p300 has a conserved domain structure and interacts functionally with the nuclear receptor SmFtz-F1

Molecular & Biochemical Parasitology 146 (2006) 180–191

Schistosoma mansoni CBP/p300 has a conserved domain structure and interacts functionally with the nuclear receptor SmFtz-F1夽 Benjamin Bertin a,1 , Fr´ed´erik Oger a , Jocelyne Cornette a , St´ephanie Caby a , Christophe No¨el b , Monique Capron a , Marcelo R. Fantappie c , Franklin D. Rumjanek c , Raymond J. Pierce a,∗ b

a Inserm U 547, Institut Pasteur de Lille, 1 rue du Professeur A. Calmette, 59019 Lille, France School of Biology, Institute for Research on Environment and Sustainability, Devonshire Building, University of Newcastle upon Tyne, NE1 7RU, UK c Instituto de Bioquimica Medica, CCS, Universidade Federal do Rio de Janeiro, Ilha do Fundao, 21941-590, Brazil

Received 15 November 2005; received in revised form 15 December 2005; accepted 15 December 2005 Available online 6 January 2006

Abstract Metazoan species diversification in general and the adaptation of parasites to their life-style in particular are due, not only to the evolution of different structural or metabolic proteins, but also to changes in the expression patterns of the corresponding genes. In order to explore the conservation/divergence of transcriptional regulation in the platyhelminth parasite Schistosoma mansoni, we are studying the structures and functions of transcriptional mediators. CREB-binding protein (CBP) and p300 are closely related transcriptional coactivators that possess histone acetyltransferase (HAT) activity that can modify chromatin to an active relaxed state. They are also thought to link transcription factors to the basic transcriptional machinery and to act as integrators for different regulatory pathways. Here we describe the cloning and functional characterization of S. mansoni CBP. SmCBP1 comprises 2093 amino acids and displays a conserved modular domain structure. The HAT domain was shown to acetylate histones with a marked activity toward H4. Functional studies showed that SmCBP1 could interact physically with the nuclear receptor SmFtz-F1 and also potentiated its transcriptional activity in the CV-1 cell line. Screening of the EST and genomic sequence databases with the SmCBP1 sequence allowed us to characterize a second CBP gene in S. mansoni. SmCBP2 shows a high degree of sequence identity to SmCBP1, particularly in the HAT domain. Phylogenetic studies show that these peptides are more closely related to each other than to either mammalian CBP or p300, suggesting that they derive from a platyhelminth-specific duplication event. Both genes are expressed at all life-cycle stages, but differences in their relative expression and structural variations suggest that they play distinct roles in schistosome gene regulation. © 2005 Elsevier B.V. All rights reserved. Keywords: CBP/p300; Schistosoma mansoni; Parasite; Transcription; Coactivator; Histone Acetyltransferase

1. Introduction Emerging evidence suggests that the number of genes does not necessarily reflect the morphological and behavioural complexity of living organisms. Indeed, it seems rather that the generation of new genetic networks, accompanied by a more

Abbreviations: RT-PCR, reverse transcriptase-polymerase chain reaction; RACE, rapid amplification of cDNA ends; bp, base pair; CBP, CREB-binding protein; HAT, histone acetyltransferase; GST, glutathione S-transferase 夽 Note: DNA sequences obtained in the course of this study have been deposited in Genbank with the accession numbers: DQ114786 and DQ212768. ∗ Corresponding author. Tel.: +33 320877783; fax: +33 320877888. E-mail address: [email protected] (R.J. Pierce). 1 Present address: Free University of Brussels, Faculty of Medicine, Laboratory of Molecular Virology, 808 route de Lennik, 1070 Brussels, Belgium. 0166-6851/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.molbiopara.2005.12.006

complex regulation of gene expression, are responsible for the generation of biodiversity and animal complexity [1]. Thus, it appears that the regulation of transcription is a central mechanism by which, for example, animal evolution can occur, but the question of how the various mediators are implicated in this process remains an important issue to decipher. An emerging theme in transcriptional regulation is the pivotal role played by remodelling of the chromatin structure. Indeed, eukaryotic genomic DNA in the nucleus is tightly packaged by basic proteins, the histones, giving a highly structured DNA–protein complex, chromatin. Within this structure, the amino termini of histones (histone tails) are accessible and susceptible to a variety of post-translational modifications such as phosphorylation [2], methylation [3], acetylation [4] or ubiquitination [5]. It now seems clear that various combinations of these modifications can remodel chromatin structure directly

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or indirectly and strongly regulate transcription according to the “histone code” hypothesis [6,7]. The acetylation of histone tails has long been correlated with transcriptional activation [8]. This modification is catalyzed by the well-characterized histone acetyltransferase (HAT) enzymes. A large number of transcriptional coactivators are now recognized to possess HAT activity and it is possible to distinguish at least six families of histone acetylase proteins [4]: (i) the GNAT superfamily (PCAF/GCN5), (ii) the MYST family, (iii) the CBP/p300 family, (iv) the nuclear receptor coactivator (SRC/ACTR/TIF) family, (v) the TAFII 250 family and (vi) the GNAT-related family (TFIIIC). Despite this, the mechanisms by which the acetylation of histone proteins contributes to transcriptional activation still remain unclear. Two models, which are not mutually exclusive, are currently proposed. The first well-accepted hypothesis is that acetylation of histone tails neutralizes positive charges, thereby decreasing DNA–histone interactions and increasing DNA accessibility to the transcriptional machinery. The second model, based on the “histone code” hypothesis, postulates that the modification marks on the histone tails should create binding sites for transcriptional effectors, given that some acetylated lysines are recognized by the bromodomain which is found in many transcriptional activators [6]. CREB-binding protein (CBP) and p300 are highly conserved and functionally related transcription coactivators. These proteins have been implicated in fundamental biological processes, such as cell growth and development, and mutations in either human protein result in a congenital development disorder named Rubinstein-Taybi syndrome (RTS) [9–11]. CBP/p300 homologues are found in many organisms, including flies, worms and plants [12]. Interestingly, the importance of CBP/p300 protein in the control of development is not restricted to vertebrates, since functional genetic alteration of the genes encoding the orthologous protein in C. elegans and D. melanogaster revealed that these transcriptional regulators play a central role in controlling key differentiation steps during embryonic development in both organisms [13,14]. The operating mode of CBP and p300 proteins is relatively complex. Indeed, in addition to their ability to acetylate histones, CBP and p300 have been shown to affect transcription by acetylating other proteins like transcription factors or coactivators [4]. Moreover, the capacity of CBP/p300 to interact both with components of the general transcriptional machinery, such as RNA polymerase II, TATA binding protein (TBP) and/or TFIIB, and multiple transcription factors allows these huge proteins to function as a physical scaffold during transcriptional activation [15]. The combination of these different functions places CBP/p300 proteins at the crossroads of multiple genetic pathways and clearly indicates the fundamental importance of this protein family. Recently, homologues of HAT proteins have been cloned and characterized in various parasitic organisms [16–18]. In order to explore the conservation/divergence of transcriptional regulation in the platyhelminth parasite Schistosoma mansoni, we report here the first cloning and characterization of CBP homologues in this organism. In silico screening and PCR-based approaches allowed us to describe two CBP genes in S. man-

