits transcriptional corepressor proteins, such as the nuclear receptor corepressor (N-CoR) and silencing mediator for the retinoid and thyroid hormone receptor (SMRT), to repress the transcription of target genes (17, 18). N-CoR and SMRT are modular proteins that contain three independent autonomous repression domains (RDs) and two separate NR interaction domains (IDs) located at their N-terminal and C-terminal regions, respectively (19, 20). IDs have been shown to directly interact with the ligand-binding domains (LBDs) of the apo form of non-steroid receptors as well as antagonist-bound steroid receptors (21–25).
As the molecular determinants required for NR interactions by corepressors, the corepressor nuclear receptor (CoRNR) box and the related extended helix motif of the consensus sequence LXX(I/H)IXXX(I/L), where X is any amino acid, have been identified within IDs of N-CoR and SMRT (26–28). Perissi et al. (28) have suggested that the corepressor motif adopts a three-turn 
-helix, as compared with the two-turn helix NR-interacting motif (LXXLL) of coactivators, and interacts with specific residues in the LBD pocket that largely overlap with those residues required for binding to the coactivator LXXLL motif. Interestingly NRs have the ability to distinguish the IDs of N-CoR and SMRT for their specific interactions, determining corepressor preference (N-CoR versus SMRT) or the ID preference (ID1 versus ID2) of a given NR (29, 30). For example, TR preferentially recruits N-CoR via its specific interaction with ID3, which is absent in SMRT (31, 32), and the orphan NR RevErb selectively interacts with N-CoR-ID1 (30). In contrast, the retinoic acid receptor (RAR) binds to ID1 of SMRT, whereas the AF2 deletion form of the retinoid X receptor (RXR
AF2) and liver X receptor (LXR) interact exclusively with the ID2 of corepressors (30, 33). Although it is quite likely that some residues within or outside of the extended helix motifs may contribute to these specificities, the molecular basis of this selective and specific binding of IDs of N-CoR and SMRT to different NRs is not fully understood.
Here we report a novel yeast genetic method, the "one- plus two-hybrid system," that efficiently selects for missense mutations that specifically disrupt a known protein/protein interaction. This system consists of dual reporter systems in which a one-hybrid system is first used for a positive selection of full-length alleles from a randomly generated mutant library and a two-hybrid reporter system is used for the second screening of interaction-defective mutants among the isolated missense mutants. In a first demonstration of this method, we successfully identified the specific amino acid residues of N-CoR-IDs that are either generally required for optimal NR binding (general determinants) or involved in the preferential interaction with a particular NR (specific determinants).
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, TR, RXR-LBDAF2, LXR, and LXRß and mouse PPAR
-LBD) (36, 37) into the appropriate sites of pRS325LexA (for mutant screening), pcDNA3HA vector (for in vitro translation), and pCMX-Gal4N (for mammalian expression) (38). For the construction of pUASGAL-HISi-1, the DNA fragment containing the upstream activating sequence of the GAL1–10 promoter (UASGAL) was obtained from pLGSD5 (39) and cloned into the EcoRI/XbaI site of pHISi-1 (MATCHMAKER, Clontech). All constructs were verified by DNA sequencing.
Yeast Strain YOK400—
Yeast strain YOK400 (MAT, leu2, trp3, ura3, lexAop-LEU2, UASGAL-HIS3) for "one- plus two-hybrid screening" was constructed by genetic manipulation of strain EGY48 (40). To generate the chromosomal reporter gene (UASGAL-HIS3) used for one-hybrid screening, EGY48 was transformed with AflII-linearized pUASGAL-HISi-1 (1 µg) and plated on galactose medium lacking histidine, resulting in the integration of the UASGAL region upstream of the chromosomal HIS3 gene. YOK400 strains generated by correct recombination were selected by their growth on galactose medium lacking histidine but not on glucose medium lacking histidine after a 4-day incubation at 30 °C. To check the 3-amino-1,2,4-triazol (3AT; a competitive inhibitor of His3p) resistance of the strain, five to six transformants were serially spotted to check their growth on galactose medium containing 25, 50, or 100 mM 3AT. The transformant showing the highest resistance to 3AT was chosen as the YOK400 strain.
Yeast Two-hybrid Test—
Yeast strain EGY48 containing the pSH18-34 plasmid (8XlexAop-LacZ reporter) (34) was co-transformed with bait plasmids expressing LexA-fused NR (cloned into pEG202 or pRS325LexA vectors) and prey vectors expressing corepressor-ID fusions between B42 and GBD by the standard lithium acetate method (41). Plate and liquid assays for ß-galactosidase activity of three or more transformants were carried out as described previously (35). Similar results were obtained in multiple repeated experiments.
