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Originally published In Press as doi:10.1074/jbc.M410465200 on February 4, 2005

J. Biol. Chem., Vol. 280, Issue 15, 14989-14996, April 15, 2005
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The Transcriptional Co-activator Protein p100 Recruits Histone Acetyltransferase Activity to STAT6 and Mediates Interaction between the CREB-binding Protein and STAT6*

Tuuli Välineva{ddagger}§, Jie Yang{ddagger}§, Riitta Palovuori{ddagger}, and Olli Silvennoinen{ddagger}||**

From the {ddagger}Institute of Medical Technology, University of Tampere, FIN-33014 Tampere, Finland, the ||Department of Clinical Microbiology, Tampere University Hospital, FIN-33521 Tampere, Finland, and the Department of Immunology, Tianjin Medical University, Tianjin 300070, Peoples Republic of China

Received for publication, September 13, 2004 , and in revised form, February 3, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
STAT6 is a critical regulator of transcription for interleukin-4 (IL-4)-induced genes. Activation of gene expression involves recruitment of coactivator proteins that function as bridging factors connecting sequence-specific transcription factors to the basal transcription machinery, and as chromatin-modifying enzymes. Coactivator proteins CBP/p300 have been implicated in regulation of transcription in all STATs. CBP is also required for STAT6-mediated gene activation, but the underlying molecular mechanisms are still elusive. In this study we investigated the mechanisms by which STAT6 recruits CBP and chromatin-modifying activities to the promoter. Our results indicate that while STAT1-interacted directly with CBP, the interaction between STAT6 and CBP was found to be mediated through p100 protein, a coactivator protein that has previously been shown to stimulate the transcription of IL-4-induced genes. The staphylococcal nuclease-like (SN)-domains of p100 directly interacted with amino acids 1099–1758 of CBP, while p100 did not associate with SRC-1, another coactivator of STAT6. p100 was found to recruit histone acetyltransferase (HAT) activity to STAT6 in vivo. Chromatin immunoprecipitation studies demonstrated that p100 increases the STAT6-p100-CBP ternary complex formation in the human Ig{epsilon} promoter. p100 also increased the amount of acetylated histone H4 at the Ig{epsilon} promoter, and siRNAs directed against p100 effectively inhibited Ig{epsilon} reporter gene expression. Our results suggest that p100 has an important role in the assembly of STAT6 transcriptosome, and that p100 stimulates IL-4-dependent transcription by mediating interaction between STAT6 and CBP and recruiting chromatin modifying activities to STAT6-responsive promoters.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Signal transducer and activator of transcription (STAT)1 family of transcription factors convert extracellular cytokine signals into diverse biological responses through modulation of gene transcription (1). Cytokines bind to their cognate receptors at the cell surface, which leads to the activation of receptor-associated JAK kinases, which in turn activate STATs by phosphorylating them at the conserved C-terminal tyrosine residue. The activated STATs undergo dimerization through a reciprocal SH2-phosphotyrosine interaction and translocate to the nucleus, where the STAT dimers bind to symmetrical TTC(N2–4)GAA DNA target elements. The specificity in cytokine signaling is conveyed by selective activation of different STATs by individual cytokine receptors, specific recognition of target DNA elements by various STATs, and through selective interaction and cooperation with other transcription factors and regulatory proteins.

The family of STAT proteins consists of seven members that mediate highly specific functions in cytokine signaling. STAT6 is mediating interleukin-4 (IL-4)- and IL-13-induced gene responses (2). IL-4 is an important regulator of immune and anti-inflammatory responses. In T cells IL-4 mediates induction of Th2 differentiation, while inhibiting Th1 development, and in B cells IL-4 triggers the immunoglobulin (Ig) isotype class switching and production of IgG1 and IgE. Several genes that are critical for these responses are directly regulated by STAT6, and e.g. the promoter regions of Ig heavy chain germline Ig{epsilon} and Ig{gamma}1, CD23, and major histocompatibility complex class II promoters contain functional STAT6 response elements (3–5).

The initiation of transcription requires sequence-specific DNA binding transcription factors and their cooperation with transcriptional coregulatory proteins and the basal transcription machinery. STAT6 cooperates with several cis-acting transcription factors such as PU.1, IRF-4, BSAP, NF-{kappa}B, and C/EBP{beta} (6–11). Transcriptional activation is also critically regulated by coactivator proteins, which are often recruited by transactivation domains (TADs). In STATs the C-terminal TAD is the most variable domain between different STATs. Recruitment of distinct coactivator complexes by various TADs may contribute to transcriptional specificity in cytokine signaling (12). We have previously identified p100 protein as a STAT6-TAD interacting protein that enhances STAT6-mediated transcriptional activation and gene expression (13). p100 protein was first identified as a coactivator for the Epstein-Barr virus nuclear antigen 2 (EBNA-2) (14), and subsequently found to interact with other transcription factors such as TFIIE, c-Myb (15), and STAT5 (16). Also a member of the p160/SRC family of steroid receptor coactivator proteins SRC-1 (also called NcoA-1) directly interacts with the TAD of STAT6 (17). SRC-1 functions as a coactivator for STAT6, as well as for STAT3 (18) and STAT5 (19). Also another member of the p160/SRC family coactivators p/CIP (NcoA-3) indirectly interacts with STAT6 via p300 (20).

