Identification and characterization of four novel ... - Wiley Online Library

Identification and characterization of four novel peptide motifs that recognize distinct regions of the transcription factor CP2 Ho Chul Kang1, Bo Mee Chung1, Ji Hyung Chae1, Sung-Il Yang2, Chan Gil Kim3 and Chul Geun Kim1 1 Department of Life Science, Hanyang University, Korea 2 Department of Pharmacology, Konkuk University, Korea 3 Department of Biotechnology, Konkuk University, Korea

Keywords CP2; gene regulation; peptide motif; phage display; protein–protein interaction; transcription factor Correspondence C. G. Kim, Department of Life Science, Hanyang University, Haengdangdong 17, Sungdong-gu, Seoul 133-791, Korea Fax: +82 2 2296 5996 Tel: +82 2 2290 0957 E-mail: [email protected] (Received 17 December 2004, revised 6 January 2005, accepted 10 January 2005) doi:10.1111/j.1742-4658.2005.04564.x

Although ubiquitously expressed, the transcriptional factor CP2 also exhibits some tissue- or stage-specific activation toward certain genes such as globin in red blood cells and interleukin-4 in T helper cells. Because this specificity may be achieved by interaction with other proteins, we screened a peptide display library and identified four consensus motifs in numerous CP2-binding peptides: HXPR, PHL, ASR and PXHXH. Protein-database searching revealed that RE-1 silencing factor (REST), Yin-Yang1 (YY1) and five other proteins have one or two of these CP2-binding motifs. Glutathione S-transferase pull-down and coimmunoprecipitation assays showed that two HXPR motif-containing proteins REST and YY1 indeed were able to bind CP2. Importantly, this binding to CP2 was almost abolished when a double amino acid substitution was made on the HXPR sequence of REST and YY1 proteins. The suppressing effect of YY1 on CP2’s transcriptional activity was lost by this point mutation on the HXPR sequence of YY1 and reduced by an HXPR-containing peptide, further supporting the interaction between CP2 and YY1 via the HXPR sequence. Mapping the sites on CP2 for interaction with the four distinct CP2-binding motifs revealed at least three different regions on CP2. This suggests that CP2 recognizes several distinct binding motifs by virtue of employing different regions, thus being able to interact with and regulate many cellular partners.

CP2 was discovered initially in mouse as a transcription factor that binds to and stimulates transcription from the a-globin promoter [1]. Initially, CP2 was known to be a homologue of Drosophila Grainyhead (Grh, also known as NTF-1 or Elf-1) [2–9]. However, recent identification of the mammalian and Drosophila homologue genes of CP2 and Grh revealed that CP2 and Grh belong to separate phylogenetic groups [10– 13]. CP2 has also been found in other organisms as

diverse as Drosophila, chicken and human, comprising a highly conserved family. CP2 is named differently depending upon species: for instance, dCP2 [10,12] in Drosophila, cCP2 in chicken [14], and LBP-1c ⁄ LSF in human [15]. Six isoforms (LBP-1a, -1b, -1c, -1d, -9 and -32) of human CP2 proteins have been identified [15– 17], whereas four CP2 isoforms (CP2a ⁄ NF2d9, CP2b, CP2c ⁄ CP2 and CRTR-1) have been reported in mice [10,18,19].

Abbreviations APP, b-amyloid precursor protein; BSA, bovine serum albumin; CKII-a, casein kinase II-a; EMSA, electrophoretic mobility shift assay; GST, glutathione S-transferase; HDAC1, histone deacetylase 1; HRP, horseradish peroxidase; LTR, long-terminal repeat; NLS, nuclear localization signal; PEG, polyethylene glycol; PKC-d, protein kinase C-d; REST, RE-1 silencing factor; PIAS1, protein inhibitor of activated STAT1; TFIIE-a, a subunit of transcription factor IIE; TS, thymidylate synthase; YY1, Yin-Yang1.

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Identification of CP2-binding peptide motifs

Numerous data point to the transcriptional activation of a range of genes by CP2, leading to functional diversity of CP2 in various species. In chicken, cCP2 regulates the lens-specific expression of the aA-crystalline gene [14]. In mammals, CP2 contributes to the preferential recruitment of the b-globin locus control region to the c-globin promoter during fetal erythropoiesis [20–25]. CP2 participates in cell-cycle regulation by binding to and thus modulating transcription of the thymidylate synthase (TS) promoter in growth-stimulated human cells of late G1 phase [26–28]. CP2 is also implicated in genetic diseases such as Alzheimer’s disease [29,30]. In addition, CP2 has been shown to be able to modulate transcription from some viral promoters; it stimulates transcription from the viral SV40 major late promoter [31], but represses transcription from HIV-1 long-terminal repeat (LTR) [16]. It has become apparent that these various functions of CP2 are mediated by its interaction with different tissue- or species-specific molecules. Lens-specific transcription of aA-crystalline appears to involve cooperation of cCP2 with a putative lens-specific factor [14]. Binding of CP2 to the stage selector element of the proximal c-globin gene promoter requires the formation of a stage selector protein [24] that is a heteromeric complex between CP2 and NF-E4, a fetal erythroid-specific partner protein [25]. Because Fe65 is a ligand of the Alzheimer’s b-amyloid precursor protein (APP) [32,33] and the aberrations of CP2 and ⁄ or APP in neuronal cells promote neuronal apoptosis in the Alzheimer’s disease brain [34,35], CP2 appears to be linked with Fe65-mediated Alzheimer’s disease. Repression of HIV-1 LTR transcription by CP2 is accomplished via the binding of CP2 to another transcription factor, Yin-Yang1 (YY1), and the resultant recruitment of histone deacetylase 1 (HDAC1) [36,37]. Despite the extensive literature, the discovery of the molecules that bind to CP2 and account for its diverse functions is far from being complete. As a strategy to reveal these molecules, we screened a phage display library that provides a pool of up to 109 random peptides. We were able to isolate a number of CP2interacting peptides and discovered that they contained some new motifs that play an important role in the interaction between CP2 and its binding proteins.