Mutagenic PCR—
Random mutagenesis of N1 and N2 fragments was performed using the Mn2+-mediated PCR mutagenesis method (42) with 30 rounds of PCR (94 °C for 30 s, 55 °C for 30 s, and 72 °C for 60 s) using Taq polymerase and pRS424UB42-N1-GBD or pRS424UB42-N2-GBD as templates in the presence of 0.1 mM MnCl2. Two oligonucleotides were designed as universal primers for mutagenic PCR of the prey gene: forward primer (oligo-SF), 5'-CC AGC CTC TTG CTG AGT GGA GAT G-3', matching the C-terminal region of the B42 activation domain, and reverse primer (oligo-SR), 5'-CGG TTT TTC TTT GGA GCA C-3', corresponding to an N-terminal portion of GBD. The mutagenic PCR products obtained with these primers commonly contained about 100 bp of flanking region at each end with sequence identities to the gap plasmid prepared by EcoRI/BamHI digestions of pRS424UB42-GBD.
One- plus Two-hybrid Screening—
To construct the mutant cell library of randomly mutated N1 or N2, we used a single step method based on the in vivo gap repair (43). Each of the mutagenic PCR products (1 µg) was co-transformed with the gap plasmid (4 µg) into strain YOK400 carrying the pSH18-34 reporter as well as the bait plasmid pRS325LexA-RAR or -RXRAF2. His+ transformants were obtained after a 4-day incubation at 30 °C on glucose medium containing 10 mM 3AT and lacking histidine. More than 1000 transformants were picked onto plate medium containing X-gal but lacking histidine, and the yeast colonies showing a white or weak blue color were isolated from the wild-type blue colonies. These candidates were retested for color phenotype by streaking on X-gal plates and subjected to the liquid ß-galactosidase assay for the selection of non-interacting mutants based on quantitative data. Prey vectors were rescued from the mutant candidates and individually transformed into the EGY48 strain expressing LexA-NR (for the test of two-hybrid interaction) as well as the EGY-LG strain containing the pLGSD5 plasmid (UASGAL-LacZ reporter) to check for intact GBD (one-hybrid test). Prey plasmids that still conferred blue color in the one-hybrid test and white color in the two-hybrid test were chosen as the final mutant candidates and subjected to DNA sequencing to identify the mutational site(s).
Preparation of Whole Cell Extracts and Immunoblot Analysis—
All protein manipulations were carried out at 4 °C in the presence of protease inhibitors (1 mM PMSF, 2 mM benzamidine, 5 µg/ml leupeptin, 5 µg/ml pepstatin). Yeast protein extracts were prepared by cultivating the cells in the appropriate medium to midlog phase followed by glass bead disruption as described previously (44). Preparation of the whole cell extract from transiently transfected HEK293 cells was performed as described previously (45). Protein concentrations of the whole cell extracts were determined using a Bradford protein assay kit (Bio-Rad). Equal amounts of protein samples (40 µg for the yeast extract and 80 µg for the mammalian cell extract) were separated on either 10% or 12% SDS-polyacrylamide gels and transferred to Hybond-ECL nitrocellulose membranes (Amersham Biosciences). Membranes were probed with monoclonal antibodies against LexA (sc-7544; Santa Cruz Biotechnology), polyclonal antibodies against GBD (sc-510; Santa Cruz Biotechnology), and monoclonal antibodies against hemagglutinin (HA) (12CA5) to detect the expression levels of the target proteins. The blots were developed with the Amersham Biosciences ECL kit according to the instruction manual.
In Vitro GST Pulldown Assay—
pGEX4T-1 derivatives expressing the wild-type and N1 or N2 mutants were introduced in E. coli strain DH5. GST alone or GST-fused proteins were overexpressed by induction for 3 h in the presence of 0.25 mM isopropyl ß-D-thiogalactopyranoside and purified with the use of glutathione-agarose beads (Promega) according to the manufacturer's instructions. NR proteins were synthesized by in vitro translation of pcDNA3HA-based NR constructs using the TNT transcription-coupled translation system (Promega). The radiolabeled NR proteins were added to similar amounts of GST or GST-fused proteins (2–4 µg) bound to glutathione-agarose beads pre-equilibrated with buffer A (50 mM Tris-HCl (pH 7.9), 5% glycerol, 1 mM EDTA, 1 mM dithiothreitol, 1x protease inhibitor, 0.01% Nonidet P-40, 150 mM KCl) in a final volume of 250 µl. The beads were washed three times in the same buffer, and the bound radiolabeled proteins were analyzed by SDS-PAGE followed by autoradiography.