An important function for the coactivator proteins is to relieve the repression of the tightly packed DNA by remodeling chromatin by post-translational modifications. Acetylation of lysine residues in the core histone proteins, particularly H3 and H4, opens the chromatin structure and allows transcription to proceed. Many transcriptional coactivators are histone acetylases (HATs), while corepressors often possess deacetylase activity (21). SRC-1 has been shown to possess some HAT activity, but the mechanism of SRC-1 coactivation in STAT signaling is unknown. Generally, the contribution of SRC-1 to transcriptional activation is considered to occur through recruitment of CREB-binding protein (CBP) and the highly homologous p300 to transcription factors.

CBP/p300 are multifunctional coactivator proteins that act as molecular integrators of several signaling pathways and mediate important functions in transcriptional regulation (22). CBP/p300 are ubiquitously expressed and they have been implicated in coactivation of all STATs (23–31). CBP/p300 have the capacity to facilitate transcription by functioning as bridging factors to the basal transcription machinery including RNA polymerase II holoenzyme, as well as by remodeling chromatin by acetylating nucleosomal histones (22). CBP/p300 can also acetylate several non-histone proteins such as transcription factor p53 (32), GATA-1 (33), coactivators of the p160 family, and components of general transcription machinery such as TFIIE and TFIIF (34).

An important question in IL-4-mediated gene regulation is the mechanism by which chromatin-modifying activities are targeted to STAT6 responsive elements and how these activities are regulated. Previous analysis of coactivators in STAT signaling has been defining the molecular interactions and the functional end point in gene regulation. This study was aimed at investigating the mechanisms by which STAT6 is recruiting histone acetyltransferase (HAT) activity to the promoter. Our results indicate that the coactivator protein p100 is mediating interaction between STAT6 and CBP, and recruits HAT activity to STAT6. p100 also increases the amount acetylated histone H4 that associates with Ig{epsilon} promoter. Taken together, these results strongly suggest that p100 protein functions as an important adapter and HAT recruiting and regulating factor for STAT6-dependent transcription.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture, Transfections, and Plasmids—HeLa cells and COS-7 cells were grown as previously reported (13). Transfection of HeLa cells was performed using the calcium phosphate precipitation method. COS-7 cells were transfected by electroporation with a Bio-Rad gene pulser at 260 V/960 microfarads.

pSG5-p100-FLAG, pSG5-p100-SN-FLAG, pSG5-p100-TD-FLAG, and pCIneo-STAT6-HA plasmids were constructed as previously described (13). The pSG5 expression plasmids containing first and second SN-like domain (SN1 + 2, amino acids 1–319), or third and fourth SN-like domain (SN3 + 4, amino acids 320–639) of human p100 protein were generated by PCR using primers containing C-terminal FLAG sequence. STAT1{alpha}, Ig{epsilon}-reporter, pSG5-SRC-1a, and pSG5-SRC-1e plasmids have been described previously (35–37).

GST-St1-TAD was constructed by cloning PCR products corresponding to amino acids 711–750 of human STAT1 into pGEX-4T-1 vector (Amersham Biosciences). GST-St6-TAD (amino acids 642–847) was constructed as previously reported (13). GST-St6-TAD-6 was constructed by cloning PCR products corresponding to amino acids 794–847 of human STAT6 into pGEX-4T-1 vector. GST-p100-SN, GST-p100-TD plasmids were constructed as previously described (13). GST-CBP-D1 (amino acids 1–452), D2 (amino acids 452–1099), D3 (amino acids 1099–1620), D4 (amino acids 1620–1877), D5 (amino acids 1877–2441), and HAT (amino acids 1099–1758) were generated by PCR amplification of the relevant domain of CBP and inserted into pGEX-4T-1 vector. pBlue-CBP-D1 (amino acids 1–452), D2 (amino acids 452–1099), D3 (amino acids 1099–1758), and D4 (amino acids 1758–2441) were constructed by PCR amplification of the related domains of CBP and inserted into BamHI and XbaI sites. All PCR products were sequenced.

GST pull-down assays—GST fusion proteins were bound on glutathione-Sepharose 4B beads (Amersham Biosciences) and incubated with in vitro translated 35S-labeled proteins, COS-7 cell lysates or HeLa cell nuclear lysates (as described in Ref. 13). Nuclear lysates of HeLa cells were prepared as previously described (13). After washing, the bound proteins were eluted from beads, separated by SDS-PAGE and analyzed by immunoblotting with anti-CBP (Santa Cruz Biotechnology) or anti-SRC-1 (Upstate Biotech) antibodies.

