SEARCH

SEARCH BY CITATION

Keywords:

  • corepressor;
  • CSN2;
  • gene repression

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

The regulation of gene repression by corepressors is a controlled process. Surface-enhanced laser desorption ionization MS proteomic analysis and a yeast two-hybrid screen showed independently that the corepressor Alien interacts with the CREB-binding protein (CBP) coactivator. This interaction was further confirmed by coimmunoprecipitation and glutathione S-transferase pull-down experiments, suggesting that Alien interacts in vivo and in vitro with the histone acetyltransferase (HAT) coactivators CBP and its paralog p300. Acetylation detection experiments indicated that Alien is acetylated in vivo. Furthermore, Alien interacts with the central region of CBP/p300 containing the HAT domain and becomes acetylated in vitro. When an inhibitor of CBP/p300 HAT activity was employed, the Alien-mediated silencing was enhanced. Thus, these findings suggest crosstalk between corepressors and coactivators, and indicate fine-tuning of corepressor function by post-translational modification through corepressor acetylation.


Abbreviations
CBP

CREB-binding protein

CoIP

coimmunoprecipitation

CtBP2

C-terminal binding protein 2

GST

glutathione S-transferase

HAT

histone acetyltransferase

HDAC

histone deacetylase

IP

immunoprecipitation

pCAF

p300/CREB-binding protein-associated factor

SELDI

surface-enhanced laser desorption ionization

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Corepressors are coregulators that are recruited by DNA-bound transcription factors and mediate gene silencing [1]. One characteristic of corepressors is the transferable silencing domain, which remains functionally active in the new protein context. The regulation of corepressor interaction with the DNA-bound transcription factors is important for normal biological function. There are many examples of aberrant binding of corepressors to DNA-bound transcription factors leading to human diseases [1].

It has been shown that, for fine-tuning of gene expression, the interplay between transcription factors, coactivators and corepressors is very important. For active silencing of gene expression, several mechanisms have been suggested. Gene silencing can be mediated by recruitment of histone deacetylase (HDAC) and/or histone methyltransferase activity, as well as by inhibition of the transcription initiation complex. Furthermore, corepressors may also compete for the binding sites of coactivators at transcription factors [2, 3].

The COPS2 gene encodes two major isoforms, Alien α and CSN2/Alien β, a subunit of the COP9 signalosome. The knock-out of the COPS2 gene causes very early embryonic lethality, with aberrant expression of cell cycle factors. Alien has the characteristics of a corepressor, mediates gene repression of nuclear hormone receptors, and interacts with the mSIN3A–HDAC1/2 complex [4, 5]. In addition, Alien binds to and inhibits E2F1-mediated transcriptional activity and represses cell proliferation [6, 7]. Interestingly, analysis of the interactome of the COP9 signalosome chromatin suggests binding of repressive factors, including the mSIN3A–HDAC1/2 complex and nucleosome assembly factors, to the COP9 signalosome [8].

Coactivators, on the other hand, increase gene expression by mediating transcription factor activity and/or relaxing the chromatin, either by intrinsic histone acetyltransferase (HAT) activity or by recruiting HAT activity. Furthermore, they can recruit the basal transcription machinery to the promoter [3]. The integral coactivators CREB-binding protein (CBP)/p300 and CBP-associated factor (pCAF)/p300 harbor intrinsic HAT activity [9]. CBP and p300 are paralogs sharing structural and functional features. Both are involved in cell type-specific and signal-specific gene expression, and therefore play a crucial role in many cellular processes, such as proliferation, embryogenesis, and apoptosis [10]. Mutations and altered activity of either of these proteins are associated with developmental abnormalities, including Rubinstein–Taybi syndrome [11, 12]. Moreover, loss-of-function or point mutations were detected in several human tumors, including colorectal, breast, ovarian, oral, gastric, lung and pancreatic carcinomas [12]. CBP/p300 not only acetylates all four core histones, but also acetylates over 70 other proteins and itself [13].

