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Human Molecular Genetics, 2003, Vol. 12, No. 4 441-450
© 2003 Oxford University Press

Loss of CBP acetyltransferase activity by PHD finger mutations in Rubinstein–Taybi syndrome

Eric Kalkhoven1,{dagger}, Jeroen H. Roelfsema2,{dagger}, Hans Teunissen1, Annemieke den Boer2, Yavuz Ariyurek2, Alt Zantema1, Martijn H. Breuning2, Raoul C.M. Hennekam3,4 and Dorien J.M. Peters2,*

1Department of Molecular Cell Biology and 2Department of Human and Clinical Genetics, MGC Centre for Biomedical Genetics, Leiden University Medical Centre, Wassenaarseweg 72, 2333 AL Leiden, The Netherlands and 3Department of Clinical Genetics and 4Department of Pediatrics, University of Amsterdam, Academic Medical Centre, Amsterdam, The Netherlands

Received November 22, 2002; Accepted December 13, 2002


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Disruption of one copy of the human CREB binding protein (CBP or CREBBP) gene leads to the Rubinstein–Taybi syndrome (RTS), a developmental disorder characterized by retarded growth and mental functions, broad thumbs, broad big toes and typical facial abnormalities. The CREB binding protein (CBP) is an essential transcriptional coactivator for many different transcription factors. CBP has the intrinsic ability to acetylate histones and other proteins, which is regarded as an important step in transcription activation. In vitro studies have shown that this enzymatic activity critically depends on the integrity of a plant homeodomain (PHD)-type zinc finger in the HAT domain of CBP. We therefore investigated whether PHD finger mutations are present in RTS patients. Mutational analysis of 39 patients revealed eight novel heterozygous mutations in the HAT domain of CBP, one of which alters a conserved PHD finger amino acid (E1278K), while a second mutation deletes exon 22, which encodes the central region of the PHD finger. Functional analysis of these RTS-associated PHD finger mutants showed that they lacked in vitro acetyltransferase activity towards histones and CBP itself and displayed reduced coactivator function for the transcription factor CREB. Importantly, in EBV-transformed lymphoblastoid cells from the exon 22 deletion patient we found ~50% less endogenous CBP HAT activity. These findings therefore underscore the functional importance of the PHD finger in vivo and imply that reduction of CBP HAT activity, as exemplified here by disruption of the PHD finger, is sufficient to cause RTS.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Regulation of transcription is the central mechanism by which cells respond to developmental and environmental cues. As a consequence, disturbances in this highly regulated process, for example by genetic alterations in the genes encoding the regulatory proteins, often result in developmental disorders and uncontrolled cell growth (1–5). One example of such a congenital developmental disorder is the Rubinstein–Taybi syndrome (RTS), which is characterized by retarded growth and mental function, broad thumbs, broad big toes and typical facial abnormalities (6). This syndrome is caused by chromosomal rearrangements, microdeletions and point mutations in one copy of the CREB binding protein (CBP or CREBBP) gene (7). Haploinsufficiency of CBP is the probable cause of RTS in humans, since no clear phenotypic differences were observed between patients in which microdeletions or truncating mutations were found (8–13).

The CBP gene encodes CREB binding protein (CBP) that, like the related protein p300, functions as an essential coactivator protein for many different transcription factors (14–17). CBP and p300 can stabilize the transcription complex by binding to several proteins simultaneously, thereby functioning as a scaffold or physical bridge. CBP and p300 harbour histone acetyltransferase activity (18,19), which is important in the regulation of transcription, as hyperacetylation of N-terminal histone tails correlates with transcriptional activity (20–22). In addition, CBP and p300 can acetylate non-histone proteins including several sequence-specific transcription factors and the basal transcription factors TFIIE and TFIIF (reviewed in 23). Within the HAT domain of CBP (amino acids 1232–1709), two functionally important regions have been identified. One of these regions (amino acids 1459–1541) is partly conserved between HAT subfamilies (24), and was subsequently postulated to be the coenzyme A (CoA) binding site (25). The second important region in the HAT domain of CBP is the plant homeodomain (PHD) type zinc finger (26), also named leukaemia-associated-protein (LAP) finger (27) or trithorax consensus (TTC) finger (28). This type of zinc-finger is characterized by a cys4-his-cys3 motif, and is predominantly found in proteins that function at the chromatin level (26). We and others have recently shown that the PHD finger of CBP (amino acid 1237–1311) is an integral part of the enzymatic core of the acetyltransferase domain (29,30).