Immunoprecipitation—Transfected COS-7 cells were resuspended in Nonidet P-40 lysis buffer (50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 0.1 mM EDTA, 0.5% Nonidet P-40, 20% glycerol, 0.1 mM sodium orthovanadate, 1 mM sodium butyrate). Protein concentrations of the lysates were measured using the Bio-Rad protein assay. The cell lysates transfected with CBP together with p100-FLAG or STAT6-HA were immunoprecipitated with rabbit polyclonal anti-CBP antibody or rabbit polyclonal IgG antibody (Santa Cruz Biotechnology) as a control. The immunoprecipitated proteins were separated by SDS-PAGE and detected by blotting with mouse monoclonal anti-FLAG M2 antibody (Sigma) or anti-HA antibody (clone 16B 12; BabCO).

Confocal Microscopy—HeLa cells were starved in serum-free medium for 4 h and treated with IL-4 (100 ng/ml) for 20 min or left untreated. Cells were fixed and permeabilized with 4% formaldehyde, 0.2% Triton X-100 in PEM buffer (100 mM PIPES, 5 mM EGTA, 2 mM MgCl2, pH 6.8) for 10 min at room temperature and postfixed thereafter with methanol for 5 min at –20 °C. Cells were stained either with the mixture of anti-STAT6 (Santa Cruz Biotechnology) and anti-p100 (14), or the mixture of anti-STAT6 and anti-CBP antibodies, followed by washing with Alexa 488 anti-mouse and Texas Red anti-rabbit secondary antibodies (Molecular Probes). Confocal images were collected using LSM510 program and Zeiss confocal microscope, equipped with an Argon laser (488 nm) and HeNe laser (543 nm) and a x63 objective. Green emission was detected using a 505-nm low pass filter and red emission using a 630-nm low pass filter.

HAT Assays with Immunopurified Proteins—Transfected COS-7 cells were lysed in Nonidet P-40 lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40, 10% glycerol, 50 mM sodium fluoride) supplemented with phenylmethylsulfonyl fluoride and aprotinin. Protein concentrations of the lysates were measured using the Bio-Rad protein assay. The lysates of cells transfected with STAT6 alone or together with CBP and/or p100-FLAG were immunoprecipitated with rabbit polyclonal anti-STAT6 antibody. The lysates of cells transfected with STAT1 alone or together with CBP were immunoprecipitated with mouse monoclonal anti-STAT1 antibody (N terminus) (Transduction Laboratories, BD Biosciences). Immunoprecipitated proteins were collected on protein A/G beads. The beads were washed with radioimmune precipitation assay buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA) and HAT buffer (50 mM Tris-HCl pH 7.5, 1 mM EDTA) supplemented with phenylmethylsulfonyl fluoride and aprotinin. The beads were then incubated with H4 substrate (synthetic peptide containing the first 23 amino acids of histone H4 coupled through a linker sequence to a biotin molecule) and [3H]acetyl-CoA (Amersham Biosciences) at 30 °C for 45 min. The acetylated substrate was collected with immobilized streptavidin (Sigma) at room temperature for 20 min and then washed with radioimmune precipitation assay buffer. Following the washing, 500 µl of the HAT buffer was added to resuspend the resin, and the contents were transferred to the scintillation vial. 1 ml of scintillation fluid (Optiphase Supermix liquid scintillation mixture; PerkinElmer Life Sciences, Wallac) was added, and the associated radioactivity was counted with 1450 MicroBeta Plus liquid scintillation counter (PerkinElmer Life Sciences, Wallac) for 5 min per vial.

To compare the protein concentrations, the amounts of STAT1, STAT6, and p100 proteins in the lysates were analyzed by blotting with anti-HA (clone 16B 12; BabCO) and anti-FLAG M2 antibodies. To compare the amount of CBP between samples, the lysates were immunoprecipitated with anti-CBP antibody, and the immunoprecipitated proteins were collected on protein A beads. The proteins were separated by SDS-PAGE and detected by blotting with anti-CBP antibody.

Chromatin Immunoprecipitation—HeLa cells were transfected with pSG5-p100-FLAG or empty vector. Formaldehyde was added to the IL-4 or mock-treated cells at a final concentration of 1%. Cross-linking was stopped by the addition of glycine to a final concentration of 100 mM. The cells were washed and harvested and the soluble chromatin was prepared according to the protocol by Nissen and Yamamoto (38). Chromatin was sonicated to an average length of 200–1000 bp using Vibra Cell 500 watt sonicator (Sonics & Materials, Inc., Newtown, CT) with microtip. After centrifugation, samples were precleared with protein A beads and immunoprecipitated with anti-STAT6, anti-CBP, anti-acetyl-histone H4 (Upstate Biotech), anti-HA (clone 16B 12; BabCO), or anti-rabbit IgG antibodies, and the immunocomplexes were collected onto protein A beads. Beads were washed three times with 10 mM Tris-HCl, pH 8, 140 mM NaCl, 1 mM EDTA, 0.1% Triton X-100 and once with TE buffer (10 mM Tris-HCl, pH 8, 1 mM EDTA). The chromatin fragments were eluted from beads with 62.5 mM Tris-HCl, pH 6.8, 200 mM NaCl, 2% SDS, 10 mM dithiothreitol, and cross-links were reverted by heating at 65 °C overnight. DNA was phenol/chloroform-extracted, ethanol-precipitated, and analyzed for human Ig{epsilon} promoter using PCR. The PCR reactions were performed in a 25-µl volume with 10 µl of immunoprecipitated material, using AmpliTaq Gold DNA polymerase (PerkinElmer Life Sciences) and 35 PCR cycles. The following primers were used: 5'-TGGGCCTGAGAGAGAAGAGA-3' and 5'-AGCTCTGCCTCAGTGCTTTC-3'. The extract aliquoted before the immunoprecipitation step (total input chromatin) was also used for PCR analysis.