Here, we show an interaction of the corepressor Alien with CBP/p300, and reveal acetylation of Alien by CBP/p300. A CBP/p300 HAT inhibitor enhanced the silencing function of Alien, indicating that acetylation of Alien reduced its repressive activity. Thus, the silencing activity of the corepressor Alien can be regulated by a post-translational modification.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

With a yeast two-hybrid screen to detect possible Alien interaction partners, not only were the nucleosome assembly protein 1 and the mixed lineage kinase 2 identified, as described previously [14, 15], but also TAFI68 factor and the coactivator CBP (Fig. 1A). Hence, a functional interaction between a corepressor and a coactivator could be potentially interesting for transcription regulation, and further analyses were therefore performed.

image

Figure 1. Alien interacts with CBP in yeast and in vitro. (A) A yeast two-hybrid screen identified several Alien α interaction partners, including the coactivator CBP. pGBKT7–hAlien α was cotransformed with the embryonic mouse cDNA library day E9.5–E10.5 day post coitum fused to the activation domain of VP16 in the yeast strain AH109. Positive clones were analyzed in fluid assays for β-galactosidase activity (Miller units indicated as fold activation as compared with the control). As a control, a yeast colony with Alien α as bait and the empty pVP16 activator vector was used, which was set arbitrarily as one. The measurements were repeated twice. The vectors were isolated from yeast, and the cDNAs were sequenced and are indicated. (B) GST and GST–Alien α were expressed in E. coli and purified with glutathione beads. GST pull-down experiments were performed with GST–Alien fusion protein and the CBP fragment (amino acids 1508–1545), which was isolated in the yeast two-hybrid screen. The latter was translated in vitro and thereby radiolabeled. The interaction was detected by fluorography SDS/PAGE. MLK2, mixed lineage kinase 2; NAP1, nucleosome assembly protein 1.

Download figure to PowerPoint

To confirm the binding between CBP/p300 and Alien, glutathione S-transferase (GST) pull-down experiments were utilized. In these, bacterially expressed and purified GST or GST–Alien was incubated with in vitro translated and labeled [35S]CBP containing the HAT domain. After stringent washing steps and SDS/PAGE, incubation with GST alone produced no signal, whereas a specific signal was obtained with GST-Alien, indicating a specific interaction between the central part of CBP containing the HAT domain and Alien (Fig. 1B).

Independently, surface-enhanced laser desorption ionization (SELDI)-MS approaches were used to detect Alien interacting factors in vivo (described in [6]). Immunoprecipitation (IP) of Alien and further analysis by MS of Alien-interacting factors identified a specific peak at ~ 267 kDa. A database search indicated CBP as a putative interaction partner (Fig. 2A). Furthermore, after IP of Alien, the coimmunoprecipitate was eluted and separated on SDS gels. The high molecular mass band was subsequently subjected to tryptic digestion, and the fragments obtained were employed in SELDI-MS analysis. The data suggested the existence of CBP fragments, and confirmed the previous results (Table S1). Coimmunoprecipitation (CoIP) and western blotting experiments with antibody against CBP or Alien, using untransfected whole cell extracts of U2OS cells, suggested an interaction between endogenous Alien and endogenous CBP (Fig. 2B), confirming previous results. Thus, these data suggest an interaction between the corepressor Alien and the coactivator CBP in vitro as well as in vivo.

image

Figure 2. Endogenous Alien interacts with CBP in vivo. (A) Protein A-coated beads were incubated with antibody against Alien or normal rabbit IgG as a negative control and U2OS cell extract. Specifically bound proteins were eluted and applied to an activated H50 ProteinChip Array. The array was then analyzed in a ProteinChip Reader by SELDI-MS. (B) CoIP was carried out with either antibody against Alien, antibody against CBP, or control antibody, bound to protein A beads, and U2OS cell extract. Bound proteins were subjected to SDS/PAGE and detected by immunoblotting with an antibody against CBP (upper panel) or an antibody against Alien (lower panel).