Microdeletions and truncating mutations in CBP have been observed in only ~20% of all RTS cases (reviewed in 9). Since in vitro studies have shown that single amino acid changes in the PHD finger of CBP result in partial or complete loss of HAT activity (29,30), we examined whether PHD finger mutations are present in RTS patients. Here we report eight novel heterozygous mutations in the exons encoding the CBP HAT domain. One of those results in alteration of a conserved PHD finger amino acid (E1278K), while the second mutation is an in-frame deletion of exon 22, which encodes the central region of the PHD finger. Functional analysis of these RTS-associated PHD finger mutants showed that both result in complete loss of HAT activity and EBV-transformed lymphoblastoid cells from the exon 22 deletion patient displayed ~50% reduced CBP HAT activity. These findings highlight the importance of the PHD finger in CBP function in vivo and indicate that a reduction in the level of CBP HAT activity, as a result of disruption of this zinc finger, is sufficient for the development of RTS.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
PHD finger mutations associated with Rubinstein–Taybi syndrome
To detect PHD finger mutations in RTS patients, the genomic DNA of 39 patients was subjected to denaturing gradient gel electrophoresis (DGGE) analysis with intronic primers flanking the exons encoding the HAT domain (exons 20–29). Since DGGE conditions were unfavourable for exon 26, this fragment was analysed by SSCP. Eight novel heterozygous aberrations were identified (Table 1). Mutations in exons 20–23, which encode the PHD finger (amino acid 1237–1311), were detected in three patients (RT198.3, RT209.1 and RT211.3). Patient RT198.3 displayed a G-to-C mutation at position +5 of the exon 21 splice donor site, which is likely to result in a splicing error and subsequently in truncation of the CBP protein at amino acid 1260. In patient RT209.1 the guanine at position 3832 was changed to adenine, resulting in an amino acid change from glutamic acid to lysine at codon 1278 (Fig. 1A). This glutamic acid is located between the second and third zinc-coordinating cysteine residues of the PHD finger and is strictly conserved among CBP and p300 proteins from different metazoans (Fig. 1C and D). In patient RT211.3 the splice acceptor site of exon 22 was mutated from AG to TG. This mutated acceptor site may no longer be recognized by the splicing machinery, resulting in fusion of exon 21 to exon 23. Since this is an in-frame fusion, the mutation is predicted to result in deletion of amino acid 1280–1305, which comprises the central part of the PHD finger (Fig. 1D). Indeed, RT–PCR of the patient mRNA revealed the presence of a 104 bp band corresponding to the exon 22 deletion and sequence analysis confirmed the in-frame fusion (Fig. 1B). Importantly, the parents of these patients did not display the same DGGE pattern as the patients (Fig. 1A and B and data not shown), indicating that these PHD finger mutations are de novo. Clinically there were no significant differences between the patients carrying a missense or in-frame deletion, both compared to each other as to the phenotype of RTS patients in general. Furthermore, the mutations observed in the PHD finger were not detected in 120 control chromosomes (data not shown).