p100 RNAi—siRNAs were generated using the Ambion SilencerTM siRNA Construction kit, according to the manufacturer's protocol. Sense and antisense primers corresponding to the following target sequences were used: p100 I 5'-AAGGCATGAGAGCTAATAATC-3' (corresponding to nucleotides 602–622 of human p100 mRNA), p100 II 5'-AAGGAGCGATCTGCTAGCTAC-3' (corresponding to nucleotides 2218–2238 of human p100 mRNA). Primers were extended with the sequence 5'-CCTGTCTC-3' at the 3'-end. Transfection of cells was performed as follows: HeLa cells were plated in 24-well tissue culture plates at a density of 3 x 104 cells per well and incubated for 24 h before transfection. Cotransfection of 100 ng of Ig{epsilon}-reporter, 50 ng of {beta}-galactosidase, and siRNAs (final concentration 60 pM) were performed with Oligofectamine (Invitrogen). 24 h after transfection cells were either stimulated with 10 ng/ml IL-4 or left untreated. Cells were lysed in Reporter Lysis Buffer (Promega, Madison, WI) 48 h after transfection. Luciferase expression was determined using the Luciferase Assay system (Promega), and the results were normalized against {beta}-galactosidase activity of the lysates.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Interaction of CBP with STAT1-TAD and STAT6-TAD—Several lines of evidence have implicated histone acetylation in IL-4-mediated gene responses. Histone acetylation has been shown to occur at the IL-4 locus during Th2 differentiation (39) and during IL-4-induced gene activation (40). Because inhibition of acetyltransferase activity of CBP by adenoviral E1A protein has been shown to repress IL-4- and STAT6-regulated gene activation (30, 31, 40), we wanted to gain further insight into the function of CBP/p300 in STAT6-mediated gene activation. The TAD domains are the most divergent domains in STATs, and it is reasonable to assume that STATs may utilize different mechanisms to interact with coactivator proteins. The TAD of STAT1 has been shown to interact directly with CBP/p300 (23, 24), and in initial experiments the interaction of CBP with STAT1 and STAT6 was compared. To investigate whether the TAD of STAT6 is interacting directly with CBP, GST pull-down assays were performed by incubating GST-STAT1-TAD and STAT6-TAD with endogenous nuclear proteins from HeLa cells. CBP was found to bind strongly with GST-STAT1-TAD while only a very weak interaction with GST-STAT6-TAD was detected in the same experiment (Fig. 1A). Similar GST pull-down experiments were also performed with COS-7 cells that overexpressed CBP. As shown in Fig. 1B, CBP strongly associated with GST-STAT1-TAD, while there was no detectable association between CBP and GST-STAT6-TAD. The expression levels of GST fusion proteins are shown in Fig. 1C. The results indicate that the lack of STAT6-CBP interaction is not caused by limitation of sensitivity in the detection methods. These results suggested that CBP/p300 may not interact directly with STAT6-TAD, but the interaction may require additional proteins as bridging factors. Alternatively the interaction may require secondary modifications in STAT6-TAD, or be mediated through N-terminal domains of STAT6.



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  		FIG. 1.
Interaction of CBP with STAT1-TAD and STAT6-TAD. A, nuclear lysates of HeLa cells were incubated with either GST or with GST fusion proteins containing the STAT1-TAD (amino acids 711–750, GST-St1-TAD) or the STAT6-TAD (amino acids 642–847, GST-St6-TAD). B, cell lysates of COS-7 cells transfected with CBP were incubated with GST, GST-St1-TAD, or GST-St6-TAD proteins. The bound proteins were subjected to SDS-PAGE and analyzed by blotting with anti-CBP antibodies. C, expression level of GST fusion proteins.

 
p100 Interacts with CBP in Vivo—p100 is a transcriptional coactivator that functions as an adapter between different transcription factors. We have previously shown that p100 protein directly interacts with the TAD of STAT6 and functions as a coactivator for STAT6 (13), and thereby we wanted to investigate whether p100 protein would interact with CBP. To this end, coimmunoprecipitation experiments were conducted to study the interaction of p100 and STAT6 with CBP in vivo. COS-7 cells were transfected with CBP and FLAG epitope-tagged p100 (p100-FLAG) or HA epitope-tagged STAT6 (STAT6-HA). Cell lysates were subjected to immunoprecipitation with anti-CBP antibody or control IgG antibody, and p100 and STAT6 were detected with anti-FLAG or anti-HA immunoblotting. As shown in Fig. 2A, upper panel, p100 was found to coimmunoprecipitate with CBP but not with the control rabbit IgG antibody. In contrast, there was no detectable coimmunoprecipitation between STAT6 and CBP under similar experimental conditions suggesting that there are no high affinity interactions between the proteins (Fig. 2B, upper panel). The middle and lower panels of Fig. 2, A and B show CBP, p100, and STAT6 protein levels. IL-4 stimulation did not induce interaction between CBP and STAT6 (data not shown) thus posttranslational modifications are not likely to provide explanation for the lack of interaction.