Download figure to PowerPoint

CBP and p300 are paralogs and can, in part, functionally replace each other. Both proteins possess intrinsic HAT activity. First, we confirmed the binding of Alien to the HAT domain of p300, as observed for CBP, by GST pull-down experiments (Fig. 3A). Furthermore, like CBP, GST–p300-HAT interacted specifically with in vitro translated 35S-labeled Alien (Fig. 3A). Next, we investigated whether functional HAT activity is required for binding. Interestingly, a p300 mutant with a catalytically inactive mutant HAT domain still interacted with Alien, suggesting that the observed binding is independent of HAT activity. Thus, Alien most likely binds to p300 or CBP via their HAT domains.

image

Figure 3. The HAT domain of p300 interacts with and acetylates Alien in vitro. (A) Bacterially expressed GST–p300-HAT wild type (wt) and GST–p300-HAT mutant (mut) were bound to GST beads and incubated with in vitro translated 35S-labeled Alien. Interaction was determined with fluorography after subjection to SDS/PAGE. Lower panel: Coomassie staining of SDS/PAGE gels used in the upper panel to ensure equal loading of GST fusion proteins. Asterixes indicate GST, GST–p300-HAT wt, and GST–p300-HAT mut (amino acids 1096–1721). (B) Alien α and Alien β were expressed as GST fusion proteins in E. coli, purified with glutathione beads, and eluted by cutting with thrombin. Acetylation of Alien was analyzed by using purified Alien, purified GST–p300-HAT, and [3H]CoA-SAc. Acetylation of Alien was assayed with fluorography. Lower panel: Coomassie staining of SDS/PAGE gels used in the upper panel to ensure equal loading of proteins. Asterixes indicate Alien α and CSN2/Alien β.

Download figure to PowerPoint

Next, we addressed the possibility of whether Alien is acetylated by CBP/p300. For this purpose, in vitro acetylation assays were performed (Fig. 3B). GST–p300-HAT (amino acids 1195–1810) and GST–pCAF-HAT (amino acids 352–832) were expressed in Escherichia coli and purified with glutathione beads. These extracts were incubated with 3H-labeled CoA-SAc and bacterially expressed Alien α or CSN2/Alien β. The latter were expressed as GST fusion proteins in E. coli, and purified with glutathione beads; the thrombin was then removed by the use of GST. After the reaction, the mixture was subjected to SDS/PAGE. Remarkably, fluorography results indicated that Alien α was acetylated by p300 (Fig. 3B). However, no detectable signal was obtained for CSN2/Alien β, indicating the absence of acetylation or weaker acetylation. The acetylation of the core histones was used as a control for the catalytic activity of p300 HAT activity (data not shown). Similar results, albeit with weaker acetylation signals, were obtained with pCAF and another HAT (Fig. S1), indicating that other HAT-containing coactivators can acetylate Alien α.

Taken together, the data suggest acetylation of Alien α through p300.

To narrow down the region of acetylation, a battery of Alien deletion mutants were generated and tested in acetylation assays with 3H-labeled CoA-SAc (tritium labeled acetyl coenzyme A) (Fig. 4A). Fluorography showed specific bands for the full-length Alien as well as for the deletion mutants Alien 128–305 and 103–185 (Fig. 4B). Therefore, we propose that the central region of Alien is the substrate for acetylation. To confirm this, a deletion mutant of Alien lacking amino acids 128–186 was generated, and, in line with the previous results, no acetylation was detectable. These data suggest that Alien is acetylated within its central region.

image

Figure 4. Alien is acetylated within the region comprising amino acids 128–186. (A) Schematic illustration of GST–Alien deletion mutants. The region of acetylation is designated by a light gray bar and a plus in the right column. (B) GST and the different GST–Alien constructs were expressed in E. coli and purified with glutathione beads. Acetylation of GST–Alien deletion mutants was investigated by using GST–Alien, incubated with purified GST–p300 extract in the presence of [3H]CoA-SAc. Acetylation of Alien mutants and full-length Alien was detected by fluorography. Full-length Alien and the deletion mutants GST–Alien (128–305) and GST–Alien (103–185) are acetylated. Lower panel: Coomassie staining of SDS/PAGE gels used in the upper panel to ensure equal loading of GST fusion proteins. Asterixes indicate GST–Alien deletion mutants as mentioned above. f.l., full length.

Download figure to PowerPoint

To analyze whether Alien is also acetylated in vivo, IP experiments on HEK293 whole cell extracts were performed. Subsequent immunoblotting with a pan-acetylation antibody gave a signal corresponding to the size of Alien α, which was confirmed with an antibody against Alien (Fig. 5).

image

Figure 5. Endogenous Alien α is acetylated in vivo. IP with antibody against Alien and normal rabbit IgG as a negative control. The antibodies were bound to protein A/G beads and incubated with HEK293 cell extracts. Immunoprecipated proteins were subjected to SDS/PAGE and detected with antibody against pan-acetylation (A) or antibody against Alien (B). Asterixes indicate immunoprecipated Alien and the putative acetylated Alien.