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  		Table 1. Novel genetic aberrations in the CBP gene of RTS patients
 


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  		Figure 1. PHD finger mutations in CBP in Rubinstein–Taybi patients. (A) DGGE analysis of exon 21 of patient RT209.1 and parents (RT209.3 and RT209.2). The arrowhead on the left indicates the band observed only in the patient. The reverse sequence of this patient is given in the bottom panel with the heterozygous mutation indicated by an arrow. (B) DGGE analysis of exon 22 of patient RT211.3 and parents (RT211.2 and RT211.1). The arrowheads on the left indicate the bands observed only in the patient. In the middle panel RT–PCR products are shown, separated on an agarose gel. The bottom panel shows the sequence of the 104 bp PCR product, with the fusion between exon 21 and 23 fusion indicated. (C) Putative structure of the PHD finger in human CBP, based on the structures of the PHD fingers in KAP-1 (59) and WSTF (60). The zinc-coordinating cysteines and histidine and the zinc atoms (Zn) are indicated, as well as the conserved glutamic acid (E) which is altered in patient RT209.1. (D) Schematic representation of CBP, showing the position of the acetyltransferase domain (HAT) and the PHD finger within this domain (PHD), the KIX domain, the bromodomain (BD), the CH3 region, the p160 binding site and the N-terminal and C-terminal transactivation domains (TAD). Numbers refer to amino acid positions in human CBP. An alignment of the PHD finger in the HAT domains of human CBP (hCBP; GenBank accession number AAC51331), mouse CBP (mCBP; P45481), human p300 (hp300; A54277), mouse p300 (mp300; ESTs BE301690, BF122259 and BE848026), frog Xenopus laevis CBP (xCBP) (61), fruitfly Drosophila melanogaster CBP (dCBP; T13828), mosquito Anopheles gambiae CBP (agCBP; EAA06516), California sea hare Aplysia california (apCBP; AAL54859) and nematode worm Caenorhabditis elegans CBP (ceCBP; P34545) is given. Grey boxes indicate the (putative) zinc-coordinating cysteines and histidine. The conserved glutamic acid (E) which is altered in patient RT209.1, and exon 22, deleted in patient RT211.3, are also indicated.

 
In addition to the PHD finger mutations, five mutations downstream of the PHD finger were found, four of which (patients RT39.1, RT231.1, RT3002 and RT2960) are predicted to result in truncation of the CBP protein at various points within the HAT domain (Table 1). In patient RT2644 a mutation was observed which is predicted to alter the conserved arginine at position 1664 to histidine. Neither the parents of patient RT2644 nor 100 healthy control individuals displayed the same DGGE pattern as the patient (data not shown), indicating that this variation is also de novo and is likely to be the disease-causing mutation.

RTS-associated PHD finger mutations result in loss of CBP acetyltransferase activity
To examine the functional consequences of the PHD finger mutations found in patients RT209.1 and RT211.3, analogous mutant forms of the highly homologous mouse CBP protein were generated. The gutamic acid E1278 in human CBP (GenBank accession number AAC51331) corresponds to E1279 in the murine protein (GenBank accession number P45481). To analyse the in vitro HAT activity of wild-type CBP and the mutants described above, we first overexpressed the CBP HAT domain (CBPHAT) fused to the DNA binding domain of the yeast transcription factor Gal4 (Gal4DBD), in U-2 OS cells. Subsequently, these proteins were isolated by immunoprecipitation with an anti-Gal4DBD antibody, and tested for their ability to acetylate purified Drosophila core histones in vitro. While wild-type CBPHAT clearly acetylated histones, with a preference for histone H3 and H4 (18,31), the E1279K mutant and the exon 22 deletion mutant lacked this activity (Fig. 2A). Furthermore, unlike wild-type CBP HAT both mutants failed to display auto-acetylation activity, as detected with an antibody against acetylated lysine residues. As a negative control, we included the C1287A mutant (which corresponds to C1286 in human CBP; Fig. 1C), in which one of the zinc-coordinating cysteines of the PHD finger is altered (30). Western blot analysis showed that comparable amounts of the Gal4DBD fusions were immunoprecipitated (Fig. 2A).