Cell Culture and Transient Transfection Assay—
HEK293 cells were grown in 24-well plates with Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum for 24 h and transiently transfected with the appropriate set of reporter and expression plasmids using SuperFect reagent (Qiagen). The total amounts of expression vectors were kept constant by adding appropriate amounts of pcDNA3HA. After 24 h, the cells were harvested and assayed for luciferase activity as described previously (35). The results from triplicate samples were averaged and normalized to LacZ expression from pSV-ß-gal. The plasmid DNAs used for transfection included the Gal4-TK-LUC luciferase reporter (200 ng/well), the pSV-ß-gal control plasmid, pCMX-Gal4N-RAR or -RXRAF2, and the pcDNA3HA-N1 or -N2 wild type or mutants for the dominant negative assay as indicated in the figure legends.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Interaction Profile of NRs with Corepressor-IDs in the Yeast Two-hybrid Assay—
First we investigated the interaction pattern between IDs of corepressors and various NRs in the yeast two-hybrid assay to confirm the biological significance of studying NR/corepressor interactions in yeast. The small fragments containing the extended helix motif of either ID1 (N1) or ID2 (N2) of N-CoR were used as prey in the two-hybrid interaction (Fig. 2A). As shown in Fig. 2B, N1 and N2 correspond to 60-amino acid polypeptides in which the conserved helix motifs involved in NR interaction are centrally located. Similarly the equivalent regions of ID1 and ID2 of SMRT (called S1 and S2, respectively) were utilized to test their interactions with various NRs. All ID fragments were inserted between the B42 and GBD regions of the pRS424UB42-GBD vector to use as prey proteins in the two-hybrid interaction assay as well as in the mutant screening assays, whereas the NR baits were expressed as LexA fusions.
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and RAR were much stronger than those of RevErb, PPAR, and especially RXR. In the case of RXR, we used the RXR mutant lacking the AF2 domain (RXRAF2) because deletion of the AF2 domain dramatically increases the ability of RXR to interact with N-CoR in vitro and in vivo (46). TR and RAR bound to both ID1 and ID2 of corepressors but interacted more strongly with ID1 (Fig. 2C). RXRAF2 and PPAR interacted primarily with N2 and S2 with similar strengths (i.e. no corepressor preference). In contrast, RevErb showed strong interaction with N1 and S1 but not with N2 or S2 (Fig. 2C). In the quantitative assays for these interactions, we obtained a pattern of NR/corepressor interactions similar to those obtained in the X-gal plate assay (Fig. 2, C and D). These results indicate an apparent linear relationship between X-gal plate and ß-galactosidase assays under our experimental conditions that would explain the significant differences (more than 10-fold) in the interactions of the corepressor-ID with RAR and RXRAF2 in a liquid ß-galactosidase assay (Fig. 2D). In particular, we observed a clearer corepressor preference of some NRs in the quantitative assay; the TR showed obvious N-CoR preference, and N-CoR interactions with RevErb and PPAR were stronger than those of SMRT (Fig. 2D). However, unlike the previous observation, RAR interacted somewhat better with N-CoR than with SMRT in the yeast system (29, 30). From these results, we concluded that the specific interaction profiles between the IDs of co-repressors and the NRs described in the mammalian system can be recapitulated in yeast.
Control Experiments for One- plus Two-hybrid Screening—
We used the one- plus two-hybrid screening system to identify loss of interaction mutants between RAR and N-CoR-ID1 (N1) and between RXRAF2 and N-CoR-ID2 (N2). Because these two examples correspond to the strongest and the weakest interactions among the ID/NR interactions we tested (Fig. 2C), it provides a means to validate the broad scope of our system for detecting protein/protein interactions with a wide range of binding affinities. Before the actual screening, a series of preliminary control experiments were performed to functionally test the bait and prey fusion proteins and the utility of the reporter constructs. We introduced the B42-N1-GBD or B42-N2-GBD prey plasmids into the EGY-LG strain carrying the episomal UASGAL-LacZ reporter (pLGSD5) to functionally test the triple fusion by checking for the blue color phenotype on X-gal plates. The absence of autonomous transactivation function by LexA-RAR or -RXRAF2 was also tested in strain EGY48 by checking for the white phenotype on X-gal plates.