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soni, respectively named SmCBP1 and SmCBP2. Each protein displays a conserved modular domain structure and phylogenetic analyses show that these peptides are more closely related to each other than to either mammalian CBP or p300, suggesting that they derive from a platyhelminth-specific duplication event. Due to its more conserved sequence identity compared to the human and fruit fly counterparts, SmCBP1 was used for functional assays. It was expressed as a GST–fusion protein and shown to acetylate histones with a marked activity toward H4. GST pull-down studies showed that SmCBP1 could physically interact with the nuclear receptor SmFtz-F1 [19]. SmCBP1 also potentiated the transcriptional activity of SmFtz-F1 on its native promoter in the CV-1 cell line. Nevertheless, the existence of subtle sequence divergences and the presence of a paralogous gene suggest that SmCBP1 and/or SmCBP2 are probably involved in schistosome-specific transcriptional pathways. 2. Materials and methods 2.1. Parasites A Puerto-Rican strain of S. mansoni was maintained in Biomphalaria glabrata snails and golden hamsters (Mesocricetus auratus). Cercariae were released from infected snails and harvested on ice. They were then washed three times by resuspension in 30 ml of Hank’s Balanced Salt Solution (Invitrogen) in a corex tube (Corning) and centrifugation for 10 min at 1500 × g. Schistosomula were obtained in vitro [20] and were maintained in culture for up to 8 days under the conditions previously described [21]. Adult worms were obtained by whole body perfusion of 6-week infected hamsters [22]. Eggs were obtained from the livers of infected hamsters and hatched out under light to obtain miracidia [23]. Primary sporocysts were obtained after overnight axenic culture of miracidia as described [23]. Parasite DNA was extracted from the free-living cercariae using standard methods [24]. Total RNA was extracted from all life-cycle stages using the guanidine thiocyanate/caesium chloride method [25] and poly A+ RNA was purified on oligo-dT cellulose [26]. 2.2. cDNA cloning In order to obtain the full-length coding sequence of CBP from S. mansoni, degenerate oligonucleotides (Table 1 in Supplementary Material online) based on mammalian and invertebrate CBPs were first used to amplify a 541 bp fragment of the coding sequence. Briefly, DNA extracted from a lambda ZAP II cDNA library was amplified using 40 pmol of forward and reverse degenerate primers in a 50 ␮l total volume with 2.5 U of platinum high-fidelity Taq DNA polymerase (Invitrogen), the supplied buffer and 1.5 mM MgCl2 . After 10 min of denaturation at 95 ◦ C, four sets of five cycles of 95 ◦ C for 15 s, an annealing temperature of respectively, 55, 50, 45 and 40 ◦ C for 30 s and 72 ◦ C for 1 min 30 s were performed, followed by 25 cycles of 95 ◦ C for 15 s, 37 ◦ C for 30s, 72 ◦ C for 1 min 30 s with an Applied Biosystems 9700 Thermocycler. Nested PCR was performed using forward and reverse primers, 2 ␮l of the previous reaction and the same

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amplification program. Analysis of the product was carried out on 1% agarose gels in TAE buffer stained with ethidium bromide. Fragments of interest were excised from the gel, purified on silica beads (Geneclean kit, BIO 101) and cloned into pCR 2.1-TOPO (Invitrogen). Top 10F competent cells were transformed according to the manufacturer’s instructions. Plasmids from positive clones were prepared by alkaline lysis [27]. Sequencing was performed on an ABI 377 automated sequencer (Applied Biosystems), using methods and reagents of the supplier. The cDNA fragment obtained was extended in both directions by performing 5 and 3 RACE using adult worm cDNA with the SMART RACE kit (Clontech) as a template, and primers derived from the sequence of the initial fragment (Table 1 in Supplementary Material online). The sequence thus obtained (1703 bp) was then compared to the TIGR gene index database (http://www.tigr.org/tigr-scripts/tgi/T index.cgi? species=s mansoni) using BLASTN. In addition, genomic sequence data comprising individual shotgun sequencing reads and contigs available from the Sanger Centre (http://www.sanger. ac.uk/Projects/S mansoni/) and TIGR (http://www.tigr.org/tdb/ e2k1/sma1/) web sites were subjected to BLASTN analysis using the BLAST Stand-Alone Programme for Win32 ( ftp://ftp.ncbi.nlm.nih.gov/blast/executables/LATEST-BLAST/) using libraries downloaded from ftp://ftp.sanger.ac.uk/pub/ pathogens/Schistosoma/mansoni/genome. The sequential use of these approaches identified the sequences listed in Table 2 in Supplementary Material online as containing CBP coding sequences and allowed us to compile a theoretical coding sequence. The extremities of this sequence, named SmCBP1, were verified by 5 and 3 RACE PCR as above and the full-length coding sequence was amplified as three fragments (see below) and fully sequenced. In order to determine whether paralogues of SmCBP1 were present in S. mansoni, we next carried out a search of the TIGR gene index database using the SmCBP1 peptide sequence and TBLASTN and the sequences obtained were used to screen the genomic sequence databases as previously. Sequences obtained are listed in Table 2 in Supplementary Material online and the compiled coding sequence of SmCBP2 was submitted to 5 and 3 RACE PCR in order to attempt to complete the sequence. 2.3. Plasmid construction and fusion proteins The cDNA sequence of SmCBP1 encoding the open reading frame (6282 bp) was cloned into the pTL-1 vector (a modified version of pSG5; Stratagene) using the BamHI/NotI restriction sites. Three different PCR fragments were produced to entirely cover the ORF. The complete sequence was recovered by ligating the PCR products into the pTL1 vector with BamH1/NotI restriction sites introduced into the oligonucleotides and unique EcoRI/HindIII restriction sites found in the SmCBP1 sequence. Briefly, reverse transcription of 5 ␮g of total RNA from adult worm was carried out using random hexamers and the ThermoscriptTM kit (Invitrogen) as described by the supplier. The resulting cDNA was used as template for each PCR. The fragment encoding amino acid residues 1–544 was amplified with SmCBPBam5 and SmCBPEco3 primers