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  		Figure 2. RTS-associated PHD finger mutations result in loss of enzymatic activity of the CBP HAT domain. (A) Ability of wild-type CBP HAT domain or various mutants to acetylate core histones (H2A, H2B, H3, H4) in vitro. An aliquot from each reaction was used for western blot analysis, using antibodies against the Gal4DBD or acetylated lysine residues, as indicated. Only the wild-type CBP HAT domain displays acetylation activity towards histones and itself, while the expression of all proteins was verified with an {alpha}-Gal4DBD antibody. (B) Transcriptional activity Gal4DBD fusions of the CBP HAT domain and various mutants in U-2 OS cells, tested in transient transfection experiments as schematically represented at the top of the figure. Activation of the luciferase reporter, normalized for ß-galactosidase activity, is shown as fold induction over Gal4DBD alone. Bars represent the average of a minimum of three independent experiments assayed in duplicate ±SEM.

 
Next, we examined the effect of these two RTS-associated PHD finger mutations on the transcriptional activity of the CBP HAT domain. Previously, Gal4DBD-CBPHAT was shown to stimulate HAT-dependent transcription of a reporter containing five Gal4 binding sites placed upstream of the adenovirus major late TATA box (5xGal4-AdMLTATA-Luc) (25). While the Gal4DBD-CBPHAT fusion gave 45-fold more transcriptional activity than the Gal4DBD alone in U-2 OS cells, both mutants failed to display any transcriptional activity (Fig. 2B). Similar results were obtained in 911 cells (data not shown), indicating that the phenotype of the mutants is not cell-type-specific. Taken together, these data show that RTS-associated PHD finger mutations ablate acetyltransferase activity of the CBP HAT domain.

RTS-associated PHD finger mutations reduce the coactivator potential of full-length CBP
Since regions other than the HAT domain are also important for CBP to function as a transcriptional coactivator (31–33), we next investigated the effect of the RTS-associated PHD finger mutations in the context of the full-length protein. For this, we stably expressed the 5xGal4-E1BTATA-Luc reporter in 911 cells (30). First, Gal4-DBD fusions of full-length CBP were tested for their transcriptional activity. While wild-type CBP activated this reporter ~80-fold more than the Gal4-DBD alone, the RTS-associated PHD finger mutations markedly reduced the ability of CBP to activate the reporter (Fig. 3A). Similar results were obtained over a range of concentrations of CBP (50–400 ng/well; data not shown).



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  		Figure 3. RTS-associated PHD finger mutations result in reduced CBP coactivator potential. (A) 911 cells stably transfected with a 5x Gal4-E1BTATA-Luc reporter (30) were transfected with expression vectors for Gal4DBD fusions of full-length CBP (FL-CBP), schematically represented at the top of the figure. Data are presented as described in Fig. 2B. (B) The ability of CBP and various mutants to potentiate PKA-dependent transcription by transcription factor CREB was assessed in 911 cells stably transfected with a 5x Gal4-E1BTATA-Luc reporter (30), schematically represented at the top of the figure. PKA-dependent phosphorylation of serine 133 of CREB is indicated by a P. Data are presented as described in Fig. 2B. Wild-type CBP potentiates PKA-activated CREB, while mutant proteins display reduced coactivator activity.

 
Next we tested the effect of the RTS-associated mutations on the ability of CBP to function as a coactivator of CREB, a transcription factor which requires CBP (32,34). For this we co-expressed a Gal4-DBD fusion of CREB with either wild-type CBP or CBP proteins containing RTS-associated mutants in 911 cells expressing the 5x Gal4-E1BTATA-Luc reporter (Fig. 3B). Coactivation of CREB by CBP required, at least in part, a functional HAT domain, because ablation of the HAT function of CBP markedly reduced its coactivator potential (mutant C1287A; Fig. 3B). The RTS-associated mutations disrupted CREB coactivation to the same extent as the HAT minus mutant (C1287A), further reinforcing the evidence that these RTS-associated mutations disrupt the function of CBP because they abrogate its acetyltransferase activity. No effect of CBP was observed in the absence of protein kinase A (PKA), because recruitment of CBP by CREB requires PKA-mediated phosphorylation of CREB at serine 133 (34–36).