Download figure to PowerPoint

To determine whether the corepressor function of Alien is regulated by HAT activity, the silencing activity of Alien was analysed with a reporter gene assay. A UAS-luciferase reporter and Gal or Gal–Alien expression vectors were used to monitor the expression in the presence or absence of the CBP/p300-specific HAT inhibitor [16] C646 (Fig. 6A). The well-established Gal-fusion system avoids interference by other transcription factors that may be affected by the CBP/p300 coactivators. In untreated or dimethylsulfoxide-treated cells, Alien showed silencing activity, depicted as fold repression in Fig. 6A. Remarkably, treatment with the HAT inhibitor C646 enhanced the silencing activity of Alien. The use of a higher concentration of the CBP/p300 inhibitor further increased Alien-mediated repression. In line with these observations, overexpression of CBP inhibited Alien-mediated repression (Fig. 6B). Furthermore, the Alien central domain (amino acids 103–187) was also employed in this assay. As compared with full-length Gal–Alien, the data indicated enhanced repression of the Alien central domain in the presence of the HAT inhibitor C646 (Fig. 6C), and suggest that one of the HAT targets is the central part of Alien.

image

Figure 6. A CBP/p300 HAT inhibitor enhances Alien-mediated repression. CV-1 cells were transfected with p4xUAS-TATA-luc and pAB–gal or pAB–gal–Alien α with or without pRC/RSV–CBP. After 24 h, cells were treated with dimethylsulfoxide or with the indicated concentrations of the CBP/p300 inhibitor C646. After 72 h, the cells were lysed, and luciferase and β-galactosidase activities were measured from the cotransfected pCMV-lacZ vector. The latter was used for normalization, and the pAB–gal sample was set as one for every treatment. The values are plotted as fold repression as compared with the values obtained with the expression vector for β-galactosidase alone. (A) Concentration-dependent enhancement of Alien-mediated epression by the CBP/p300-specific HAT inhibitor C646 (HAT-i). (B) Cotransfection of a CBP expression vector reduces Alien-mediated repression. The empty expression vector (pRSV) was use as a negative control. (C) HAT-i enhances the Alien deletion mutant-mediated repression more than it does the repression mediated by full-length Gal–Alien.

Download figure to PowerPoint

In conclusion, the data suggest functional interference of Alien-mediated silencing by CBP/p300.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Although post-transcriptional modifications appear to be a common feature of coregulators and are important for the fine-tuning of gene expression [17], the acetylation of corepressors and its functional consequences have rarely been described. The two corepressors nuclear receptor corepressor and SMRT were shown to be acetylated [18], but the impact on transcription has yet to be discovered. Receptor-interacting protein 140 is acetylated at various sites, affecting both nuclear localization and repressive activity [19]. However, the acetyltransferase that is responsible for the acetylation of receptor-interacting protein 140 is not yet known. Another example is the acetylation of the corepressor C-terminal binding protein 2 (CtBP2), which affects nuclear localization and its repressive function. In this case, it was shown that CtBP2 is acetylated by the coactivator p300 [20], suggesting that acetylation of CtBP2 by p300 weakens the silencing activity of CtBP2.

Our data suggest that CBP/p300 interacts with Alien and attenuates Alien-mediated silencing. This indicates crosstalk between coactivators and corepressors at the level of post-translational modification.

Moreover, using another HAT coactivator, pCAF, in a similar experimental setup, we observed that pCAF also specifically acetylates Alien α (Fig. S1). Alien α is a shorter isoform of Alien β, and they have identical sequences in the central region. There may be several reasons for the lack of detection of acetylation of Alien β. One of them could be that the C-terminus of Alien β masks the acetylation sites in the central domain. As pCAF acetylates Alien α, this suggests a more general molecular mechanism, such as functional crosstalk among coregulators. Additionally, these findings also suggest that analysing the expression levels of coregulators, such as with microarrays or RNA sequencing, may not be necessarily reveal the activity of a particular coregulator.