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  		FIG. 2.
p100 coimmunoprecipitates with CBP in vivo. COS-7 cells were transfected with plasmids encoding for HA-tagged STAT6 (STAT6-HA) or FLAG-tagged p100 (p100-Flag) together with CBP. Cell extracts were immunoprecipitated with anti-CBP antibody and immunoblotted with anti-FLAG antibody (A, upper panel) or anti-HA antibody (B, upper panel). 10% of total cell lysate (TCL) from transfected cells was included as a control. The immunoprecipitated CBP was detected by blotting with anti-CBP antibody (A and B, middle panel). The expression level of p100 (A, lower panel) or STAT6 (B, lower panel) from different lysates was detected by anti-FLAG or anti-HA immunoblotting.

 
STAT6 but Not p100 Interacts with SRC-1—Next we wanted to gain further insight into the specificity of p100 interactions in the assembly of STAT6 transcriptosome. SRC-1 is known to stimulate STAT6 gene responses and to directly interact with both CBP and STAT6-TAD (41, 17). To investigate whether SRC-1 interacts with p100, GST pull-down experiments were performed. Cell lysates of COS-7 cells transfected with either SRC-1a or SRC-1e were incubated with either GST or with fusion proteins GST-p100-SN, GST-p100-TD, or GST-St6-TAD-6 (amino acids 794–847). The bound proteins were subjected to SDS-PAGE and analyzed by blotting with anti-SRC-1 antibodies. As previously reported, SRC-1 was found to interact with STAT6-TAD. In contrast, there was no detectable interaction between p100-SN or -TD and SRC-1 (Fig. 3A). The expression levels of GST fusion proteins are shown in Fig. 3B.



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  		FIG. 3.
STAT6 but not p100 interacts with SRC-1. A, cell lysates of COS-7 cells transfected with either SRC-1a or SRC-1e were incubated with either GST or with GST fusion proteins GST-p100-SN, GST-p100-TD, or GST-St6-TAD-6 (amino acids 794–847). The bound proteins were subjected to SDS-PAGE and analyzed by blotting with anti-SRC-1 antibodies. B, expression level of GST fusion proteins.

 
Mapping the Interaction Domains of p100 and CBP—The predicted structure of p100 indicates that the protein is composed of four repeats homologous to the staphylococcal nuclease (SN)-fold, followed by a C-terminal tudor domain (42). The structure of CBP/p300 consists of three cysteine-histidine-rich domains (CH1–3), a KIX domain, a bromodomain, and a HAT domain (22). In order to investigate whether the interaction between p100 and CBP is direct and to identify the specific interaction domains between the proteins, we performed in vitro GST pull-down analysis. GST fusion protein plasmids encompassing the different functional domains of CBP and p100 were constructed (Figs. 4A and 5A). Purified and bead-bound GST-CBP-D1 (amino acids 1–452), GST-CBP-D2 (amino acids 452–1099), GST-CBP-D3 (amino acids 1099–1620), GST-CBP-D4 (amino acids 1620–1877), GST-CBP-D5 (amino acids 1877–2441), or GST protein alone were incubated with in vitro translated full-length p100 protein, SN-like domains, or the TD domain of p100 separately. Fig. 4B shows the expression levels of different GST-CBP fusion proteins. As shown in Fig. 4C, both CBP-D3 (amino acids 1099–1620) and CBP-D4 (amino acids 1620–1877) interacted with the p100 protein (upper panel) and the SN-like domain of p100 (middle panel), but did not bind with the TD domain of p100 (lower panel). GST protein alone or other domains of CBP did not interact with p100 protein. The in vitro translated, full-length STAT6 consistently showed only extremely weak binding to CBP (Fig. 4D). These results suggest that CBP binds directly to p100 but not to STAT6, and that the SN domains of p100 are directly interacting with the D3 and D4 regions of CBP. The D3-D4 region contains the HAT domain and CH3 domain.



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  		FIG. 4.
Mapping the interaction domain of CBP to p100 protein. A, schematic structure of CBP protein and CBP domains of GST fusion proteins. B, expression level of different GST-CBP domains. C, region from amino acids 1099–1877 of CBP binds with full-length p100 protein (upper panel) and with SN domains of p100 (middle panel) but not with TD domain (lower panel). The full-length p100, SN, or TD domains of p100 were in vitro translated and 35S-labeled and then incubated with beads loaded with different domains of GST-CBP-(D1-D5) or GST separately. D, different domains of GST-CBP-(D1-D5) or GST alone were incubated with in vitro translated and 35S-labeled STAT6. The bound proteins were subjected to SDS-PAGE and visualized by autoradiography. 20% of the in vitro translated proteins were included as a control.