In summary, these experiments show that RTS-associated PHD mutants of CBP lack acetyltransferase activity, leading to a marked reduction in the ability of CBP to activate transcription.

Reduced CBP HAT activity in RTS patient-derived cells
We next determined whether PHD finger mutations disrupt the HAT activity of CBP in cells derived from RTS patients. To this end we established an Epstein–Barr virus (EBV)-transformed lymphoblastoid cell line from patient 211.3, who is predicted to bear a 26 amino acid deletion in CBP due to the deletion of exon 22 (Table 1 and Fig. 1). The small size of the protein deletion that results from the mutation in this patient made it impossible to distinguish between wild-type and mutant CBP in these cells by western blotting (data not shown). In order to assess the impact of the RTS-associated PHD finger mutation on CBP acetyltransferase activity, therefore, we immunoprecipitated CBP from a cell line derived from a healthy control individual and the cell line derived from patient 211.3. We then compared equal amounts of CBP derived from each cell line in an acetylation assay, and observed that CBP isolated from patient RT211.3 displayed ~50% less acetyltranferase activity, when compared with roughly equal amounts of CBP isolated from a healthy individual (Fig. 4A and B). This reduction of endogenous CBP acetyltransferase activity is most likely due to the inability of the mutant CBP protein (predicted to be 50% in a heterozygous patient cell) to acetylate histones, as a result of the heterozygous PHD finger mutation observed in this patient (Fig. 1).



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  		Figure 4. Reduced CBP acetyltransferase activity in cells derived from an RTS patient. (A) Ability of endogenous CBP to acetylate core histones in vitro. CBP immunoprecipitated from increasing amounts of EBV-transformed lymphoblastoid cell lysate from a healthy control individual or patient RT211.3 (exon 22 deletion) were used to acetylate histones in vitro. An aliquot from each reaction was used for western blot analysis, using an antibody against CBP. (B) Quantification of the data presented in (A) by PhosphoImager analysis. Acetylation levels were corrected for small differences in the amount of CBP protein isolated from a healthy control individual and RT211.3 at each concentration, using a LumiImager. The level of acetylation observed in lane 3 was set at 100%. CBP protein isolated from patient RT211.3 showed ~50% of the HAT activity detected with CBP isolated from a healthy control individual.

 
Collectively these findings show that mutations in the PHD finger of CBP abrogate its acetyltransferase activity and support the idea that inhibition of CBP acetyltranferase activity is sufficient to cause RTS.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Using in vitro approaches, we and others have recently shown that the PHD-type zinc finger of CBP, which is located at the very N-terminus of the CBP HAT domain, is essential for its acetyltransferase activity (29,30). Since various heterozygous microdeletions or truncating mutations in the CBP gene result in RTS in humans (7–9,37), we hypothesized that functional inactivation of the HAT function by PHD finger mutations could give rise to the same phenotype. Indeed we identified in two RTS patients a mutation that specifically affected the PHD finger of CBP, which was not observed in their parents. In addition, we identified several novel mutations which lead to truncation of the CBP protein as well as one missense or splice mutation in the HAT domain (Table 1). Both PHD finger mutations result in loss of acetyltransferase activity and therefore clearly underscore the in vivo relevance of this zinc finger in CBP function. The exon 22 splice site mutant ( patient RT211.3) results in deletion of amino acids 1280–1305, including three cysteines and a histidine residue of the PHD finger involved in zinc coordination (Fig. 1D). As expected, this RTS-associated deletion mutant displays no acetyltransferase activity, since mutation of individual zinc-coordinating residues abolishes the ability of CBP to acetylate histones and non-histone substrates (29,30). Alteration of the glutamic acid at position 1278 to lysine as found in patient RT209.1 also abolished the HAT activity, most likely due to an alteration of the PHD finger domain structure resulting from the conversion of a conserved negatively charged amino acid to a positive one. Like the E1278K mutation (Fig. 2B), a double mutation of the hydrophobic residues F1281 and V1282 in mouse CBP completely abolishes the HAT-dependent transcriptional activity (29), while mutation of the three lysine residues at position 1271–1273 had no effect (unpublished data). These findings indicate that some, but not all, residues located in the loop between the second and third cysteine residues contribute to the integrity of the PHD finger and, consequently, the acetyltransferase function of the CBP protein (Fig. 1C and D).