(Table 1 in Supplementary Material online). The fragment encoding residues 545 to 1325 was amplified with SmCBPEco5 and SmCBPHind3 . The fragment encoding residues 1326 to 2093 (C-terminal end including the stop codon) was amplified with SmCBPHind5 and SmCBPNot3 . Each PCR product was first cloned into pCR4 (TOPO TA Cloning; Invitrogen) and sequenced. In order to obtain a clone encoding the complete coding sequence, sequence-validated clones containing each of the above inserts were first digested with the appropriate enzymes and the purified inserts included in a ligation mixture containing the BamHI/NotI digested pTL-1 vector, giving the SmCBP1 pTL-1 construct. The GST-SmCBP1 construct encoding the full-length SmCBP protein in frame with GST, was obtained by digesting the SmCBP1 pTL-1 construct with BamHI/NotI and cloning the resulting insert into the BamHI/NotI cloning sites of the pGEX4T3 vector (Amersham). To generate the GST HAT SmCBP1 construct, we amplified the region encoding amino acid residues 675–1387 with primers SmCBPHAT5 and SmCBPHAT3 , using the SmCBP1 pTL-1 construct as a template. After sequence verification, the PCR product was cloned in frame with GST into the BamHI/EcoRI cloning sites of the pGEX-4T3 vector. Constructs pTL1FF1 (encoding the full-length SmFtz-F1 protein) and GST-SRC-1 were previously described in [19] and in [28], respectively. The GST HAT p/CAF vector is described elsewhere [29]. 2.4. Phylogenetic analyses Accession numbers of the sequences included in the data set are listed in the legends of Figs. 3 and 4. Amino acid sequences were aligned with the use of the BioEdit v7.0.1 package (http://www.mbio.ncsu.edu/BioEdit/bioedit.html) and phylogenetic inference was restricted to sites that could be unambiguously aligned (435 shared sites, corresponding to the histone acetyltransferase domain, were analysed). Full-length alignments and sites used in the analysis are shown in Fig. 3. Phylogenetic analysis of the data set was carried out using MrBAYES v3 0b4 [30]. Bayesian analysis was performed using the JonesTaylor-Thornton (JTT) amino acid replacement model [31] + Γ (gamma distribution of rates and four rate categories) + I (proportion of invariant sites), with proportion of invariant sites and the shape parameter alpha of Γ distribution estimated from the data. The JTT Model was estimated using ProtTest [32]. Briefly, starting trees were random, four simultaneous Markov chains were run for 2 million generations, burn-in values were set at 50,000 generations (based on empirical values of stabilizing likelihoods), and trees were sampled every 100 generations. Bayesian posterior probabilities were calculated using a Markov chain Monte Carlo (MCMC) sampling approach [33] implemented in MrBAYES v3 0b4. 2.5. Quantitative real-time PCR Total RNA (2 ␮g) from miracidia, sporocysts, cercaria, schistosomula and adult male and female worms were reverse transcribed using the ThermoscriptTM RT-PCR System (Invitro-

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gen). cDNA (50 ng) were used as templates for PCR amplification using the SYBR Green Master Mix and the ABI PRISM 7000 sequence detection system (Applied Biosystems). Primers (Table 1 in Supplementary Material online) specific for S. mansoni ␣-tubulin (Genbank accession number M80214; [34]), for SmCBP1, for SmCBP2 and for SmGCN5 (Genbank accession number AY337317; [18]) were designed by the Primer Express Program (Applied Biosystems) and used for amplification in triplicate assays. In order to determine the efficiency of the PCR reactions with each primer pair, Ct values were obtained for cercarial cDNA in amounts ranging from 40 pg to 100 ng. The standard curves obtained (not shown) showed high linearity (Pearson correlation coefficient r > 0.99). The real time PCR efficiency (E) of one cycle in the exponential phase was calculated according to the equation: E = 10[−1/slope] [35]. The investigated transcripts all showed very high and comparable efficiency rates; SmCBP1, 2.05; SmCBP2, 2.05; SmGCN5, 2.07; S. mansoni ␣tubulin, 2.00. For graphical representation of quantitative PCR data, Ct values were obtained by deducting the raw cycle threshold (Ct values) obtained for ␣-tubulin mRNA, the internal standard, from the Ct values obtained for SmCBP1, SmCBP2 and SmGCN5 in miracidia, sporocysts, cercaria, schistosomula and adult male and female worms. The efficiency rates of the PCR reactions allow the ratios of expression to be calculated using the 2−Ct ratio [36] compared to the life-cycle stage showing the lowest relative level of expression in each case. 2.6. Purification of GST fusion proteins and GST pull-down assay GST alone and GST fusion proteins were expressed in the E. coli BL21 strain. Protein expression was induced with 0.1 mM isopropyl ␤-d-thiogalactopyranoside (IPTG) at 30 ◦ C for 4 h. Cells (corresponding to a culture of 200 ml) were harvested, resuspended in 4.5 ml of cold PBS triton X-100 1% containing protease inhibitors (complete EDTA free, Roche) and then sonicated (pulses of 4 s during 5 min). The suspensions were clarified by centrifugation and the supernatants were immediately placed in contact with 160 ␮l of a 50% suspension of glutathione-Sepharose 4B beads (Amersham) in PBS. Incubation was performed overnight under rotation at 4 ◦ C. Proteins bound to the agarose beads were washed three times with cold PBS Triton X-100 1%, three times with cold PBS and finally resuspended as a 50% suspension in PBS. Equivalent amounts of GST and GST fusion proteins bound to glutathione-Sepharose beads were used in pull-down assays. Three microliters of [35 S]-methionine-labelled SmFtz-F1 proteins translated in vitro, using the Quick Coupled Transcription/Translation System (Promega), were added to the beads in a final volume of 200 ␮l of buffer Z (25 mM HEPES pH 7.5, 12.5 mM MgCl2 , 0.1% NP-40, 150 mM KCl, 20% glycerol and protease inhibitors) supplemented with BSA (final concentration 1 mg/ml) and DTT (1 mM final) and incubated for 2 h at 4 ◦ C under rotation. Beads were collected by centrifugation, washed five times with NETN (20 mM Tris–HCl pH8, 100 mM NaCl, 1 mM EDTA, 0.5% NP-40 and protease inhibitors), resuspended in SDS-PAGE sample buffer and boiled 5 min prior to loading