Results Isolation of CP2-binding phage clones through a phage display library screening In an attempt to identify targets with which CP2 could potentially interact, we screened a highly complex M13 1266

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phage library that contains 1.9 · 109 different recombinant phage clones. Each of these phage displays a random 12-mer peptide on its coat, by virtue of its capsid pVIII protein being tethered to the peptide by a (Gly4Ser)3 peptide linker. Each peptide is composed of 12 amino acids randomly encoded by the NNK codon (N ¼ A, C, G, T; K ¼ G, T). To enhance identification of CP2-interacting peptides, a full-length CP2 protein was used as bait for this screening. Several rounds of amplified selection were undertaken on phage that exhibited the ability to bind to GST–CP2. Following each round of selection, the titer of GST–CP2-bound phage (measured as pfu) increased consecutively (data not shown), indicating successful biopanning. A number of phage clones was obtained from this screening. Because GST–CP2 fusion protein was used in this screening (Fig. 1A), we eliminated the phage that may have been bound to GST rather than CP2. To this end, the abilities of each phage to bind to GST–CP2 and GST were compared in ELISA assays as described in Experimental procedures. As shown in Fig. 1B,C, some phage (e.g. clones 01, 04, 09, 11 and 12) bound to GST–CP2 no better than to GST. However, many phage clones, such as 05, 08, 13, 21 and 31, exhibited stronger binding to GST–CP2, indicating that they bound specifically to CP2. Identification of CP2-binding motifs Approximately 100 clones showed highly specific binding to CP2 and were subjected to DNA sequencing for their expressed peptides. Only two clones were found three times; the remaining clones were diverse in terms of their sequences. Searching the PIR-PSD database (http://pir.georgetown.edu/pirwww/search/patmatch. html) revealed that none of these sequences exactly matched those of any known proteins. Interestingly, however, 15 sequences could be categorized into four classes with some common motifs using the clustal w multiple sequence alignment program (http://clustalw. genome.jp); the remainder did not show any common motif. The deduced amino acid sequences of those 15 peptides are shown in Table 1. Class I mostly contains His-X-Pro-Arg (HXPR) amino acids with minor conserved variations between aromatic and imidazole groups His (H), Tyr (Y) and Trp (W). X, indicating any amino acid, was frequently found to be Pro or His. The second motif, found in several other phage clones, is Pro-His-Leu (PHL), usually flanked by Leu, Ala, or Ser. The third and fourth classes exhibit an Ala-Ser-Arg (ASR) and Pro-X-His-X-His (PXHXH) motif, respectively. These data suggest that CP2 can interact with a variety of peptides, each of which FEBS Journal 272 (2005) 1265–1277 ª 2005 FEBS

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A


B

C

Fig. 1. Isolation of phage expressing CP2-binding peptides from a phage display library. (A) Coomassie Brilliant Blue-stained gel of GST–CP2 used as bait in the screening. GST–CP2 was purified by glutathione–agarose affinity chromatography as described in Experimental procedures. (B) Abilities of some phage clones isolated from a random phage display library to bind to GST-CP2. Each phage is designated by a number listed at the bottom of the graph. Considerable numbers of phage clones, including 03, 05, 08, 10, 13, 17, 21, 31 and 54, show specific binding to GST–CP2, whereas others such as 01, 04, 09, 11 and 12 bind equally to GST–CP2 and GST. In each experiment 1.23 · 1012 phage were used and all assays were performed in triplicate. Specific binding to CP2 was obtained by subtracting the absorbance value of background GST binding from that of GST–CP2 binding. The results are presented as mean ± standard error. (C) CP2-binding activity is proportionate to the number of phage. The extent of binding to GST–CP2 or GST was determined by measuring absorbance at 405 nm from ELISA. Table 1. Deduced amino acid sequences of the 12-mer peptides in phage clones that showed highly specific binding to CP2. Standard single letter amino acid abbreviations are used. Conserved amino acids are indicated in bold. Selected clone number