Strain YOK400 expresses a very low level of His3p even in glucose medium (repressive condition). To establish the growth conditions that minimize the basal level of growth for the YOK400 strain and permit the growth of cells expressing only the intact triple fusion prey, we determined the optimal concentration of 3AT, a competitive inhibitor of His3p, for the actual screening. Strain YOK400 was transformed with plasmids expressing B42-N1-GBD, B42-N2-GBD, or B42 alone, and the resulting transformants were grown in a series of synthetic glucose/His– medium containing different concentrations of 3AT (0, 5, 10, and 20 mM). In the presence of 10 mM 3AT, the growth of yeast cells expressing B42-N1-GBD or B42-N2-GBD was barely affected, whereas the growth of cells expressing the B42 domain alone was completely inhibited, indicating that 10 mM was the optimal 3AT concentration for the positive selection of the intact prey fusion (His+ phenotype).
Validation for the Enrichment of Full-length Clones after One-hybrid Selection—
To verify how effectively our one-hybrid system eliminates truncation mutants and enriches for full-length alleles, a randomly mutagenized allele library for the N2 fragment was constructed in the YOK400 strain. We adopted a PCR-mediated random mutagenesis and gap repair-recombination method to generate the mutant cell library in one step, which dramatically accelerates the entire screening process (42, 43). Mutagenic PCR products of N2 were generated using specific primers corresponding to portions of the B42 and GBD regions in the presence of 0.3 mM MnCl2 and co-transformed along with the linearized gap plasmid into strain YOK400. The introduced mutagenic PCR products served as templates for copying into the gap plasmid by the in vivo gap repair system of Saccharomyces cerevisiae (43). The transformants were selected on minimal medium plates containing histidine, and 600 transformants were patched onto duplicate medium plates containing histidine or onto plates lacking histidine but containing 10 mM 3AT. Among 600 transformants, only 92 colonies (15%) were not viable on medium plates lacking histidine (His– phenotype) under these conditions. Plasmids were successfully rescued from 50 colonies displaying the His+ phenotype and from 43 colonies showing the His– phenotype. All the plasmids were retransformed into the EGY-LG strain to confirm the one-hybrid interaction (intactness of prey) and subjected to DNA sequencing.
Fig. 3 shows the overall distribution of the mutations detected within the mutagenized open reading frame (102 amino acids) of the N2 fragment partially flanked by B42 and GBD. Among the 50 clones conferring the His+ phenotype, 27 clones had single or double substitution mutations (including five silent mutations), and 23 clones were identified as wild type (Fig. 3A). The mutations were uniformly distributed, and the mutation frequency was 2.3 mutations/kb of DNA under these PCR conditions. Importantly there were no nonsense or frameshift mutations among the 50 clones recovered from the colonies showing the His+ phenotype. Conversely 42 of the 43 clones conferring the His– phenotype had nonsense or frameshift mutations. Among these mutations, 24 and 10 mutations were single base pair deletions and insertions, respectively, and eight mutations were nonsense mutations (Fig. 3B). However, one clone from 43 colonies showing the His– phenotype (2%) turned out to be a substitution mutant (false positive). The prey for the false positive clone was found to be intact by retransforming the clone into EGY-LG and YOK400 strains, suggesting that the false positive clone might be due to spontaneous inactivation of the HIS3 reporter system in this cell. All these results clearly demonstrate that our modified one-hybrid system designed for the selection of full-length alleles effectively eliminates uninformative nonsense or frameshift mutations that constitute the bulk of the unwanted mutations generated by random mutagenesis.
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AF2—AF2 plasmids and the episomal two-hybrid reporter (lexAop-LacZ) plasmid. As a control, mutagenic PCR products generated with or without 0.3 mM MnCl2 were also co-transformed. The transformants were grown in synthetic glucose medium containing 10 mM 3AT but lacking histidine for the positive selection of intact prey fusions. Among the surviving transformants, non-interacting mutants could be easily selected by isolating white colonies on X-gal plates. Table I shows the results of screening for interaction-defective mutants of N1 and N2. As predicted, in both cases, the data revealed a gradual increase in the mutation rate (as inferred from the occurrence of white colonies) with increased concentrations of MnCl2 in the PCR. Consistent with this, the total number of transformants decreased with the introduction of PCR products generated under increased MnCl2 concentrations, confirming the elimination of uninformative mutations in this step. In the control transformations, all of the tested colonies transformed with the supercoiled wild-type prey plasmid were blue in color. In addition, the number of colonies transformed by the gap plasmid alone was very small compared with the number co-transformed with PCR products (less than 0.1%), and all were blue in color, suggesting they were generated by the incompletely digested supercoiled plasmid. These observations enabled us to conclude that our system works as designed.