Various chromosomal translocations (37–40) or inversions (7,38,41) and point mutations (7,8,12,13) in the CBP gene have previously been reported in RTS patients, most of which are predicted to result in truncated CBP proteins. This would imply that loss of the region C-terminal to these truncation points is an essential event in the development of RTS, either because a specific function (e.g. HAT activity) is lost or because the truncated CBP proteins are unstable. Our analysis of RTS-associated PHD finger mutants supports the idea that at least in these patients the primary effect of the mutations is to disrupt CBP HAT activity. Firstly, unlike wild-type CBP, the RTS-associated PHD finger mutants were unable to acetylate histones in vitro or to activate a HAT-dependent reporter. Secondly, the mutant proteins displayed reduced coactivator potential towards CREB, a function that is critically dependent on the HAT domain. Finally, the acetyltransferase activity of CBP derived from patient cells ( patient RT211.3; {Delta}exon22) is 50% less than an equal amount of CBP derived from healthy control cells, as would be predicted if the mutation specifically disrupted the HAT function of CBP. The recently described arginine-to-proline missense mutation at position 1379 in an RTS patient, which also abolishes in vitro HAT activity, supports this hypothesis (8). In addition, as was also reported in previous studies (8,10), the patients with the mutations described in this study do not display characteristics that are strikingly different from the general RTS phenotype. Taken together, these findings strongly argue that the full dosage of CBP HAT activity is required to prevent RTS. The consequence of the mutation found in patient RT2644 is currently unclear. The mutation is unlikely to result in a splicing error, since we were able to detect wild-type and mutant transcripts by RT–PCR (data not shown). The CBP HAT domain bearing this mutation displayed in vitro acetyltransferase activity towards histones comparable to the wild-type protein (data not shown). It is, however, possible that the mutant protein might fail to acetylate a relevant non-histone substrate or that the mutation affects essential posttranslational modifications of the endogenous protein. Furthermore, our hypothesis does not rule out missense mutations outside the HAT domain, which would for example disrupt HAT–substrate interactions or destabilize the CBP protein, giving rise to the same phenotype. Interestingly, the importance of the HAT function of CBP in development is not restricted to humans, since inactivation of the CBP gene or inactivation of only its HAT function gives rise to the same developmental defects in C. elegans (42,43).

At present no acetyltransferase other than CBP is linked to a congenital syndrome in humans. However, mutations in the RSK2 gene (RPS6KA3) result in Coffin–Lowry syndrome (CLS), an X-linked disorder characterized by mental retardation, facial and digital dysmorphims and progressive skeletal deformations (44). The RSK2 protein can phosphorylate the N-terminal tail of histone H3 at serine 10, a modification linked to acetylation since this phosphorylated H3 tail is a better substrate for HATs then the non-phosphorylated form (45,46). Interestingly, RSK2 can bind to the CH3 region of CBP in a dynamic fashion, an interaction which affects the enzymatic activity of both proteins (47). Although the overlap in clinical features between patients with RTS and CLS is small, it seems possible that CBP and RSK2 share in part the same substrates, either histones or non-histone proteins.

It is currently unclear how the loss of one functional copy of CBP results in the well-defined RTS, given the plethora of transcriptional events in which CBP has been implicated (14–17). Both RTS patients and heterozygous CBP knock-out mice (48–51) indicate that the regulation of a subclass of developmentally important genes is extremely sensitive to the total CBP gene dosage. The identification of RTS-associated HAT mutants would argue that specific regulatory acetylation events on the promoters of these genes are essential in this respect. An important goal for the future will therefore be to identify these CBP-specific target genes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Patients
In this study we analysed DNA samples from 39 individuals diagnosed as having RTS. DNA was isolated from peripheral blood using standard procedures (52).