Amino acid sequences

13 26 32 43 50 53 54 61 74 81

HKSHLHFH PPHKH H H H HHFH HQRHH HKPHMH ILGAH HTKEFH

IB

05 69

HERRESNYPQRP AHRSRWSPRPSY

II

21 24 29 35 64

HKFHQHR HKFHQHL H HFKHHKS

L A S L S

P P P P P

H H H H F

L L L L L

A A SHRHLLR G SHRHYVPH

III

03 08 49 18

H H H YDLW

S K Q P

Y H H F

V S H S

A A A A

SRPS SRIH SRVT S

IV

20 31 33 99

W VWKH W GHTHK

P P P P

H H Y Q

H H H H

H H W F

Classes IA

I N T W

T M P N

Y H S S


P H V N P V H G H P

P P P P P P P P P P

R R R R R R R R R R

P Q M A G S K P P G

P H E H N S W I I

H H H H

A K S L O P A K W

T Y P R

M H S V S E

NLQM SHLR PTTP P

P

RLSTV KR AMKKN S

possesses at least one of the four distinct CP2-binding motifs. Despite this carefully controlled screening procedure we remained concerned about the potential existence of false positives in the selected set of peptides, which might originate from the highly complex phage sequences. Therefore, as a second approach to confirm the bona fide association of CP2 with the peptides presented in Table 1, we performed in vitro binding assays using purified peptides instead of phage. Six peptides were chosen for this experiment: five representing one of the four putative CP2-binding motifs and one internal control peptide which had a high binding affinity to GST rather than CP2. To facilitate expression and purification in Escherichia coli, peptides were fused to the C-terminus of GST (Fig. 2A). Whole-cell extracts were prepared from cells expressing HA-tagged CP2, and incubated with each GST-fused peptide. Complex formation between the GST–peptide and HA–CP2 was determined by GST pull-down and immunoblot analyses. Although control peptide was not able to bind to CP2, all the other peptides containing the putative CP2-binding motifs consistently showed a strong interaction with CP2 (Fig. 2B). Taken together, these results indicate that the phage clones presented in Table 1 genuinely bind to CP2 through their coatdisplayed exogenous peptides with some common motifs. 1267


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A

B

Fig. 2. Purified peptides possessing the proposed CP2-binding motifs bind to CP2 in vitro. (A) Amino acid sequences of five representative GST-tagged peptides that contain CP2 (05, 08, 13, 21 and 31) and one GST-binding peptide (04). Residues underlined in black correspond to the 12-mer peptide sequences. (B) In vitro binding between HA–CP2 and the purified peptides. Whole-cell extracts from 293T cells expressing HA–CP2 were mixed with each of the purified GST-tagged peptides and the complex formation was analyzed by GST pull-down and immunoblotting against anti-HA serum. Coomassie Brilliant Blue staining and anti-HA blotting of the input materials ascertain the equivalent amounts of extracts and peptides between each pull-down samples being employed. Arrow indicates the position of HA–CP2.

Mapping the sites of interaction between CP2 and each CP2-binding motif Because four CP2-binding motifs were identified as above, we were interested in whether these distinct motifs recognize the same region in CP2. CP2 is composed of 502 amino acids, whose structure can be divided into six regions: N-terminal transcriptional activation (amino acids 1–63), N-terminal Elf-1 (amino acids 63–244), the basic (amino acids 244– 250), SPXX (amino acids 250–403), Q ⁄ P (amino acids 403–413) and C-terminal acidic (amino acids 413–502) regions [17,38]. To date, the functional role of each region has not been fully studied, although the carboxyl-flanking region of SPXX has recently been shown to be involved in interactions with YY1 [36]. We mapped the binding site of each CP2-binding peptide on CP2. Several N- and ⁄ or C-terminally deleted CP2 mutants were generated (Fig. 3A) and their abilities to bind either with GST-tagged YY1 (Fig. 3B) or with representative phage clones each displaying one of the CP2-binding motifs (Fig. 3C) were examined. GST pull-down assays showed that YY1 bound only to those CP2 proteins that contained a region corresponding to amino acids 306–396. This 1268

result was anticipated, as this region of CP2 has been shown to be involved in the interaction with YY1 [36]. This putative CP2-binding region was then further confirmed in ELISA experiments in which phage clone 13 (containing an HXPR motif) and other CP2 mutant constructs were employed (Fig. 3C). This phage was able to bind only to those CP2 mutant proteins with amino acids 306–396. Interestingly, this region of CP2 was found to be involved in interactions with not only phage clone 05, which has YXQR, a subset of HXPR motif, but also clone 08, which possesses an ASR motif. These results indicate that two CP2-binding motifs, HXPR (found in phage clones 13 and 05) and ASR (found in phage clone 08) recognize a region of amino acids 306–396 that corresponds to the carboxyl-flanking region of the SPXX region. However, phage clones 21 and 31 were found to bind to regions other than SPXX. Clone 21, which represents a PHL motif, bound to the C-terminal part of the Elf-1 domain (amino acids 134–243). The Elf-1 domain was also found to provide an interacting site for clone 31, a representative of the PXHXH motif. For interaction with clone 31, however, the N-terminal part of Elf-1 (amino acids 67–134) was required. FEBS Journal 272 (2005) 1265–1277 ª 2005 FEBS

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A EIf-1

BD

SPXX

Q/P

Net Acidic

B

C

Fig. 3. Identification of the domains on CP2, with which CP2-binding peptides interact. (A) Schematic representation of full-length CP2 composed of six regions and various N- and ⁄ or C-terminally deleted CP2 mutants. Regions recognized by CP2-binding peptides were determined by the experiments shown in (B) and (C). (B) GST pull-down assays showing the binding of YY1 to various CP2 deletion mutants. Lysates (500 lg of protein) from cells over-expressing HA–CP2 deletion mutant proteins were incubated for 2 h with GST–YY1 immobilized on glutathione–Sepharose beads. Bound proteins were eluted with SDS sample buffer, resolved by SDS ⁄ PAGE, and analyzed by immunoblotting with anti-HA and anti-GST sera. None of the CP2 deletion mutant proteins showed detectable level of binding to GST itself (GST panel). Western blot using an anti-HA serum shows that similar amounts of CP2 deletion mutants were used in each lane (INPUT panel). (C) Binding of various CP2 deletion mutants to phage clones in which each displays a 12-mer peptide containing a representative CP2-binding motif as in Fig. 1. Binding of each phage clone to the GST-tagged CP2 proteins was determined by ELISA as described in Experimental procedures. Absorbance at 405 nm was used to determine extent of binding. Shown are the specific bindings to CP2, obtained after subtracting the nonspecific bindings to GST. Similar amounts of CP2 deletion mutants were used in each assay, as confirmed by Western blot using an antiGST serum (subset figure in a box).