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Identification of Diverse N-CoR Mutants Defective in RAR or RXR Interactions—
Table II shows the mutational sites and the changed amino acids found in the isolated N1 and N2 mutants defective in interactions with RAR and RXRAF2, respectively. In the case of N-CoR-ID1, almost all of the identified amino acid residues required for RAR interaction were located within the extended helix motif (Leu+1, Ile+5, Cys+6, Ile+8, Ile+9). Interestingly residue Phe+13 was also identified as an absolute requirement for the RAR interaction, although it is located downstream of the core motif. Among these residues, Cys+6 appeared to act as a minor determinant for RAR binding because the C+6S mutant had significant binding activity (Fig. 4A). Although proline substitution mutants were also isolated as the sole mutants for His+4 and Gln+7 residues, we did not regard them as informative because proline is known to act as a helix breaker (data not shown). Importantly most of the N1 mutations were recovered multiple times, and more than one amino acid change was observed at Leu+1 (to His, Arg, or Pro), Ile+5 (to Asn or Thr), and Ile+9 (to Phe or Thr), indicating that the screen was carried out under or near saturating conditions. In the case of the N2 mutants, although the number of final mutants was somewhat smaller than for N1, we could identify specific residues located within the extended helix motif (Glu+2, Ile+5, Arg+6, Ala+8, and Leu+9) as determinants of the RXR interaction (Table II and Fig. 4B). Among these, changes to multiple amino acids were observed at residues Ile+5 (to Ala, Asn, Thr, or Val) and Ala+8 (to Thr or Val). In addition to these, an M+10P mutant was also isolated but was regarded as uninformative as described above. In conclusion, using our unbiased genetic selection system, we successfully identified multiple, informative N-CoR-ID mutations that disrupt NR interactions and found that almost all of them are located within the conserved NR interaction motif of the N-CoR as predicted.
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We also evaluated the interactions of N2 mutants with RAR, TR, and PPAR in addition to RXR in the yeast two-hybrid assays (Fig. 4B). Intriguingly we observed a similar, but not identical, binding pattern relative to that of N1. First we consistently observed strong and general effects on NR binding by substitutions at Ile+5 and Arg+6, indicating that these residues are general determinants of NR binding by N-CoR-ID2. In contrast, mutations at residues Glu+2 and Ala+8 showed differential effects on NR binding depending on the NR member. This was shown by the observation that the E+2V and A+8T mutants were able to interact to some extent with TR, RAR, and PPAR but not with RXRAF2 (Fig. 4B). Interestingly all of the tested NRs except for TR showed greatly reduced interactions with the L+9I mutant, although Leu or Ile is generally found in this location of corepressors. We consider the residue Leu+9 to be a general rather than a specific determinant because another substitution mutant of this residue (L+9A) has been reported to have a complete and general defect on NR binding (28). The expression level of each N2 mutant was also confirmed by Western blot analysis (Fig. 4D).
In Vitro Interactions of N-CoR-ID Mutants with Various NRs in GST Pulldown Assays—
To verify the molecular determinants for N-CoR/NR interactions, we investigated the in vitro interactions between N-CoR-ID mutants and NRs using GST pulldown analysis (Fig. 5). We prepared NRs as 35S-labeled proteins by in vitro translation and examined their binding to GST-fused N1 or N2 derivatives. As shown in Fig. 5A, N1 mutants carrying a substitution at residue Leu+1, Ile+5, Ile+8, or Ile+9 displayed no detectable interactions with the tested NRs, confirming the yeast two-hybrid data identifying these residues as general determinants of ID1/NR interactions (Fig. 4A). Moreover the F+13S mutant partially interacted with TR and RevErb but not with RAR, defining the Phe+13 residue as a specific determinant for NR binding. Inconsistent with the yeast data, however, the mutation at residue Cys+6 had only a partial or negligible effect on interactions with TR and RevErb, suggesting that the Cys+6 residue acts as minor determinant of the NR interaction in vitro (Fig. 5A).