Experimental procedures

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Yeast two-hybrid screen

Yeast two-hybrid screening (Matchmaker Two-Hybrid System 3; Clontech, Heidelberg, Germany) was performed according to the manufacturer's protocol and as described previously [14]. pGBK-T7–hAlien α was cotransformed with the embryonic mouse cDNA library embryonic day (E)9.5–E10.5 days post coitum fused to the activation domain of VP16 in the yeast strain AH109. After transformation, cells were spread on selection medium for growth selection with two stable integrated selection genes driven by two different promoters. In addition, the expression of LacZ, as the third selection marker, was assayed with the colony filter lift method. Recovered plasmids from positive colonies were retransformed into another yeast strain, Y187, and subjected to LacZ liquid culture assay for quantification of the interaction [4]. Mouse CBP cDNA encodes amino acids 1508–1545 as a VP16 fusion protein.

GST pull-down

GST pull-down experiments were performed essentially as described elsewhere [5]. In brief, bacterial expression of GST or GST fusion proteins was induced with 0.2 mm isopropyl thio-β-d-galactoside for at least 3 h at 37 or 18 °C, respectively. Purification of the GST or GST fusion proteins and interaction studies with in vitro translated [35S]methionine-labeled constructs (TNT kit; Promega, Mannheim, Germany) were employed according to the manufacturer's protocol. Mouse CBP was translated from cDNA encoding amino acids 1508–1545.

The SDS/PAGE gels were stained with Coomassie brilliant blue to ensure equal loading of GST fusion proteins. The bound and labeled proteins were visualized by autoradiography.

CoIP

The CoIP assays were carried out as previously described [6]. Briefly, specific antibody against Alien (rabbit polyclonal [5]), antibody against CBP or, as a negative control, normal rabbit IgG (Pepro Tech, Rocky Hill, NJ, USA) were bound on protein A–agarose beads (Sigma, Munich, Germany). Crude extract (150 μL) from U2OS cells was incubated with the antibody-loaded beads for 1 h at 4 °C. The resins were then washed three times with CoIP buffer containing 20 mm Hepes/KOH (pH 8.0), 50 mm KCl, 0.1 mm EDTA, and 0.05% Chaps. Bound proteins were subjected to 10% SDS/PAGE and detected by immunoblotting. To detect high molecular mass proteins such as p300 or CBP by immunoblotting, instead of the routinely used 10% methanol, 5% methanol was used in the transfer buffer to transfer proteins from the gel to the membrane.

Protein–protein complex identification assay

The protein–protein complex identification assay has been described previously [6]. Protein A was bound to IDM beads (Ciphergen Biosystem, Fremont, CA, USA) and incubated with antibody against Alien or normal rabbit IgG (Pepro Tech) as a negative control. The beads were then incubated with 200 μL of crude U2OS cell extract overnight at 4 °C and unbound proteins were washed away by sequential washes in NaCl/Pi, 0.5 m sodium chloride, 0.05% Triton X-100 in NaCl/Pi, NaCl/Pi, and aqua bidest. Specifically bound proteins were eluted from the IDM beads with 50% acetonitrile/0.5% trifluoroacetic acid, and the eluates were applied to an activated H50 ProteinChip Array (Ciphergen Biosystem, Fremont, CA, USA). The array was then analyzed in a ProteinChip Reader (series 4000; Ciphergen, Bio-Rad, Munich, Germany), according to an automated data collection protocol, by SELDI-MS. This includes an average of 265 laser shots for each spot, with laser intensities of 2300 and 3500 nJ (2–200 kDa), depending on the measured region (low, 2.5–20 kDa; high, 20–200 kDa), and automatically adapted detector sensitivity.

Tryptic digestion

The protein bands were transferred to reaction tubes, and incubated in 50% methanol with 10% acetic acid for 2 h. Subsequently, the gel pieces were passed through a graded series of buffers for dehydration. The dehydrated pieces were incubated with 20 ng·mL−1 trypsin overnight at 37 °C, and the resulting digest was applied to N20 ProteinChips and analyzed by SELDI MS. The resulting spectra were interpreted by using a search of the ProFound database (http://prowl.rockefeller.edu).