Recognition of proteins containing CP2-binding motifs and verification of the CP2-binding ability of REST and YY1 that contain the HXPR motif As stated above, none of the 12-mer peptide sequences exactly matched those of any known proteins. Having discovered common 3–5 amino acid motifs on these FEBS Journal 272 (2005) 1265–1277 ª 2005 FEBS

peptides, we decided to see if there were any proteins in the database that had these motifs. We searched iProClass (PIR ± Swiss-Prot ⁄ TrEMBL) and the PIRPSD database using the PIR pattern ⁄ peptide match search tool. As a result, several proteins were recognized, including REST, YY1, protein inhibitor of activated STAT1 (PIAS1), protein kinase C-d (PKC-d), 1269


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Table 2. Proteins containing the putative CP2-binding motifs. Putative CP2-binding proteins

Conserved motifs

Functions

REST Yin-Yang1 PIAS1 PKC-d CKII-a ETS-1 TFIIE, a subunit

HXPR HXPR PHL PHL and ASR PHL and ASR PHL and HXPR PXHXH

Transcriptional repressor of neuronal genes in non-neuronal tissues [39,40] Transcriptional repressor and activator [36,41] Transcriptional repressor and activator, SUMO-E3 ligase [42–44] Commitment to terminal erythroid differentiation [45,46] Commitment to terminal erythroid differentiation [47,48] Erythroid-specific gene activation [49] Transcription factor IIE, a subunit (TFIIE a) [50]

casein kinase II-a (CKII-a), ETS-1 and the a subunit of transcription factor IIE (TFIIE-a) (Table 2). REST and YY1 have one HXPR motif and PIAS1 has a PHL motif, whereas PKC-d, CKII-a and ETS-1 have two motifs. The two protein kinases, PKC-d and CKII-a, have both PHL and ASR motifs, whereas ETS-1 has PHL and HXPR. TFIIE-a contains a PXHXH motif. Among these proteins, YY1 is the only one that has been shown to interact with CP2, whereas the remaining proteins are novel in that their ability to interact with CP2 has not been reported, although their physiological functions and roles are well characterized. Of the proteins that have CP2-binding motifs, we further investigated the CP2 binding of the two HXPR-motif-containing proteins, REST and YYI. Both REST and YY1 function as a transcription modulator by recruiting and utilizing HDAC family proteins [36,37,39]. REST acts largely as a repressor for expression of neuronal genes in non-neuronal tissues [51,52]. YY1 regulates the transcription of a variety of genes [53,54], but its activity could be activating or inhibiting, depending upon the promoter context and cellular environment [55,56]. It is known that HDAC1 recruitment requires prior interaction of YY1 with CP2 [36,37], and the first zinc finger domain is crucial for this interaction [36,57,58]. Importantly, we found that the HXPR motif is located within this region. In REST, a HXPR motif is found to reside in the DNA-binding domain clustered with eight zinc fingers [40]. We investigated whether REST as well as YY1 can associate with CP2. To this end, 293T cells were cotransfected with EGFP–CP2 and HA–REST (Fig. 4A) or CP2 and HA–YY1 (Fig. 4B) and the cell lysates were immunoprecipitated with appropriate antibodies. A significant amount of REST was shown to be present in anti-EGFP immunoprecipitates and conversely, CP2 was found in anti-HA immunoprecipitates (Fig. 4A). Likewise, YY1 and CP2 could be coimmunoprecipitated with the counterpart anti-HA or antiCP2 sera (Fig. 4B). These results indicate that both REST and YY1 bind to CP2 in vivo. 1270

A

B

Fig. 4. REST and YY1 strongly interact with CP2 in vivo. (A) Coimmunoprecipitation assays. 293T cells were cotransfected with EGFP–CP2 and HA–REST or HA–REST(P272A ⁄ R273A) plasmids and total cell lysates were immunoprecipitated against either antiHA or anti-EGFP sera. Bound proteins were eluted with SDS sample buffer, resolved by electrophoresis on SDS)8% polyacrylamide gels. The presence of coimmunoprecipitated proteins was detected by probing with the appropriate antibodies. (B) Coimmunoprecipitation of CP2 and HA-YY1 was similarly assayed by using anti-HA and -CP2 antibodies. Input, one-tenth of the cell extracts used for IP; IP, immunoprecipitation.