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in the analysis for N-CoR-ID2 (data not shown). In the case of RXRAF2 and TR, N2 binding was absolutely dependent on residues Ile+5 and Arg+6, again defining these residues as general requirements for the N-CoR-ID2/NR interaction. In contrast, the mutations at residues Glu+2, Ala+8, and Leu+9 showed a negligible effect (Glu+2) or partial defects (Ala+8 and Leu+9) in TR binding as well as significant defects in the RXRAF2 interaction, which is roughly consistent with the yeast two-hybrid data (Figs. 4B and 5B). Because it was also reported that LXR interacts with ID2 of corepressors (33), we performed GST pulldown analysis to examine the interactions of LXRs with the isolated N2 mutants. As shown in Fig. 5B, similar bindings were observed between RXRAF2 and LXR and between TR and LXRß. This was shown by the fact that both LXR isoforms had residues Ile+5 and Arg+6 as general requirements for N2 interactions, whereas residues Glu+2, Ala+8, and Leu+9 acted as either minor or specific determinants for N2/LXR interactions depending on the LXR isoform. Collectively both the in vivo and in vitro results consistently indicated that residues Leu+1, Ile+5, Ile+8, and Ile+9 of N1 and Ile+5, Arg+6, and Leu+9 of N2 are commonly required for optimal NR binding (general determinants), whereas residues Cys+6 and Phe+13 of N1 and Glu+2 and Ala+8 of N2 are differentially important for NR binding, depending on the specific NR member (specific determinants).
Functional Tests of the Isolated N-CoR-ID Mutants in Mammalian Cells—
To establish the functional properties of the individual N-CoR-ID mutants in the relevant biological system, we tested their effects on NR function in mammalian cells. Because the predominant role of corepressors is associated with the repressive function of unliganded NRs, we examined the effects of overexpression of N-CoR-ID fragments on NR-mediated repression of a reporter gene (dominant negative effect). In a control experiment, we transiently co-transfected pCMXGal4-RAR and the luciferase reporter gene driven by the upstream Gal4-binding site (Gal4-TK-LUC) into HEK293 cells. As expected, Gal4-RAR showed a 2.5-fold repression of reporter gene expression, and this repressive effect was reversed by the overexpression of the wild-type N1, indicating that the N1 fragment specifically competes with endogenous corepressors (Fig. 6A). In contrast, the N1 fragments containing single mutations at residues Leu+1, Ile+5, Ile+8, Ile+9, or Phe+13 had almost no effect on RAR-mediated repression, indicating that these mutants are unable to interact with RAR. Interestingly overexpression of the C+6S mutant resulted in derepression of the reporter gene activity, implying that this mutant specifically interacts with RAR as in the GST pulldown assay (Figs. 5A and 6A). In similar experiments with N2 mutants, we observed a significant dominant negative effect on RXRAF2-mediated repression of the reporter gene by the overexpression of the N2 fragment (Fig. 6B). In contrast, none of the tested N2 mutants could relieve the repressive activity of Gal4-RXRAF2, suggesting that none of these mutants are able to interact with RXRAF2 in mammalian cells (Fig. 6B). Western blot analysis revealed that the differences in reporter gene activity were not due to differences in the expression levels of the N1 or N2 mutants (Fig. 6, C and D). All these results clearly demonstrate that N-CoR-ID mutants isolated by the yeast one- plus two-hybrid system show identical functional properties in a mammalian system.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Molecular Determinants of N-CoR-IDs for NR Interactions—
Two NR interaction motifs of N-CoR share the consensus sequence LXX(I/H)IXXX(I/L), which has amphipathic properties. These motifs are predicted to adopt an extended helix by comparison with the known LXXLL motif of coactivators (28). We have identified the residues within the extended helix motif of N-CoR-IDs (Ile+8 for ID1 and Arg+6 for ID2) as novel general determinants for NR binding in addition to the previously identified residues located at the +1, +5, and +9 positions (28, 47). Moreover some specific residues within or flanking the extended helix motifs (Cys+6 and Phe+13 for ID1 and Glu+2 and Ala+8 for ID2) were found to be involved in the preferential interaction with a specific NR. These results confirm that certain specific residues within or outside the extended helix motifs are differentially recognized by the LBD structures specific to NR members, providing a molecular basis for corepressor preference or ID preference shown by target NRs.
Although the crystal structure of the NR·ID1 complex is not currently available, the extended helix motif of N-CoR-ID1 (+1LADHICQII+9) is thought to form a three-turn helical conformation. It has been proposed that residues Leu+1, Ile+5, and Ile+9 of ID1 are on the same face of the helix so that they can make core contact with a common structure imbedded in NR-LBDs (28). We have classified Leu+1, Ile+5, Ile+8, and Leu+9 as the general determinants for the interaction of ID1 with NRs. Consistent with our findings, these residues are conserved within either the extended helix motif (Leu+1, Ile+5, and Leu+9) or the CoRNR motif (Ile+5, Ile+8 and Leu+9) of N-CoR-ID1, reinforcing the general importance of these residues for NR binding. It is unexpected that the Phe+13 residue of ID1 was identified as a specific determinant for NR binding because it lies far outside the extended helical motif. Interestingly Phe+13 is conserved among N-CoR-IDs of different species but not among SMRT-IDs (Tyr+13), suggesting a role for this residue in the N-CoR preference shown by some NRs.