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on a 10% denaturing polyacrylamide gel. The gel was fixed (10% acetic acid, 10% ethanol), dried and exposed to Hyperfilm (Amersham Pharmacia) under an amplifying screen. 2.7. Histone acetyltransferase (HAT) assay The HAT assay was done using recombinant GST fusion proteins expressed and purified as described above. GST fusion proteins bound to glutathione-Sepharose beads were washed once with buffer A (50 mM Tris–HCl pH8, 1 mM DTT, 0.1 mM EDTA, 10% glycerol and protease inhibitors) before starting the acetylation reaction. Reaction mixtures (20 ␮l) were set up in buffer A as follows: 2 ␮g of crude histone mixture (Sigma; calf thymus, type IIa), 0.25 ␮Ci of [3 H] acetyl CoA (4.40 Ci/mmol; Amersham Pharmacia) and an equivalent amount of GST fusion proteins. Tubes were incubated with agitation at 30 ◦ C for 30 min. The reactions were stopped by adding SDS sample buffer and the reaction products were resolved by SDS-PAGE (20%). Following fluorography (1 h with Amplify, Amersham Pharmacia), the gel was dried and exposed to Hyperfilm (Amersham Pharmacia) under an amplifying screen. 2.8. Cell culture and transfection CV-1 cells were maintained in DMEM (Invitrogen) supplemented with 10% fetal calf serum and gentamycin 50 ␮g/ml at 37 ◦ C and 5% CO2 . Cells were plated 1 day before transfection in six-well plates at a density of 200,000 cells per well. Transfections were performed with linear PEI ExGen 500 (Euromedex, France), under the conditions recommended by the supplier. Cells were lysed 48 h after transfection and assayed using a luciferase assay kit (Promega) in a Wallac Victor2 1420 multilabel counter (Perkin Elmer). For all experiments, protein content was used to normalize luciferase results. The SFRE-Luc and promo-FF1 reporter plasmids are described in [19] and in [37], respectively. Each experiment represents at least three sets of independent triplicates and the statistical significance of the results was determined using Student’s t-test. 3. Results 3.1. Molecular cloning and characteristics of SmCBP1 and SmCBP2 The complete cDNA sequence of SmCBP1 was obtained by a combination of a PCR-based strategy, RACE-PCR and use of the EST and genomic databases (see Section 2). The putative coding sequence thus obtained was confirmed by amplifying three contiguous segments of the cDNA, which were cloned and completely sequenced. 5’ and 3 untranslated regions were confirmed by RACE PCR followed by cloning and sequencing of the fragments obtained. The complete cDNA sequence obtained comprises 7479 bp and encodes a protein containing 2093 amino acids. SmCBP2 was identified by screening the S. mansoni EST and genomic databases with the SmCBP1 sequence. The sequence obtained was completed at the 3 end using RACE PCR, but repeated attempts to extend the 5 end of

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Fig. 1. (A) Comparative domain structure of CBP family proteins. The domain structures of SmCBP1 and SmCBP2 are compared to human CBP (HsCBP, accession number: S39162) human p300 (Hsp300, AAA18639) and Drosophila melanogaster CBP (DmCBP, AAB53050). The identity of each domain is as indicated and the scale indicates the number of amino acid residues. Arrows indicate the positions of the “NR boxes”. (B) Alignment of CBP “NR box” I. The SmCBP1 NR box I was aligned using the BioEdit v7.0.1 package (http://www.mbio.ncsu.edu/BioEdit/bioedit.html) with those of HsCBP, Hsp300, DmCBP, Aplysia californica CBP (AcCBP, AAL54859) and Tetraodon nigroviridis CBP (TnCBP, CAF96470). Identical residues are shaded in black and similar residues in grey. Asterisks indicate the conserved leucine residues of the LXXLL motif.

the sequence have failed. The partial sequence obtained comprises 5849 bp and encodes a protein of 1884 amino acids. The cDNA sequences of both SmCBP1 and SmCBP2 predicted in silico were further verified by PCR on adult worm cDNA using primers spanning the exons containing the coding sequence (Table 1 in Supplementary Material online). The identities of the fragments obtained were verified by sequencing the extremities. All the major domains typical of the CBP/p300 protein family are present in SmCBP1 and are shown diagrammatically in Fig. 1A. Moreover, the same is true for SmCBP2, even though the sequence is incomplete at the N-terminal extremity. These domains include the KIX domain and three Cys–His rich regions, respectively containing the TAZ1, PHD and TAZ2 and ZZ zinc finger domains all involved in binding transcriptional activators (reviewed in [38]), a bromodomain which recognizes acetylated lysine residues [39], and the HAT domain. Not shown on Fig. 1A is the C-terminal Q-rich domain which is poorly conserved in SmCBP1. Table 1 shows the degree of conservation of each of the above domains in SmCBP1 and SmCBP2 with respect to human CBP and p300 as well as Drosophila CBP. It is noticeable that, apart from the HAT domain, each of the domains of SmCBP2 is less well conserved compared to the human and

fruit fly counterparts than are those of SmCBP1. However, it is likely that each of the domains is functionally conserved. For instance, in the case of the bromodomain, both SmCBP1 and SmCBP2 have the conserved PXDLS motif (P734 MDLT in SmCBP1, P457 MDLS in SmCBP2) shown to be crucial, not only for binding to acetylated histones and transactivation, but also for binding the transcriptional repressor, C-terminal binding protein by mammalian p300 [40]. In addition to the above domains, CBP/p300 members contain three “NR-boxes” that determine interactions with nuclear receptor ligand-binding domains via LXXLL motifs (where L represents leucine and X any amino acid) [41]. In human CBP these motifs are spread throughout the sequence (motif I: L70 XXL73 L74 ; motif II: L358 XXL361 L362 ; motif III: L2057 XXL2070 L2071 ). SmCBP1 also contains three LXXLL motifs (motif I: L124 XXL127 L128 ; motif II: L210 XXL213 L214 ; motif III: L1450 XXL1453 L1454 ) but motif III is situated within the second TAZ zinc finger domain in contrast to the human motif III which is well downstream of this domain. In the partial SmCBP2 sequence, motifs II and III are also present, but in this case motif III at the C-terminal extremity of the protein. It is now clear that although all three motifs in mammalian CBP can be

Table 1 Levels of sequence identity in CBP domains.