The HXPR motif is important in the interaction of YY1 and REST with CP2 To verify that YY1 and REST interact with CP2 via the HXPR motif, we generated mutants for each protein that had a double (PRfiAA) or single (HfiA) amino acid substitution on HXPR and determined their binding to CP2. We performed GST pull-down experiments in which GST-tagged YY1 proteins were incubated with 293T whole-cell extracts expressing HA–CP2 (Fig. 5A). HA–CP2 was retained by GST– YY1 but not by GST itself (GST-pull down panel), indicating that YY1 binds to CP2 in vitro. Under conditions in which equivalent amounts of wild-type YY1 and the two YY1 mutants were expressed (Input panel), the single point mutant of YY1 possessed FEBS Journal 272 (2005) 1265–1277 ª 2005 FEBS

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A

B

Fig. 5. A HXPR motif mediates the interatcion of YY1 with CP2. (A) GST pull-down assays. Whole-cell extracts (500 lg of protein) containing HA–CP2 protein were incubated with glutathione–Sepharose bead-immobilized GST, GST–YY1, GST–YY1(P322A ⁄ R323A) or GST– YY1(H320A) proteins for 2 h. Bound proteins were eluted with SDS sample buffer, resolved by electrophoresis on SDS)8% polyacrylamide gels, and analyzed by immunoblotting with anti-HA or anti-GST sera. (B) Effect of YY1 point mutants and a competitor peptide, on CP2-driven luciferase activity. A pGL3-based luciferase plasmid was engineered to contain a region of the a-globin promoter with CP2-binding elements. 293T cells were cotransfected with this reporter and one, some or all of the following plasmids as indicated at the bottom of the plot: HA–CP2, HA–YY1, HA–YY1(P322A ⁄ R323A), HA–YY1(H320A) and FLAG–peptide 13 plasmids. To eliminate DNA concentrationdependent variation of transfection efficiency and reporter expression between transfections, total amount of plasmid DNA was adjusted to 2 lg by supplementing the pcDNA3–FLAG vector to test vectors. All other conditions are described in detail in Experimental procedures. Experiments were performed three times and the results were analyzed statistically using the Student’s t-test. P values were obtained by comparison of bar 5 with bar 2 (*P £ 0.001), bar 5 with bar 6 (**P £ 0.01) and bar 14 with bar 16 (**P £ 0.01).

CP2-binding activity that was approximately one third that of wild-type YY1. The double-point mutant of YY1 did not show any detectable level of CP2 binding. We also demonstrated that the REST double-point mutant (PRfiAA) exhibited much less ability to bind to CP2 in a coimmunoprecipitation assay (Fig. 4A, right panel). These results clearly indicate that the binding of YY1 and REST to CP2 are mediated by the HXPR motif. That HXPR is a motif for binding of YY1 to CP2 was further confirmed by competition with an HXPR motif-containing peptide (i.e. peptide 13) and by YY1 FEBS Journal 272 (2005) 1265–1277 ª 2005 FEBS

point mutants. The extent of the CP2–YY1 interaction was indirectly measured by assaying the transcriptional activity of CP2, because YY1 decreases the transcriptional activity of CP2 by forming a complex with it [36]. A luciferase assay was employed using a synthetic promoter that contains four copies of a CP2-binding element in the mouse a-globin promoter [21]. Cells were transfected with CP2, YY1, YY1 double- or single-point mutant, and peptide 13 plasmids in various combinations and the CP2 promoter-driven luciferase activity was measured (Fig. 5B). CP2 stimulated luciferase activity quite well, whereas YY1 and peptide 1271


13 alone did not (Fig. 5B; lane 1 vs. lanes 2, 9 and 10), showing that specific transcription is driven by CP2. YY1 was able to suppress this CP2-driven luciferase activity in a dose-dependent manner (Fig. 5B; lane 2 vs. lanes 3–5). When a plasmid encoding peptide 13 was cotransfected, however, the suppressing effect of YY1 on CP2’s transcriptional activity was reduced in proportion to the amount of peptide 13 (Fig. 5B; lane 5 vs. lane 6 and lane 4 vs. lanes 7–8). Furthermore, we found that the YY1 double-point mutation on HXPR could not suppress CP2’s transcriptional activity (Fig. 5B; lane 5 vs. lane 13) but the YY1 single-point mutation showed significant suppression of CP2 activity although it was not completely inhibited (Fig. 5B; lane 5 vs. lane 16). Taken together, these results indicate that the HXPR motif can confer on YY1 or REST the ability to bind to CP2 and the physiological role of CP2 is mediated by the HXPR motif.

Discussion Since CP2 was first isolated almost 15 years ago, much effort has been put into finding its functional roles. As a result, it is now known that CP2 is not only a ubiquitous transcription factor expressed in most tissues, but is also involved in tissue- or stage-specific transcription activation of some genes. It remains, however, unclear how CP2’s activity is regulated and how CP2 exerts tissue- and stage-specific functions. This regulation and specificity can certainly be achieved by various mechanisms, one of which would be interaction with some other proteins, as found for a number of cellular examples including NF-jB with IjB. An M13 phage display library that provides up to 109 peptides composed of 12 random amino acids was screened in this study. Approximately 100 phage clones that express CP2-binding peptides on their coats were isolated, and specific binding of some of these clones to CP2 was verified using purified 12-mer peptides. Sequence analysis showed that some clones could be grouped into four classes each having a unique consensus sequence of 3–5 amino acids, whereas the remainder did not display a consensus sequence (Table 1). The consensus motifs are as follows: HXPR for Class I clones, PHL for Class II, ASR for Class III and PXHXH for Class IV. Class I clones can have some substitutions at positions 1 and 3; Y or W is found at position 1 in two clones and Q at position 3 in one clone. For Class IV clones, positions 2 and 4 show a preference for H, although Y, Q W, and F are also tolerated. We became aware of the presence of these CP2binding motifs in REST, YY1, PIAS1, PKC-d and 1272