 



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  		FIG. 5.
Mapping the interaction domain of p100 to CBP. A, schematic structure of p100 protein and the p100 constructs. B, expression level of different GST-p100 domains. C, p100-SN domains interact only with the HAT domain (amino acids 1099–1758) of CBP. Different domains of CBP were 35S-labeled by in vitro translation and then incubated with beads loaded with GST-p100-SN, GST-p100-TD, or GST separately. The bound proteins were subjected to SDS-PAGE and visualized by autoradiography. 20% of the in vitro translated proteins were included as a control. D, SN domain of p100 associates with HAT domain of CBP. SN, SN1 + 2, SN3 + 4, or TD domains of p100 protein were 35S-labeled by in vitro translation and then incubated with beads loaded with GST-CBP-HAT or GST separately. The bound proteins were subjected to SDS-PAGE and visualized by autoradiography. 20% of the in vitro translated proteins were included as a control.

 
To further delineate the interaction domain in p100, GST pull-down assay was performed by incubating GST-p100-SN, GST-p100-TD fusion proteins or GST alone with in vitro translated different domains of CBP-D1 (amino acids 1–452), -D2 (amino acids 452–1099), -D3 (amino acids 1099–1758), and -D4 (amino acids 1758–2441). Fig. 5B shows the expression level of GST-p100 fusion proteins. As shown in Fig. 5C, only the SN-like domains of p100 interacted with CBP-D3 (amino acids 1099–1758), but no interaction was detected with other domains of CBP. TD domain of p100 protein did not interact with any domain of CBP.

To characterize which part of the SN domain associates with CBP, we performed GST pull-down assay by incubating GST-CBP-HAT domain (amino acids 1099–1758) or GST protein alone with in vitro translated SN1 + 2 (amino acids 1–319), SN3 + 4 (amino acids 320–639) or TD (amino acids 640–885) domain of p100 protein. As shown in Fig. 5D, both SN1 + 2 (containing the first two SN domains) and SN3 + 4 domains (containing the last two SN domains) interacted with CBP-HAT domain equally. However, the interaction was weaker compared with the entire SN domain. To further delineate the interacting domain, we performed similar GST pull-down experiments with in vitro translated individual SN-domains (SN 1, 2, 3, and 4). In these experiments we could no longer detect interaction between any of the SN domains and CBP or STAT6-TAD (data not shown). This suggests that the affinity of individual SN domains for CBP is low, and the functional interaction requires an interphase formed by multiple SN domains.

Intracellular Localization of STAT6, CBP, and p100—Intracellular localization of endogenous STAT6, p100, and CBP was also investigated in HeLa cells by confocal microscopy. Cells were starved and then mock-treated or treated with IL-4 for 20 min before fixing and staining. As shown in Fig. 6, A–C, in unstimulated cells STAT6 and p100 were distributed throughout the cells, only partially colocalizing with each other. IL-4 stimulation induced significant nuclear translocation of STAT6, and STAT6 was uniformly distributed in the nucleus, except in nucleoli. The merged images show significant nuclear colocalization of STAT6 and p100 in IL-4-stimulated cells (Fig. 6, D–F). As expected CBP showed nuclear staining that was not affected by IL-4 treatment. Colocalization of STAT6 with CBP was less evident than with p100 (Fig. 6, G–L), possibly because only a small fraction of total CBP is involved in interaction with STAT6.



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  		FIG. 6.
Intracellular localization of STAT6, p100, and CBP. HeLa cells were starved in serum-free medium and treated with IL-4 (D–F, J–L) or mock-treated (A–C, G–I). Cells were fixed and stained with anti-p100, anti-STAT6, and anti-CBP antibodies, followed by Alexa 488 and Texas Red-conjugated secondary antibodies. Confocal images were collected using LSM510 program and Zeiss confocal microscope with a x63 objective. A and D, STAT6; B and E, p100; C and F, merged images of STAT6 and p100; G and J, STAT6; H and K, CBP; I and L, merged images of STAT6 and CBP.

 
p100 Recruits STAT6 to CBP in Vivo—Next we wanted to study, whether p100 protein could function as a bridging factor between CBP and STAT6 in vivo. COS-7 cells were transfected with CBP and STAT6-HA, with or without p100-Flag. CBP was immunoprecipitated from total cell lysate with anti-CBP antibody, and the presence of STAT6 was detected by anti-HA immunoblotting. As shown in Fig. 7A, STAT6 did not coimmunoprecipitate with CBP, but STAT6 was detected in the CBP immunocomplex in cells ectopically expressing p100 protein. IL-4 stimulation did not affect the interaction. The protein level of CBP, STAT6 and p100 in different lysates is shown in Fig. 7, B–D. These results suggest that p100 protein functions as a bridging factor between STAT6 and CBP.