The extended helix motif of N-CoR-ID2 (+1LEDIIRKAL+9) is thought to represent the C-terminal extension form of the CoRNR2 motif (LEDII). Among these, we identified Ile+5, Arg+6, and Leu+9 residues as general determinants for NR binding, whereas Glu+2 and Ala+8 proved to be specific determinants. In the crystal structure of the PPAR-LBD complexed with the antagonist GW6741 and a SMRT-ID2 peptide (47), the extended helix motif (+1LEAIIRKAL+9) of SMRT-ID2 forms a three-turn helix in which Leu+1, Ile+5, and Leu+9 are aligned on the same face of the helix to form the core hydrophobic interaction with the receptor. Considering the near sequence identity between the extended helix motifs of N-CoR-ID2 and SMRT-ID2, this observation supports our conclusion that the residues Ile+5 and Leu+9 of N-CoR-ID2 are of general importance in NR binding. In particular, our screening did not find any mutations at residue Leu+1 probably because of the small number of N2 mutants available, although Leu+1 has been shown by others to be a general determinant for NR binding (26, 28, 47). We generated an L+1R substitution mutant by site-directed mutagenesis and identified this residue as an absolute requirement for the binding of all tested NRs (data not shown).
It is intriguing that residue Arg+6 of N-CoR-ID2 was identified as being a general requirement for NR binding in contrast to the NR-specific role of Cys+6 in N-CoR-ID1. The crystal structure of the PPAR·SMRT-ID2 complex reveals that residue Arg+6 of S2 forms a strong intramolecular hydrogen bond with Asn-303 located in helix 4 of the PPAR-LBD (47). Consistent with this, the Asp-295 residue of RXR, which is equivalent to Asn-303 of PPAR, is also required for optimal interaction of S2 with RXR (48). These observations strongly support our finding that the Arg+6 residue of N2 is also generally required for NR binding in addition to the residues involved in core hydrophobic interactions with NRs (Leu+1, Ile+5, and Leu+9).
We hope that these corepressor mutants showing differential binding specificities for various NRs will facilitate the elucidation of the physiological roles and the molecular basis of the repressive functions of specific NRs. In addition, identification of specific residues that mediate high affinity binding between NRs and corepressors will be helpful in dissecting the complex network of corepressor/NR interactions in the absence of the crystal structures for NR·corepressor complexes.
One- plus Two-hybrid System—
The one- plus two-hybrid system is designed for the rapid and efficient selection of missense mutations that specifically disrupt known protein/protein interactions and has three major advantages over the existing methods. First, this system allows rapid and efficient genetic selection for missense mutants. This advantage originates from the simultaneous operation of dual reporter systems in the same cell and the use of the gap repair system for the one-step construction of a mutant cell library containing only missense mutations. For the reverse two-hybrid systems, several alternative strategies have been used to isolate full-length alleles by adding an easily detectable C-terminal fusion such as ß-galactosidase (7), green fluorescent protein (9–11), or epitope tag (12). In these modified systems, truncation of the prey by an uninformative mutation is indicated by white color phenotype on X-gal plates, absence of green fluorescence, or defective epitope/antibody interactions in immunoblot analysis, indicating that these methods cannot positively select for the non-interacting full-length alleles. To circumvent this technical obstacle, Gray et al. (13) developed a novel method specifically designed to generate full-length, high coverage allele libraries. In this system, mutagenized prey proteins are expressed as C-terminal fusions of kanamycin resistance gene, enabling the construction of full-length allele libraries in E. coli by the positive selection of antibiotic-resistant colonies. To operate, this system requires two in vitro recombinational cloning steps and the generation of a full-length allele library in E. coli prior to isolating the non-interacting mutants by the yeast reverse two-hybrid system. This indicates that this strategy has more methodological complexity and difficulties in the construction of a full-length allele library compared with our system using the modified one-hybrid system in yeast. Thus, our one- plus two-hybrid system is the first positive selection system for full-length alleles fortified with the one-step construction of full-length allele library in yeast.