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three other proteins (Table 2). GST pull-down and coimmunoprecipitation assays indicate that REST and YY1 indeed bind to CP2 in vitro and in vivo (Fig. 4). Although YY1 is known to interact with CP2 [36], REST binding to CP2 is a novel finding. We also provide the first evidence that the HXPR motif mediates the binding of these two proteins to CP2. YY1 and REST mutants having substitutions of H to A or PR to AA on the HXPR motif had significantly reduced or virtually no CP2-binding capability (Figs 4A and 5A). In addition, an HXPR motif-containing peptide was able to repress the interaction of YY1 and CP2 (Fig. 5B). It is noteworthy that HXPR encloses a newly discovered PR motif [59] with which the WW-A domain has been shown to interact. Because Fe65, which binds to CP2 [33] and is thought to be pathogenically associated with Alzheimer’s disease [60], has a WW-A domain [59] it might be of interest to explore the possible association of Fe65 with YY1 and REST. Using various deletion mutants of CP2, we have shown that the carboxyl-flanking region (more specifically, amino acids 306–396) of the SPXX region on CP2 is involved in the binding of the ASR motif as well as HXPR (Fig. 3). However, we do not know whether these two motifs recognize the same sequences in CP2, and thus further study is required to dissect this region. If the two motifs bind to overlapping, or the same, residues, the question might be raised as to whether the two motifs can compete with each other for binding to CP2. Because several proteins, including PKC-d and CKII-a, possess the ASR motif (Table 2), it will be interesting to know whether PKC-d and CKII-a can also alter the effect of YY1 on CP2. We also demonstrated that, unlike the HXPR and ASR motifs, PHL and PXHXH motifs bind to the N- and C-terminal parts of the Elf-1 domain on CP2, respectively (Fig. 3). Because the Elf-1 domain has been thought to primarily mediate DNA binding by CP2 [38], this is the first time that an additional, novel function has been assigned to the Elf-1 domain of CP2. Our list of polypeptides that bind to CP2 shows a preponderance of histidines, implying the importance of this amino acid residue in the CP2-binding motif of proteins that interact with CP2. Indeed, two or three histidine residues are found around the HXPR motif of both YY1 and REST. We can exclude possible nonspecific interactions between polypeptides and the plastic material of the plate well during the phage screening, which may result in enrichment of histidine-rich peptides, as it has been reported that plastics prefer tyrosine and tryptophan but not histidine [61]. FEBS Journal 272 (2005) 1265–1277 ª 2005 FEBS

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Taken together, our findings suggest that CP2 is able to receive a variety of single and ⁄ or combined regulatory inputs by utilizing its different regions to interact with various CP2-binding motifs. Thus, this might be one of the mechanisms by which CP2 exerts multifunctional roles in various cellular processes including apoptosis, proliferation and differentiation via differential transcriptional regulations of target genes.

Experimental procedures Cell culture and luciferase assay 293T cells were maintained in Dulbecco’s modified Eagle’s medium (Invitrogen-Gibco ⁄ BRL, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT, USA). For luciferase assays, cells (5 · 104) were plated on 24-well plates and transfected with various combinations of reporter and expression vectors (total 2 lg DNA) using a calcium phosphate method [62]. At 48 h after transfection, cells were harvested in NaCl ⁄ Pi and resuspended in passive lysis buffer (Promega, Madison, WI, USA) and dual (firefly and renilla) luciferase activities were measured using a luminometer Lumat LB9501 (Berthold, Gaithersburg, MD, USA). Firefly luciferase expression was normalized against renilla luciferase. Each experiment was performed in duplicate and repeated at least three times.

Plasmid constructs CP2 cDNA fragments were cloned in-frame with the GST coding sequence in prokaryotic pGEX-4T1 vectors (Amersham-Pharmacia, Piscataway, NJ, USA) and also cloned in-frame with the HA tag sequence in the eukaryotic pCMV-HA vector (Clontech, Palo Alto, CA, USA). EGFP–CP2 was generated by PCR cloning into pEGFP-N1 (Clontech) using following primers; 5¢-GAAGCTTAT GGCCTGGGCTCTGAAG-3¢ and 5¢-CGGTACCGCCTT GAGAATGACATGATAG-3¢, which contain HindIII and KpnI sites, respectively (underlined). GST-tagged CP2deletion constructs were generated by PCR from pGEX4T1–CP2 or pCMV-HA–CP2. GST- or FLAG-tagged 12-mer peptides were prepared as follows: phage DNA was amplified by PCR using primers 5¢-CTTTAGTGGTACCTTTC TATTC-3¢ and 5¢-GTATGGGATTTTGCTCGAGAACTT TC-3¢, which introduced KpnI and XhoI sites, respectively (underlined). The PCR products were then inserted into the pGEM-Teasy vector (Promega). The EcoRI ⁄ XhoI fragments from these pGEM-Teasy–peptide constructs were cloned into pGEX-4T1 (Amersham-Pharmacia) or pcDNAFLAG vectors. Two oligonucleotides containing the nuclear localization signal (NLS) and EcoRI and KpnI restriction sequences (underlined) were inserted into the respective sites



of pcDNA-FLAG)12-mer peptide vector: 5¢-CGGAATTC CCCCCAAAAAAGAAGAGAAAGAT-3¢ and 5¢-GGGG TACCCCGTCTTCTATCTTTCTCTTCTTT-3¢. The REST cDNA fragment was generated by PCR from the pcDNAFLAG–REST vector with specific primers (see below) and then cloned in frame into the pCMV–HA vector. The pcDNA–FLAG–REST vector was kindly provided by G Thiel (University of Saarland Medical Center, Germany), and HA–YY1 and GST–YY1 vectors were from Y Shi (Harvard Medical School, Cambridge, MA, USA) and E Seto (University of South Florida, Tampa, FL, USA), respectively.