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  		FIG. 7.
A–D, p100 protein enhances the in vivo association of STAT6 with CBP. COS-7 cells were transfected with plasmids encoding for STAT6-HA and CBP, with or without p100-FLAG as indicated. Lysates were immunoprecipitated with anti-CBP antibodies and blotted with anti-HA antibody (A), anti-CBP antibody (B). The expression levels of STAT6 and p100 protein were analyzed by blotting with anti-HA (C), or anti-FLAG antibodies (D). E, p100 enhances the association of STAT6 and CBP with Ig{epsilon} promoter HeLa cells were transfected with pSG5-p100-FLAG or empty vector. Cells were treated with IL-4 for 20 h or left untreated. Immunoprecipitation of cross-linked chromatin was performed with anti-STAT6, anti-CBP, or as control anti-rabbit IgG antibodies. DNA was extracted and analyzed for the region of human Ig{epsilon} promoter that contains a STAT6 binding site. The PCR products were visualized on an ethidium bromide gel. The lower panel shows the PCR products of the total input chromatin-aliquoted before the immunoprecipitation step. The figure is a representative of five experiments performed.

 
In Vivo Binding of STAT6, p100, and CBP Complex to the Ig{epsilon} Promoter—To confirm that p100 and CBP are recruited to IL-4-inducible Ig{epsilon} promoter by STAT6, chromatin immunoprecipitation assays were performed. HeLa cells were transfected with pSG5-p100-FLAG or empty vector and stimulated with IL-4 for 20 h or left untreated. Immunoprecipitation of cross-linked chromatin was performed with anti-STAT6, anti-CBP or as a control with anti-rabbit IgG antibodies. DNA was extracted and analyzed for the STAT6 binding site containing region of human Ig{epsilon} promoter.

As shown in Fig. 7E, upper panel, IL-4 stimulation increased the binding of endogenous STAT6 to the promoter. Ectopic expression of p100 enhanced the promoter binding of STAT6 after IL-4 stimulation. Immunoprecipitation of endogenous CBP demonstrated the enhanced formation of a ternary complex between CBP, p100, and STAT6 in Ig{epsilon} promoter after IL-4 stimulation. The lower panel shows the PCR products of the total input chromatin aliquoted before the immunoprecipitation step.

p100 Bridges the HAT Activity of CBP with STAT6—Next we wanted to investigate whether p100 could functionally link the HAT activity of CBP to STAT6. COS-7 cells were transfected with plasmids encoding for STAT6-HA, p100-FLAG, and CBP as indicated. STAT6 was immunoprecipitated with anti-HA antibody, and the beads were incubated with H4 substrate and [3H]acetyl-CoA. The acetylated substrate was purified with immobilized streptavidin, and the associated radioactivity was analyzed. The presence of exogenous CBP or p100 slightly enhanced the STAT6 copurifying histone H4 acetylation activity when compared with the activity of cells transfected only with STAT6. However, coexpression of STAT6, together with p100 and CBP elicited a marked increase in the STAT6 associated HAT activity (Fig. 8A). As a control we compared the abilities of STAT1 and STAT6 to associate with histone acetylation activity. A similar level of HAT activity copurified with both STAT1 and STAT6, but coexpression of CBP markedly increased only the STAT1 associated histone H4 acetylation activity (Fig. 8B). The protein levels of CBP, STAT1, STAT6, and p100 in different lysates are shown in the panels. These results suggest that a ternary complex of STAT6, p100, and CBP is formed in cells, and that p100 can bridge the HAT activity of CBP to STAT6.



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  		FIG. 8.
A and B, p100 couples the HAT activity of CBP to STAT6. A, COS-7 cells were transfected with plasmids encoding STAT6-HA, with or without p100-FLAG and CBP. Lysates were immunoprecipitated with anti-HA antibody. B, COS-7 cells were transfected with plasmids encoding STAT1 or STAT6 with or without CBP. Lysates were immunoprecipitated with anti-STAT1 or anti-STAT6 antibodies as indicated in the figure. The beads were incubated with H4 substrate (synthetic peptide containing the first 24 amino acids of histone H4 coupled through a GSGS linker sequence to a biotin molecule) and [3H]acetyl-CoA. The acetylated substrate was purified with immobilized streptavidin. The associated radioactivity (cpm) was counted with liquid scintillation counter. The figures are representatives of eight (A) and six (B) independent experiments. STAT1, STAT6, and p100 protein in the lysates were detected by blotting with anti-STAT1, anti-STAT6, or anti-FLAG antibodies, respectively. CBP was detected from immunoprecipitates by blotting with anti-CBP. C, p100 enhances the acetylation of histone H4 at Ig{epsilon} promoter HeLa cells were transfected with pSG5-p100-FLAG or empty vector. Cells were treated with IL-4 for 4 h or left untreated. Immunoprecipitation of cross-linked chromatin was performed with anti-acetyl-histone H4 or as control anti-HA antibodies. DNA was extracted and analyzed for the region of human Ig{epsilon} promoter that contains a STAT6 binding site. The PCR products were visualized on an ethidium bromide gel. The lower panel shows the PCR products of the total input chromatin aliquoted before the immunoprecipitation step. The figure is a representative of seven experiments performed.