SmCBP1 SmCBP2 HsCBP HsP300 DmCBP a

SmCBP1

SmCBP2

HsCBP

HsP300

DmCBP

100 44/31/50/63/73/34 66/53/56/54/55/53 67/53/55/57/59/53 47/51/56/55/45/56

44/31/50/62/73/34a 100 40/28/45/53/52/43 40/26/45/54/52/41 34/28/45/55/45/43

66/53/56/53/55/53 40/28/45/52/52/43 100 95/91/96/84/93/97 48/76/74/68/66/84

67/53/55/55/59/53 40/26/45/53/52/41 95/91/96/84/93/97 100 51/77/75/67/68/84

47/51/56/54/45/56 34/28/45/55/45/43 48/76/74/68/66/84 51/77/75/67/68/84 100

TAZ1/KIX/Bromo/HAT/ZZ/TAZ2. The figures indicate the percentage of identical amino acid residues in each domain.

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Fig. 2. SmCBP1 and SmCBP2 mRNA are differentially expressed during the schistosome life-cycle. The relative expression levels of mRNA encoding (A) SmCBP1, (B) SmCBP2 and (C) SmGCN5 ([18]; AY337317) were compared using quantitative RT-PCR using S. mansoni ␣-tubulin ([34]; M80214) as a reference gene. Results are expressed as the 2−Ct ratio (see Section 2) compared to the life-cycle stage showing the lowest relative expression level in each case. Life-cycle stages compared were adult male worms (AMW), adult female worms (AFW), miracidia (Mir) sporocysts (Sporo), cercariae (Cer) and schistosomula (schisto).

shown to interact with nuclear receptor ligand-binding domains [42], motif I is the most important. In vertebrate CBPs motif I is within a conserved region of the protein and flanking sequences are also similar to the NR box II of mammalian SRC1 [42] which we have shown to interact with SmFtz-F1 [28]. The alignment of SmCBP1 motif I and flanking regions with human CBP and p300, Tetraodon nigroviridis CBP, Aplysia californica CBP and Drosophila CBP is shown in Fig. 1B and demonstrates that, apart from the LXXLL motif itself, this region is not well conserved in invertebrate CBPs. This suggests that the mode of interaction between SmCBP1 and nuclear receptor partners may differ from that characterized in the case of mammalian CBP [43]. The absence of the N-terminal extremity of SmCBP2 (containing the LXXLL motif I) and the overall lower level of conservation of its functional domains, led us to carry out subsequent functional assays using SmCBP1. A further difference between SmCBP1 and SmCBP2 and mammalian CBPs is evident in the C-terminal region, downstream from the second TAZ zinc finger domain. In human

CBP a domain interacting with SRC1 and other factors and called the SRC1-interacting domain (SID) has been localized between residues 2058 and 2130 [44]. This region contains four ␣-helices and repeated QPGM/L motifs and no equivalent domain is detectable in either SmCBP1 or 2 but a similar domain, albeit lacking the QPGM/L repeated motif, is detectable in CBP from the marine gastropod Aplysia californica (not shown). This suggests that the schistosome CBPs may interact with a different set of transcriptional coactivators, specific to platyhelminths and indeed, no homologue of SRC1 or the p160 group of transcriptional coactivators (see [45] for review) is identifiable in the schistosome genomic or EST databases (R.P., unpublished). 3.2. Expression of SmCBP1 and SmCBP2 during the S. mansoni life-cycle CBP family proteins are involved in the transcription of a wide variety of genes [9] and it would be expected that

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Fig. 3. Alignment of CBP HAT domains. HAT domain peptide sequences of SmCBP1, SmCBP2, HsCBP, Hsp300, AcCBP, DmCBP and Caenorhabditis elegans CBP1 (CeCBP-1, P34545) were aligned using the BioEdit v7.0.1 package (http://www.mbio.ncsu.edu/BioEdit/bioedit.html). Peptide sequence residue numbers are indicated on the right-hand side. Black asterisks designate conserved cysteine and histidine residues implicated in the PHD zinc finger [47] and the phenylalanine residue essential for HAT activity is indicated by a diamond. Motifs A, B and D are conserved between GNAT family HATs and the CBP family and are involved in CoA binding [48]. Conserved residues (identical or similar) within the motifs are designated by grey asterisks. In the overall alignment identical residues are shaded in black and similar residues in grey.

they would be expressed constitutively. However, two members of this family are present in schistosomes and SmCBP1 and SmCBP2 showed significant structural differences, implying different functions in the parasite. We therefore determined the levels of expression of their respective mRNAs at different stages of the schistosome life-cycle using real-time quantitative PCR and S. mansoni ␣-tubulin as a reference. For purposes of comparison, we also analysed the expression of another ubiquitous transcriptional coactivator possessing HAT activity, S. mansoni GCN5 [18]. Results are shown in Fig. 2 expressed as the value of 2−Ct , the ratio of the relative expression level compared to the reference gene (Ct ) to that of the stage showing the lowest relative expression for each gene. This representation shows that all three genes are indeed expressed at each stage with miracidia showing the highest relative levels mRNA for each of the genes tested. However, marked differences in expression do exist, particularly between SmCBP1 (Fig. 2A) and SmCBP2 (Fig. 2B). The

relative expression of SmCBP1 and SmCBP2 are compared directly in Fig. 1 in Supplementary Material online, demonstrating that SmCBP2 is expressed at a higher level than SmCBP1 at each life-cycle stage except in cercariae, in which SmCBP1 shows a two-fold greater level of expression. Interestingly, SmGCN5 (Fig. 2C) also shows variations in expression level that are independent of the other two transcription cofactors. Thus although, as expected, all three coactivators are ubiquitously expressed during the schistosome life-cycle, the variation in their relative levels of expression point to distinct functional properties and roles in the expression of different sets of genes. 3.3. Phylogenetic analysis of the HAT domain of SmCBP1 and 2 The ability of CBP to function as a coactivator of transcription is due in part to its intrinsic HAT activity. The HAT