Site-directed mutagenesis A PCR-based site-directed mutagenesis approach was used to generate two YY1 point mutants, YY1(H320A) and YY1(P322A ⁄ R323A) that have amino acid substitution of the His residue at position 320 or both Pro and Arg residues at positions 322 and 323 to Ala. For cDNA construction of each mutant, two products were generated covering bases 1–975 and 949–1245 of the respective mutant YY1 cDNA. pCMV–HA–YY1 was employed as a template and the following primers (bases changed for point mutations are underlined) were used: forward primer (5¢-GGAATT CTCATGGCCTCGGGCGACACC-3¢) corresponding to bases 1–18 of YY1 cDNA; reverse oligomer (5¢-GCTCGA GTCACTGGTTGTTTTTGGCC-3¢) for bases 1227–1245; reverse (5¢-GTGGACTGCGGCTCCGTGGGTGTG-3¢) and forward (5¢-CACACCCACGGAGCCGCAGTCCAC-3¢) oligonucleotides for Pro ⁄ Arg to Ala ⁄ Ala mutations in bases 952–975; reverse (5¢-GTGGACTCTGGGACCGGCTGTG TGCAG-3¢) and forward (5¢-CTGCACACAGCCGGT CCCAGAGTCCAC-3¢) oligonucleotides for His to Ala mutation in bases 949–975. The two overlapping PCR fragments were mixed and added as a template in the second PCR, which used 1–18 and 1227–1245 base oligomers as a forward and a reverse primer, respectively. This resulted in the generation of 1245 bp products containing the point mutations PRfiAA or HfiA of YY1. These products were directly cloned into pGEM-T Easy vector (Promega). Following digestion with EcoRI and XhoI, fragments were cloned into pGEX-4T2 or pCMV–HA vectors and sequencing analysis was performed to confirm the point mutations. A plasmid containing point mutation in REST, pCMV–HA–REST(P272A ⁄ R273A), was generated by the same method. pcDNA–FLAG–REST was used as a template and the following primers (bases changed for point mutations are underlined) were used: forward primer (5¢-GGAATTCTCATGGCCACCCAGGTAATGG-3¢) corresponding to bases 1–19 of REST cDNA and reverse primer (5¢-CTCGAGTTACTCCTGCCCTTGAGCTGC-3¢) for bases 3274–3294; reverse primer (5¢-GTGTATACTT TCGCTGCAAAATGGTTTC-3¢) and forward primer

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(5¢-GAAACCATTTTGCAGCGAAAGTATACAC-3¢) for Pro ⁄ Arg to Ala ⁄ Ala mutations in bases 803–830. The two overlapping PCR fragments were mixed and added as a template in the second PCR that used 1–19 and 3274–3294 base oligomers as a forward and a reverse primer, respectively. This resulted in the generation of a 3294 bp product containing the PRfiAA mutation of REST. This product was directly cloned into pGEM-T Easy vector (Promega). Following digestion with EcoRI and XhoI, the fragment was cloned into the pCMV–HA vector.

Purification of GST fusion proteins GST-fused proteins were expressed in E. coli BL21 (pLys). Cell extracts were incubated with glutathione–agarose beads (Sigma-Aldrich, St. Louis, MO, USA), and the beads were washed extensively with NaCl ⁄ Pi. Proteins were eluted with 20 mm reduced glutathione. The protein concentration was determined using the Bradford-based protein assay (BioRad, Hercules, CA, USA) and the purity was checked by SDS ⁄ PAGE.

Peptide phage library screening GST–CP2 or GST itself was immobilized onto microtiter plates by directly coating the wells with 10 lL of 100 lgÆmL)1 proteins in TBS (50 mm Tris ⁄ HCl, pH 7.5, 150 mm NaCl) for 8 h at 4 C in a humidified container. The wells were rapidly washed six times with TBST [TBS, 0.1% (v ⁄ v) Tween-20]. To remove any potential GST-binding phage, the library was precleared three times using GST. Approximately 1.2 · 1012 phage (New England Biolabs, Beverly, MA, USA) in 100 lL TBS were added sequentially to two GST-coated wells and incubated for 10 min at room temperature in each round of preclearing. Precleared phage were then added to GST–CP2-coated wells in 200 lL TBS ⁄ 0.1% (w ⁄ v) bovine serum albumin (BSA) ⁄ 0.05% (w ⁄ v) GST and incubated for 2 h at 4 C. Unbound phage were removed by washing the wells with cold TBS ⁄ 0.05% (v ⁄ v) Tween 20. Bound phage were eluted with 100 lL solution of glycine ⁄ HCl (0.2 m, pH 2.2) and 0.1% (w ⁄ v) BSA. After immediate neutralization with 10 mm Tris ⁄ HCl (pH 9.1), the eluted phage were amplified by infecting log-phase E. coli (strain ER2738). The supernatants of the bacterial culture were treated with polyethylene glycol (PEG) and NaCl for 8 h on ice, and the phage precipitates were recovered by centrifugation. To kill any residual bacteria, the phage resuspension in TBS was heattreated at 70 C for 15 min. The collected phage were then subjected to five more rounds of amplification as above. The phage titer of every round was determined by infecting 200 lL of a mid-log phase culture of E. coli with 10 lL of serially diluted phage and plating on LB ⁄ IPTG ⁄ Xgal plates. The number of resulting colonies was used to determine plaque-forming units per milliliter. The ability of the