Second, no or a low level of background of false positives was generated in the first positive selection of the full-length alleles as well as in the second screening of the non-interactors by the one- plus two-hybrid system. There was no truncation mutant among the 50 transformants with the His+ phenotype during the selection of the full-length allele (Fig. 3A), indicating that our modified one-hybrid system works well and is relatively stable. We recommend that the YOK400 strain be made freshly or carefully checked for the His– phenotype before initiating each round of screening to minimize the generation of false positives in the first selection step of the full-length clones. In a second screening step for interaction-defective prey mutants, we found that only six of 42 N1 and three of 17 N2 mutant candidates were false positives (less than 20%). This result is in contrast to reports indicating that reverse two-hybrid systems generally create a high background of false positive (more than 65%). This discrimination might be due to differences in the stabilities of two-hybrid reporter systems utilized by these methods.
Third, the one- plus two-hybrid system can be generally applied for the characterization of a wide spectrum of protein/protein interactions without knowledge of the specific system or mechanism involved in the interactions. For example, we have successfully identified residues that are important for NR-specific interactions within the NR interaction motifs of various coactivator proteins.2 In addition, we have discovered novel motifs within RD3 of corepressors that mediate the interactions with class II histone deacetylases3 and have mapped the amino acid residues required for the direct interaction between the two subunits of the activating signal cointegrator 2-containing complex (45).4 These (unpublished) examples using the one- plus two-hybrid system prove that our system is suitable for general applications as well as for systematic applications on a larger scale. Notably our system can also be used to analyze the interaction interfaces of DNA-binding proteins using dual one-hybrid reporter systems: one for the generation of full-length allele library and the other for the isolation of interaction-defective mutants.
However, our system may have two potential limitations. First, because the prey protein is expressed as a triple fusion between B42 and GBD, a complex protein whose proper three-dimensional structure is impaired by B42 and GBD fusions may not be a suitable target for mutant screening. Second, another potential limitation for the general use of our system is a restriction in the length of the prey protein because of the difficulty to maintain the optimal mutation rate with increasing size of the prey. Thus far, we have successfully analyzed prey proteins of up to 200 amino acids.
Finally our "one- plus two-hybrid method" could be improved by introducing a positive selection system for non-interactors as has already been tried in the reverse or split hybrid systems. For example, instead of using the episomal LacZ reporter, a yeast counterselectable marker, such as URA3 or CYH2, could be used in the two-hybrid screen for simultaneous double positive selection of missense mutations and non-interacting mutants.
In conclusion, the one- plus two-hybrid system rapidly and efficiently generates missense mutations that can specifically disrupt a known protein/protein interaction without knowledge of the specific system or mechanism involved in the interaction. The use of loss-of-interaction mutants will be very helpful in determining the functional significance of their interactions in the relevant biological system and understanding the underlying molecular mechanism for their interactions. More importantly, information regarding the residues involved in the interactions and the effects of various mutations on these residues will be critical for the molecular modeling of the protein/protein interactions.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Published, MCP Papers in Press, July 2, 2007, DOI 10.1074/mcp.M700079-MCP200
1 The abbreviations used are: DBD, DNA-binding domain; NR, nuclear receptor; N-CoR, nuclear receptor corepressor; SMRT, silencing mediator for the retinoid and thyroid hormone receptor; LBD, ligand-binding domain; AF2, activation function 2; RD, repression domain; ID, interaction domain; CoRNR, corepressor nuclear receptor; GBD, Gal4 DNA-binding domain; N1, N-CoR-ID1; N2, N-CoR-ID2; S1, SMRT-ID1; S2, SMRT-ID2; RAR, retinoic acid receptor; TR, thyroid hormone receptor; RXR, retinoid X receptor; LXR, liver X receptor; PPAR, peroxisome proliferator-activated receptor; UAS, upstream activating sequence; 3AT, 3-amino-1,2,4-triazol; X-gal, 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside; HA, hemagglutinin; HEK, human embryonic kidney. 
2 Y. L. Son and Y. C. Lee, unpublished data.
3 M. J. Park and Y. C. Lee, unpublished data.
4 M. J. Kang, Y. C. Lee, and J. W. Lee, unpublished data.
* This work was supported in part by Korea Research Foundation Grant KRF-2002-070-C00068) and Chonnam National University Program 1999 (to Y. C. L.) and National Institutes of Health Grant DK064678 (to J. W. L.). 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.
Both authors contributed equally to this work.
¶ Supported in part by the Second Stage BK21 Program.
** To whom correspondence should be addressed: Hormone Research Center, School of Biological Sciences and Technology, Chonnam National University, 300 Yongbong-dong, Buk-gu, Gwangju 500-757, South Korea. Tel.: 82-62-530-0909; Fax: 82-62-530-0500; E-mail: yclee{at}jnu.ac.kr
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