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domains of SmCBP1 and SmCBP2 are globally highly conserved (Table 1) and alignment of the SmCBP HAT domains to those of other species (Fig. 3) shows that this is true in particular of the PHD zinc finger domain and the central core A, B and D motifs. The plant homeodomain type (PHD) zinc finger [46] is an integral part of the CBP acetyltransferase domain, essential to the enzymatic activity and therefore a key contributor to the transcriptional activity of the CBP protein [47]. All the zinc-coordinating cysteine and histidine residues of the PHD domain are perfectly conserved in SmCBP1 and 2 (Fig. 3). However, the phenylalanine 76 (in the human CBP HAT domain, Fig. 3), preceding the last two conserved cysteine residues which was shown to be essential to the maintenance of HAT activity is conserved in SmCBP2, but is a tyrosine in SmCBP1 (Fig. 3). Similarly, the central core motifs A, B and D that have been shown to bind the CoA cofactor in GNAT family HATs (reviewed in [48]) and which are conserved in human CBP [49] are also conserved with minor differences in SmCBP1 and 2 (Fig. 3). On the other hand, both SmCBP1 and 2 contain an insertion, corresponding to residues 1181–1202 on the SmCBP1 peptide sequence, compared to mammalian and invertebrate CBPs. It is probable that these minor differences in HAT domain sequences determine species-specific variations in enzyme activity toward different substrates. The comparison of the different conserved domains between CBP family proteins (Table 1) indicated that SmCBP1 and SmCBP2 were globally more similar to each other than to CBPs from other species, but less so than human CBP and p300. In order to determine whether SmCBP1 or SmCBP2 represented orthologues of vertebrate CBP and p300, or on the contrary, if they had been generated by a different duplication event, we carried out phylogenetic analysis of metazoan CBP family proteins. This was based on the HAT domain and done using Bayesian inference (Mr. Bayes) and rooted with Aribidopsis thalania HAC4 (Fig. 4). All the main groupings are supported by high BPP values (2 million generations). The tree obtained confirms the close relationship between the vertebrate CBP and p300 proteins and infers a vertebrate-specific duplication event generating the corresponding genes. SmCBP1 and 2 cluster together on a branch with Caenorhabditis elegans CBP-1, but separate from another “invertebrate” branch containing two insect CBPs, along with A. californica CBP. The tree therefore supports a platyhelminth-specific duplication event generating SmCBP1 and 2 but suggests that this was more ancient than that generating vertebrate CBP and p300, or that the schistosome CBPs have undergone a more rapid evolution. 3.4. HAT activity of SmCBP1 CBP family proteins display histone acetyltransferase activity and are able to acetylate core histones in vitro [50] thereby potentiating transcriptional activity. To determine whether the highly conserved HAT domain of SmCBP1 possesses this activity, we used recombinant proteins to conduct a histone acetylation assay. Amino acids 675–1387 of SmCBP1, which contain the HAT domain, were fused to GST and the HAT domain

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Fig. 4. SmCBP1 and SmCBP2 derive from a schistosome-specific gene duplication. Phylogenetic analysis of CBP family HAT domains was carried out by Bayesian inference restricted to sites that could be unambiguously aligned (435 shared sites, corresponding to the histone acetyltransferase domain, were analysed). Phylogenetic analysis of the data set was carried out using MrBAYES v3 0b4 (see Section 2). Numbers at branch points are Bayesian posterior probabilities. Sequences used were as in Figs. 1B and 3, with the addition of Anopheles gambiae CBP (AgCBP; EAA06516), Xenopus laevis CBP (XlCBP; AAH86282), Mus musculus CBP (MmCBP; AAB28651), M. musculus p300 (Mmp300; NP 808489) and T. nigroviridis p300 (Tnp300; CAG04516). Arabidopsis thalania HAC4 (AtHAC4; NP 564706) was used as an outgroup.

of the human p/CAF protein fused to the GST was used as a positive control. The corresponding recombinant proteins were expressed and purified for use in the assay. Equivalent amounts of GST alone and both fusion proteins (Fig. 5, lower panel) were incubated with calf thymus crude histone mixture and [3 H]-acetyl-CoA. The results showed that SmCBP1 acetylates histones in vitro (Fig. 5, upper panel). As expected, GST alone did not show any HAT activity, and the HAT domain of human p/CAF strongly acetylated free histones. Under the same conditions, SmCBP1 seemed to preferentially acetylate histone H4, and to a lesser extent, H3 and H2. No signal was detected when histone proteins were absent from the reaction. Thus, these data clearly demonstrate that the HAT domain of SmCBP1 is active in vitro and can acetylate free histone proteins.

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Fig. 5. The HAT domain of SmCBP1 acetylates free histones in vitro. The HAT assay was done using recombinant GST fusion proteins (see Section 2 for details). Upper panel: autoradiography of the acetylation assay. Histones H2, H3 and H4 are indicated. Bottom panel: coomassie blue stained gel of the acetylation reactions showing that equal amounts of each fusion protein were used in the assay.