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isolated phage to bind CP2 was confirmed by an ELISA assay where GST–CP2-bound phage in the microtiter wells were colorimetrically detected using a horseradish peroxidase (HRP)-conjugated anti-M13 serum (AmershamPharmacia) and 2,2¢-azino-bis (3-ethylbenzothiazoline-6sulfonic acid) buffer (Sigma-Aldrich). To eliminate the false-positive signals, control experiments with GST (10 lg protein) coated wells were performed concurrently.

Deduced peptide sequence determination To amplify individual phage clones for DNA sequencing analysis, single colonies were isolated from the titering plates and used to infect 1 mL of a 100-fold diluted overnight culture of ER2738 E. coli. The cultures were incubated for 5 h with shaking at 37 C and the phage-containing supernatant was cleared by centrifugation (13 000 g, 30 s at 4 C). For DNA sequencing, 500 lL of the phage stock was precipitated with 200 lL of PEG ⁄ NaCl and the phagecontaining pellet was resuspended in 100 lL of iodide buffer (10 mm Tris ⁄ HCl, pH 8.0, 1 mm EDTA, 4 m NaI). The preferential precipitation of single-stranded phage DNA was accomplished by ethanol precipitation. The DNA pellet was then resuspended in 30 lL of Tris ⁄ EDTA buffer (pH 8.0). DNA sequencing of recombinant phage inserts was carried out using Dideoxy-Terminator Sequencing Chemistry from CEQ 2000 XL DNA Analysis System (Beckman, Urbana, IL, USA) with the primer 5¢-CCCTCA TAGTTAGCGTAACG-3¢.

Coimmunoprecipitation and GST pull-down assays For coimmunoprecipitation assays, 293T cells were transfected with either CP2 alone or CP2 together with HA– REST, HA–REST(P272A ⁄ R273A) or HA–YY1 expression vectors. Forty-eight hours after transfection, cells were solubilized with precooled lysis buffer A (30 mm Hepes, pH 7.4, 100 mm NaCl, 1 mm EGTA, 1% (v ⁄ v) Triton X100, 0.1% (w ⁄ v) BSA, protease inhibitor cocktail [SigmaAldrich] and 1 mm phenylmentanesulfonyl fluoride]. To these extracts (200 lg of protein), appropriate antibodies and 10 lL of protein A–Sepharose beads (Sigma-Aldrich) were added. After incubation for 1 h at room temperature, the beads were washed three times with NaCl ⁄ Pi ⁄ 0.6% (v ⁄ v) Tween 20 (PBST), and proteins were eluted by boiling in SDS sample buffer [50 mm Tris ⁄ HCl, pH 6.8, 100 mm dithiothreitol, 2% (w ⁄ v) SDS, 0.1% (w ⁄ v) bromophenol blue, and 10% (v ⁄ v) glycerol]. Proteins were fractionated by SDS ⁄ PAGE and transferred to a nitrocellulose membrane in transfer buffer [25 mm Tris, 40 mm glycine, 0.05% (w ⁄ v) SDS, 20% (v ⁄ v) methanol]. The membrane (BioRad) was blocked with PBST ⁄ nonfat dried milk for 1 h, followed by 1 h incubation with the appropriate antibodies and finally treated with a secondary HRP-conjugated goat


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anti-mouse IgG (Pierce, Rockford, IL, USA) at 1 : 10 000 in PBST for 1 h. All of these immunoblotting procedures were performed at room tempertaure, and membranes were washed several times with PBST for 15 min between steps. Membrane were then treated with ECL reagents (Amersham-Pharmacia) and exposed to X-ray film for 5–20 s. For GST pull-down assays, glutathione–Sepharose beads (Amersham-Pharmacia) bound with GST–YY1, GST– YY1(P322A ⁄ R323A), GST–YY1(H320A), GST)12-mer peptide or GST were incubated with the extracts prepared from 293T cells transiently expressing HA–CP2 in buffer B [50 mm potassium phosphate, pH 7.5, 100 mm KCl, 10% (v ⁄ v) glycerol, 0.1% (v ⁄ v) Triton X-100] for 2 h at 4 C. The beads were washed four times with buffer B omitting glycerol and Triton X-100 [53]. Beads were then boiled for 5 min in SDS sample buffer, analyzed by 10% SDS ⁄ PAGE and immunoblotted with specific antibodies.


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Acknowledgements We thank Drs Seto, Mu¨ller, Shi, Shank and Thiel for their donations of the plasmid constructs and technical inputs. We also thank Brian Watson for his critical reading and comment on the manuscript. This work was supported by a Korea Research Foundation Grant (KRF-2002-015-CP0283). HCK and JHC were supported by the Brain Korea 21 Project from the Ministry of Education and Human Resources of Korea through the Research Group on Stem Cells and Early Development.

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