Mutation detection
PCR amplified fragments were analysed by DGGE. For each fragment optimal electrophoresis conditions were based on time travel experiments. Amplified fragments were analysed on a 9% polyacrylamide gel (37.5:1; Mallinckrodt-Baker, Deventer, The Netherlands) with various linear denaturing gradients. An acrylamide mixture with 40% formamide (Mallinckrodt-Baker) and 7 M urea (USB, Amersham Pharmacia Biotech, Roosendaal, The Netherlands) was defined as 100% and without formamide and urea the acrylamide solution was defined as 0%. Electrophoresis was performed at a constant temperature of 60°C, at 70 V for 16 h. After electrophoresis, gels were stained with ethidium bromide for 30 min and photographed on an UV transilluminator. Primer sequences, annealing temperatures and DGGE conditions are listed in Table 2.


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  		Table 2. Primer sequences and DGGE conditions
 
Exon 26 was analysed by single-strand conformation polymorphism (SSCP). For this, primers were selected to amplify exon 26 with adjacent splice sites and branch site (Table 2). The products were separated on 10% non-denaturing polyacrylamide gels (49:1, acryamide:bis-acrylamide) without glycerol and on 1x sequagel MD gels (Biozym, Landgraaf, The Netherlands), at 10 W for 16 h at room temperature, essentially according to Orita et al. (53).

Sequencing
PCR products, purified using the Qiaquick PCR purification kit (Qiagen, Westburg, Leusden, The Netherlands), were sequenced using the Big-Dye Terminator cycle sequence reaction kit and an ABI-3700 (Perkin Elmer, Applied Biosystems, Nieuwerkerk a/d IJssel, The Netherlands). Mutations were designated according to the recommendations of Den Dunnen and Antonarakis (54) using the CBP sequence (GenBank accession number U85962). If mutations caused the loss or gain of a restriction site, a digestion with the appropriate enzyme was performed to confirm the sequence data (data not shown). To eliminate the possibility that mutations were PCR artefacts, a second PCR was performed. This PCR product was used either for sequencing, restriction analysis or a second DGGE.

RT–PCR
RNA was isolated from peripheral blood using the GenElute Kit from Sigma-Aldrich (Sigma-Aldrich, Zwijndrecht, The Netherlands). Traces of genomic DNA were removed with DNaseI (Sigma-Aldrich). For each sample 1 µg RNA was reverse-transcribed using Superscript reverse transcriptase (Life Technologies, Breda, The Netherlands) with random hexamer primers according to the manufacturers protocol. Each PCR was performed on 10% of the produced cDNA. The primers are listed in Table 2.

Cell culture, plasmids and transient transfection experiments
The human osteosarcoma cell line U-2 OS and the adenovirus type 5 transformed human embryo retina cell line 911 stably transfected with a 5xGal-E1BTATA-Luc reporter (30) were routinely maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (Invitrogen Life Technologies, Breda, the Netherlands), 100 µg/ml penicillin and 100 µg/ml streptomycin. EBV-transformed lymphoblastoid cell lines were established according to standard procedures (55) and maintained in RPMI1640 medium containing 10% fetal bovine serum, 100 µg/ml penicillin and 100 µg/ml streptomycin.

All recombinant DNA work was performed according to standard procedures (56). The mammalian expression vectors containing the Gal4 DNA binding domain (Gal4DBD), Gal4DBD–CBPHAT (amino acid 1099–1758) (25), Gal4DBD–full-length CBP (30), full-length CBP (30), Gal4DBD–CREB (57), and the catalytic subunit of PKA (58) as well as the 5xGal-AdMLTATA-Luc reporter (30) have been described previously. Point mutations were generated with the Quick Change mutagenesis kit (Stratagene, La Jolla, CA, USA) according to the manufacturer's instructions and verified by sequencing.