3.5. SmCBP1 interacts with SmFtz-F1 and potentiates its transcriptional activity In order to determine whether SmCBP1 can act as a transcriptional coactivator, we decided to study its transcriptional properties with a well-characterized schistosome transcription factor, the SmFtz-F1 nuclear receptor. Indeed, CBP/p300 is thought to integrate various signaling pathways and can interact with a large number of transcription factors, including nuclear receptors [51,52], the interaction occurring with the ligand-binding domain [43]. Moreover, it was also demonstrated that CBP can mediate the transcriptional activity of SF-1, one of the human homologues of the Ftz-F1 family [53]. Thus, we first decided to test the capacity of SmCBP1 to interact in vitro with SmFtzF1. For this purpose, the GST fusion protein of the full-length SmCBP1 was bound to glutathione-Sepharose and incubated with 35 S-labelled SmFtz-F1 protein. The receptor-interacting domain (RID) of the human SRC-1 coactivator fused to GST (GST SRC1RID construct; Fig. 6A) was used as a positive control, since we previously demonstrated that this fragment strongly interacts with SmFtz-F1 in identical GST pull-down experiments [28]. The results (Fig. 6A) showed that SmCBP1 interacts with SmFtz-F1. Compared to the signal obtained with the SRC1 RID, the interaction between SmCBP1 and SmFtz-F1 is weaker but remains specific since no signal was detected with GST alone. This result led us to study the biological activity of SmCBP1 in a cellular context by using cotransfection experiments. We have previously shown that the CV-1 mammalian cell line represents a satisfactory model to test the transcriptional activity of schistosome nuclear receptors and of SmFtz-F1 in particular [19,28]. Our previous work also led to the characterization of various reporter plasmids that contain the luciferase gene

under the control of different promoters responsive to SmFtzF1. Here, we used two such reporter plasmids. One contains the SF-1 response element (SFRE) repeated three times upstream of the thymidine kinase minimal promoter and the second is the so-called “promo-FF1 Luc” vector that contains a 300 bp region upstream of exon 2 of the Smftz-f1 gene from which SmFtzF1 is strongly capable of transactivating the transcription of the reporter gene [37]. We therefore cotransfected plasmids expressing full-length SmFtz-F1 and SmCBP1 into CV-1 cells, along with one or the other reporter vector. Using the SFRE reporter vector (SFRE-Luc; Fig. 6B) SmFtzF1 alone led to a 2–3 fold increase in luciferase activity compared to the background with the reporter vector alone. SmCBP1 had no effect on transcription from this promoter when used on its own. However, when we cotransfected the cells with increasing amounts of the plasmid expressing SmCBP1 a slight, significant increase in transcriptional activity was observed (Fig. 6B) and this activity increased slightly, but not significantly, with increasing doses of SmCBP1. This suggested that the latter could potentiate the transcriptional activity of SmFtzF1, but that this effect was maximal at the lowest dose of SmCBP1. In contrast, when we used the Promo-FF1 Luc reporter vector, in line with our previous observations, when transfected alone SmFtz-F1 led to an approximate 5-fold induction of the reporter gene (Fig. 6C, [37]). No significant transactivation of the luciferase gene above background was detected when the highest amount of the vector-expressing SmCBP1 (three times that used for the vector-expressing SmFtz-F1) was transfected alone in CV-1 cells, indicating that SmCBP1 had no effect on its own on the Smftz-f1 promoter. In contrast, when SmCBP1 was coexpressed with SmFtz-F1, a significant increase in transcription was noted and this was dose-dependent, suggesting that SmFtzF1 and SmCBP1 interact functionally to transactivate transcription from promoter containing response elements for the SmFtzF1 nuclear receptor. It should be noted that on Fig. 6C the significance levels given compare results obtained with SmFtz-F1 and SmCBP1 to those obtained with the receptor alone. However, the increases in transcriptional activity obtained with increasing amounts of SmCBP1 (for instance the 3× level compared to the 2× level) were also statistically significant (p < 0.01). The marked and dose-dependent activation obtained using the Smftz-f1 gene promoter probably reflects a less artificial context for transcriptional activity, compared to the SFRE-Luc promoter, which may allow supplementary factors to amplify the effect of the interaction between SmFtz-F1 and SmCBP1. Moreover, it should be underlined that SmCBP1 requires the presence of SmFtz-F1 in order to coactivate transcription from this promoter (Fig. 6C). Taken together, our results demonstrate that SmCBP1 can cooperate functionally with a schistosome nuclear receptor to transactivate a reporter gene in cotransfection experiments. 4. Discussion We have described the molecular cloning of two members of the CREB-binding protein (CBP) family from the platy-

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Fig. 6. SmCBP1 interacts with SmFtz-F1 and potentiates its transcriptional activity. (A) SmCBP1 interacts with SmFtz-F1 in vitro. A GST pull-down assay was used to test the interaction between SmCBP1 and SmFtz-F1 in vitro (see Section 2 for details). Upper panel: autoradiography of the GST pull-down reactions. Five percent of the in vitro synthesized SmFtz-F1 protein engaged in the reactions was loaded in the first well. Bottom panel: coomassie blue staining of the gel showing the quantities of GST fusion proteins used for the assays. (B and C) SmCBP1 potentiates the transcriptional activity of SmFtz-F1. As indicated above the bar graphs, CV-1 cells were cotransfected with 500 ng of reporter vector (“SFRE-Luc” in panel B and “Promo FF1-Luc” in panel C), 500 ng of plasmid expressing full-length SmFtz-F1 and increasing amounts of plasmid expressing full-length SmCBP1 (500, 1000 and 1500 ng). When necessary, the DNA quantity was kept constant using the empty expression vector (pTL1). Results are expressed as normalized luciferase units (RLU) and the statistical significance of increases in luciferase activity of cells transfected with SmFtz-F1 and SmCBP1 compared to cells transfected with SmFtz-F1 alone was determined using Student’s t-test (* p < 0.05; ** p < 0.01; *** p < 0.001).

helminth parasite S. mansoni, and the functional characterization of one of them. Both SmCBP1 and SmCBP2 show highly conserved domain structures, and a high level of peptide sequence conservation within the domains. This suggests that each of the domains (ZZ, TAZ, KIX, bromodomain, PHD and HAT domains) is likely to be functional and indeed we have shown that SmCBP1 can acetylate histones, physically interact with a schistosome nuclear receptor and coactivate transcription in a heterologous assay system. Therefore, schistosome nuclear receptors use at least some of the components of coactivator complexes that link transcription factors to the basal transcriptional machinery and facilitate transcription via chromatin modification (reviewed in [54]). However, detailed analysis shows that certain aspects of CBP function in schistosomes are likely to differ from those characterized in vertebrates, as was the case for the schistosome nuclear receptor, SmFtz-F1 [28]. Moreover, this would be expected since variations in the regulation of gene transcription represent the principal motor for the generation of diversity in evolution [1].