HAT assay

pGEX-2TK–p300 (amino acids 1096–1721) (kindly provided by U. Bauer, Marburg, Germany), pGEX-2TK–p300 D1399Y (referred to as p300 mut) and pGEX-5X–pCAF (amino acids 352–832) (kindly provided by S. Berger, The Wistar Institute, Philadelphia, PA, USA [21]) were bacterially expressed, purified over GST beads (GE Healthcare Europe, Munich, Germany), and eluted with glutathione. Each extract was checked in a HAT assay with histones (Roche, Mannheim, Germany) as substrate to evaluate the HAT activity. The HAT assay was performed in a buffer containing 50 mm Tris (pH 8), 10% glycerol, 10 mm sodium butyrate and 1 mm dithiothreitol in the presence of 1 μCi of 3H-radiolabeled CoA-SAc with p300-containing or pCAF-containing extracts. The reaction was incubated for 30–60 min at 30 °C. Proteins were separated with SDS/PAGE, and the gel was subsequently incubated in NAMP100V (GE Healthcare Europe, Munich, Germany) to amplify the 3H signal. The acetylated proteins were visualized by autoradiography (Kodak, Germany).

Generation of Alien deletions

All restriction enzymes, CIAP, Klenow fragment, T4 kinase and ligase were purchased from Fermentas (St. Leon-Rot, Germany). pGST–Alien 1–183 was generated by cutting pHIL–GST–Alien with DpnI and ligating the Alien 1–183 fragment into pGEX-2T opened with EcoRI and filled in with Klenow fragment. pGST–Alien 186–305, pGST-linker and pABgal94–TRIP15 [4] were digested with EcoRI and BamHI. The fragment containing amino acids 186–305 was afterwards ligated into the opened pGST-linker. The plasmid containing Alien 103–185 was constructed by cutting pABgal94–TRIP15 103–185 with SalI and BamHI, and ligated into pGST-linker, cut with the same enzymes. The internal deletion of Alien (Δ128–186) was generated by digestion of pGST–Alien [4] and pGST–Alien 186–305 with EcoRI and subsequent ligation of the fragment containing amino acids 1–128 into pGST–Alien 186–305.

Detection of Alien acetylation

HEK293 cells were lysed [20 mm Tris, pH 7.5, 200 mm NaCl, 0.5% NP-40, complete protease inhibitor cocktail (Roche, Munich, Germany)], and the extract was incubated with a 1 : 1 mixture of protein A–Sepharose and protein G–Agarose coupled with Alien-specific antibody. After several washing steps (20 mm Tris, pH 7.5, 100 mm NaCl, 0.25% NP-40), the retained proteins were run on an SDS/PAGE gel and blotted, and this was followed by immunodetection with either antibody against Alien or pan-acetylation recognizing acetyl groups fused to lysines (Abcam, Cambridge, UK).

Reporter gene assays

CV1 cells were grown in DMEM with 10% fetal bovine serum (Gibco-Invitrogen, Carlsbad, CA, USA) at 37 °C with 5% CO2. Cotransfection experiments were performed with a modified CaPO4 method as described previously [22]. In total, 2 × 105 cells were seeded on six-well dishes, and transfected 6 h later with 0.5 μg of p4xUAS–TATA–luc, 0.3 μg of pAB–gal, pAB–gal–Alien α, pAB–gal–Alien α 103–187, pSG424–puc, or pSG424–puc–Alien β, 2.5 μg of pRC/RSV–CBP, and 0.2 μg of pCMV–LacZ for internal normalization. Cells were treated with dimethylsulfoxide or C646, and harvested 72 h after treatment for measurement of both luciferase and β-galactosidase activity. Independent duplicate experiments were performed and were repeated at least twice.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

We are grateful to S. Bartsch for critical reading of the manuscript. This work was supported by DFG 1457/3 from the German Research Council.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information
FilenameFormatSizeDescription
febs12211-sup-0001-TableS1-FigS1.zipZip archive844K

Table S1. SELDI-MS of Alien immunoprecipitation and detected masses corresponding to CBP fragments.

Fig. S1. pCAF acetylates Alien α but not CSN2/Alien β in vitro.

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.