For luciferase reporter assays, cells were plated into 24-well microtitre plates and transfected using the calcium phosphate co-precipitation method with Gal4DBD expression plasmids at 200 ng/well, 150 ng of pcDNA-LacZ plasmid (Invitrogen, Groningen, the Netherlands) as an internal control, and 1 µg reporter plasmid or pBluescript SK-, in the case of the 911 reporter cells. Transfections with CREB in 911 reporter cells contained 100 ng Gal4DBD-CREB, 100 ng PKA expression vector, 100 ng pcDNA-CBP and 1 µg pBluescript SK-. After 24 h, the cells were washed with 1x HEPES-buffered saline and then maintained in medium for another 24 h. The cells were then washed with PBS and harvested in lysis buffer [25 mM trisphosphate pH 7.8, 2 mM dithiothreitol (DTT), 2 mM 1,2-diaminocyclohexane-N,N,N'N' tetraacetic acid, 10% glycerol, 1% Triton X-100]. Extracts were assayed for luciferase activity, according to the manufacturer's protocol (Promega, Leiden, The Netherlands), and ß-galactosidase activity (56), as a control. For immunoprecipitation experiments, cells were plated in 9 cm dishes and transfected with Fugene (Roche Diagnostics Corp., Indianapolis, IN, USA) with 10 µg expression plasmid.

Acetyltransferase assays and western blotting
In vitro acetyltransferase assays were performed as described (30). In short, Gal4DBD–CBPHAT proteins were overexpressed in U2-OS cells, isolated by immunoprecipitation with a polyclonal antibody against the Gal4DBD (SC-570; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA). Subsequently, the enzymes were incubated with 5 µg Drosophila core histones in AIPH buffer (20 mM Tris–HCl pH 8.0, 60 mM NaCl, 2 mM EDTA, 0.2% NP-40, 40 µM PMSF) containing [14C]-acetyl CoA (0.05 µCi) for 40 min at 30°C. Reactions were stopped by the addition of 2x SDS–PAGE sample buffer. Proteins were separated on 15% SDS–PAGE gels, fixed and stained with Coomassie Brilliant Blue. Subsequently, gels were enhanced with Amplify (Amersham plc., Little Chalfont, UK), dried and labelled proteins were visualized by fluorography. Radiolabelled histones were quantified with a PhosphoImager using ImageQuant software. The same method was used to test endogenous CBP acetyltransferase activity from EBV-transformed lymphoblastoid cells, only here the A22 polyclonal antibody against CBP (SC-369; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) was used.

Western blotting was performed as described (30). Blots were incubated with 1:1000 dilutions of a monoclonal antibody against the Gal4DBD (SC-510) or a polyclonal antibody against CBP (A22; both Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) and immunoreactive bands were visualized by enhanced chemoluminescence. Blots were subsequently stripped and acetylated CBPHAT was detected with a polyclonal antibody against acetylated lysine residues (06-933; Upstate Biotechnology, Lake Placid, NY, USA) as described above. Western blots were quantified on a LumiImager (Roche Diagnostics Corp., Indianapolis, IN, USA).


   ACKNOWLEDGEMENTS
 
We would like to thank patients and their parents for participation in this research. We also thank Professor J. Burn (Newcastle-upon-Tyne, UK) for DNA of patient 39.1 and Dr N.C. Jones and Dr T. Kouzarides for plasmid constructs. We are grateful to G. Chalkley for Drosophila core histone preparations, F. Petrij for sample collection, P. van der Bent and J.G. Dauwerse for technical assistance and Professor C.P. Verrijzer, T. Mahmoudi and Dr D.A. Baker for critical reading of the manuscript. This work was supported by grants from the Dutch Cancer Society (KWF) (E.K., J.H.R, H.T. and Y.A.) and a fellowship from the Royal Netherlands Academy of Arts and Sciences (E.K.).


   FOOTNOTES
 
* To whom correspondence should be addressed. Tel: +31 715276048; Fax: +31 715276075; Email: d.j.m.peters{at}lumc.nl. Back

{dagger} The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors. Back


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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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