The presence of two isoforms of CBP in S. mansoni is an example of just such a diversification of the regulation of transcription and raises questions both about their evolutionary origin and their respective roles. In vertebrates, two isoforms, CBP and p300, are also present with considerable redundancy in their functional characteristics. However, subtle differences in levels of expression of CBP and p300 during development [55] and distinct phenotypes produced by knockouts of the two coactivators in mice [56] show that although their functions overlap to a degree, they are not redundant. In ecdysozoan invertebrates, only one isoform of CBP/p300 is present (reviewed in [9]) and in the lophotrochozoan A. californica, again only one isoform has so far been described [57]. However, since similar PCR-based strategies including the use of degenerate oligonucleotide primers were used both in the latter study and in our work, initially leading in each case to the cloning of only one isoform, no conclusion can be drawn concerning the eventual existence of another isoform in A. californica. The presence of two isoforms in S.

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mansoni thus led us to question whether they were orthologues of vertebrate CBP and p300, or derived from a separate gene duplication event. A phylogenetic study using Bayesian inference showed conclusively that the duplication events leading, in vertebrates to CBP and p300 and in platyhelminths to SmCBP1 and SmCBP2, were separate. Moreover, branch lengths seem to indicate that the latter duplication is more ancient, but only the sequencing of further lophotrochozoan genomes such as those of Biomphalaria glabrata and A. californica now in progress (http://www.genome.gov/10002154) will determine whether this duplication is basal to the Lophotrochozoa. The existence of paralogous forms of a large number of genes was highlighted by the large-scale sequencing of schistosome ESTs [58] which the authors ascribe in part to a possible adaptation to the parasitic life-style, acting as a mechanism for generating antigenic variability in the absence of highly variable gene families such as those in Plasmodium. However, in the case of regulatory molecules such as SmCBP1 and SmCBP2 this seems unlikely. Another emerging feature of the schistosome genome seems to be the absence of certain gene families that are essential components of vertebrate regulatory complexes or signaling networks (R.P., unpublished) although it is not yet clear whether this is due to the specific evolution of certain genes in vertebrates, or to gene loss from schistosomes. One example is provided by the p160 steroid receptor coactivator (SRC) family, for which no orthologues are present in the current S. mansoni genomic or EST sequence releases (R.P., unpublished). Although it is theoretically possible that the corresponding genes may be present in fragments of the genome that have not been sequenced, this is unlikely considering the overall genome coverage (>6×) of the current release. SRC family cofactors are components of CBPcontaining nuclear receptor coactivator complexes [45] and bind to a conserved ␣-helical motif within the C-terminal SID domain of CBP [44]. The lack of conservation of this domain and motif in SmCBP1 and SmCBP2 supports the absence of the SRC family in schistosomes, whereas its relative conservation in A. californica CBP suggests that this absence from schistosomes may represent a specific gene loss and not that the SRC family is a vertebrate synapomorphy. However, the presence of SRC family orthologues has yet to be demonstrated unequivocally in any invertebrate and, again, the question will only be resolved by genome sequencing in progress. Whether the absence of SRC family genes from schistosomes is due to gene loss or their vertebrate-specific evolution, the duplication of the schistosome CBP gene permits the diversification of the function of the gene products. That they do not fulfil redundant functions is evidenced by their expression patterns throughout the schistosome life-cycle. Although both genes are expressed at all life-cycle stages, relative expression levels vary, clearly suggesting distinct functions. This is further supported by peptide sequence divergences, notably in the C-terminal region. The SmCBP2 C-terminal region shows richness in glutamine residues much more typical of the CBP family than that of SmCBP1. This may suggest that SmCBP2 binds a distinct subset of cofactors from SmCBP1, although the absence of the SRC family indicates that these are likely

to be schistosome-specific. This diversification in the detailed structure and function of the schistosome orthologues of central components of the metazoan transcriptional machinery is in favour of the view that such diversification is one of the most important motors for evolution. Moreover, a wide variety of studies are currently being undertaken into transcriptional regulation and chromatin dynamics in the context either of normal development or in various pathologies including cancer. The current enormous input into the schistosome sequence databases will permit the identification of a panel of proteins, probably including schistosome-specific molecules that interact with SmCBPs, involved in these mechanisms in the parasite and these will open up opportunities for the development of specific and novel therapeutic strategies Acknowledgements The work was supported by the Institut National de la Sant´e et de la Recherche M´edicale (U547), the Institut Pasteur de Lille, the Centre National de la Recherche Scientifique and the Microbiology program of the Minist`ere de l’Education Nationale, de la Recherche et de la Technologie (MENRT). BB was supported by the MENRT. FO is supported by INSERM-Region Nord Pas de Calais. We thank Dr. Franc¸ois Fuks (from the Free University of Brussels) for kindly providing the GST HAT p/CAF vector. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.molbiopara.2005.12.006. References [1] Levine M, Tjian R. Transcription regulation and animal diversity. Nature 2003;424:147–51. [2] Nowak SJ, Corces VG. Phosphorylation of histone H3: a balancing act between chromosome condensation and transcriptional activation. Trends Genet 2004;20:214–20. [3] Zhang Y, Reinberg D. Transcription regulation by histone methylation: interplay between different covalent modifications of the core histone tails. Genes Dev 2001;15:2343–60. [4] Sterner DE, Berger SL. Acetylation of histones and transcription-related factors. Microbiol Mol Biol Rev 2000;64:435–59. [5] Zhang Y. Transcriptional regulation by histone ubiquitination and deubiquitination. Genes Dev 2003;17:2733–40. [6] Jenuwein T, Allis CD. Translating the histone code. Science 2001;293:1074–80. [7] Margueron R, Trojer P, Reinberg D. The key to development: interpreting the histone code? Curr Opin Genet Dev 2005;15:163–76. [8] Allfrey VG, Faulkner R, Mirsky AE. Acetylation and methylation of histones and their possible role in the regulation of RNA synthesis. Proc Natl Acad Sci USA 1964;51:786–94. [9] Goodman RH, Smolik S. CBP/p300 in cell growth, transformation, and development. Genes Dev 2000;14:1553–77. [10] Petrij F, Giles RH, Dauwerse HG, et al. Rubinstein-Taybi syndrome caused by mutations in the transcriptional co-activator CBP. Nature 1995;376:348–51. [11] Roelfsema JH, White SJ, Ariyurek Y, et al. Genetic heterogeneity in Rubinstein-Taybi syndrome: mutations in both the CBP and EP300 genes cause disease. Am J Hum Genet 2005;76:572–80.

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