 
Acetylation of Histone H4 at the Ig{epsilon} Promoter—To investigate whether p100 regulates histone acetylation at natural promoters, we performed chromatin immunoprecipitation assays at the Ig{epsilon} promoter in cells transfected with p100-FLAG or empty vector and stimulated with IL-4 for 4 h or left untreated. Immunoprecipitation of cross-linked chromatin was performed with acetyl-histone H4 antibody or as a control anti-HA antibody. DNA was extracted and analyzed for the STAT6 binding site containing region of human Ig{epsilon} promoter.

As shown in Fig. 8C, upper panel, IL-4 stimulation increased the acetylation of the H4 histones at the Ig{epsilon} promoter. Ectopic expression of p100 increased the acetylation, and the enhancement was more prominent after IL-4 stimulation. The lower panel shows the PCR products of the total input chromatin aliquoted before the immunoprecipitation step.

Down-regulation of p100 by siRNA Inhibits Ig{epsilon} Reporter Gene Expression—To confirm the significance of p100 in STAT6-linked transcription activation complex, we performed knockdown experiments with siRNAs directed against p100 in Ig{epsilon} reporter gene assays (Fig. 9). IL-4 induced strong transcriptional activation in the control cells that were not treated with siRNA or treated with siRNA directed against scrambled sequence. siRNA against STAT6 completely abrogated Ig{epsilon} reporter activation, and both the siRNAs directed against p100 caused inhibition of Ig{epsilon} reporter activation. In separate experiments the p100 siRNAs reduced the amount of p100 protein by ~60%.



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  		FIG. 9.
p100 siRNA inhibits Ig{epsilon}-reporter gene expression. HeLa cells were transfected with Ig{epsilon}-reporter, {beta}-galactosidase, and siRNAs directed against p100 (p100 I and II), STAT6 or scrambled sequence. 24 h after transfection cells were either stimulated with IL-4 or left untreated. Cells were lysed 48 h after transfection, and luciferase expression was determined. The results were normalized against {beta}-galactosidase activity of the lysates.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Sequence-specific transcription factors recruit distinct sets of coregulatory proteins through protein-protein interactions to the promoters. Activation of gene expression is likely to involve the ordered assembly of multiprotein complexes, in which coactivators function as bridging factors to recruit components of the basal transcription machinery and as chromatin-modifying enzymes. Interaction with coregulatory proteins has been shown to facilitate STAT-mediated RNA pol II-dependent transcription. However, the precise mechanisms by which these events are connected to activation of transcription are still unknown. This study was aimed to investigate the mechanism by which chromatin modifying activities are targeted to STAT6 response elements and how these activities are regulated.

Several lines of evidence indicate that histone acetylation, as well as histone deacetylation activities critically modulate the function of STAT factors (43). Most prevailing evidence is implying a functional role for CBP/p300 coactivators in the STAT-mediated transcriptional activation. However, the precise mechanisms and functions of CBP in STAT responses are not known and each STAT appears to interact with a different domain of CBP.

In this report, we investigated the relationship between STAT6 and CBP, and our results suggest, that the interaction between STAT6 and CBP is mediated by adaptor proteins. These findings are consistent with an earlier report by McDonald and Reich (30), which did not support physical interaction between STAT6 and CBP/p300, although CBP was required for STAT6 function. Interaction between CBP and STAT6 has been observed in certain experimental settings indicating that low affinity interaction may occur between the proteins (17, 29, 31). However, we did not detect direct interaction between STAT6 and CBP in the experimental settings where p100 interacted strongly with both proteins. Our results indicate that p100 interacts directly with CBP. The p100 protein is one of the first proteins shown to interact with the HAT domain of CBP between amino acids 1099 and 1758. This result is consistent with a previous report showing that p100 is present in a purified HAT preparation containing CBP, p300, and pCAF (44). Furthermore, overexpression of p100 protein enhanced the association between STAT6 and CBP suggesting that p100 protein functions as a bridging protein between STAT6 and CBP. The finding that p100 physically links CBP to STAT6 was confirmed by chromatin immunoprecipitation assay data in natural Ig{epsilon} promoter. Furthermore, functional assays demonstrated that p100 connects the HAT activity of CBP to STAT6, and importantly in chromatin immunoprecipitation assays p100 increased the amount of acetylated histone H4 at Ig{epsilon} promoter. The chromatin immunoprecipitation assays suggested that p100 stabilizes the IL-4-regulated STAT6 enhanceosome in the promoter, and provide a molecular explanation for our previous finding that p100 enhances the STAT6-mediated Ig{epsilon} gene expression in B cells. The role of p100 in IL-4-mediated induction of Ig{epsilon} transcription was confirmed using siRNAs.

STAT6-TAD mediates the interaction between p100 and STAT6. SRC-1 is another STAT6-TAD interacting coactivator that selectively regulates STAT6-mediated gene responses (17). The mechanism of SRC-1 in IL-4 gene activation is currently unknown, but the coactivator function of p160/SRC proteins is considered to be mediated through bridging CBP/p300 with sequence-specific trancription factors (45, 41). In our experiments SRC-1 did not interact with either p100-SN or -TD, whereas a strong interaction was detected between SRC-1 and STAT6-TAD as previously reported (17). Although p160/SRC family coactivators NcoA-2 and p/CIP are closely related to SRC-1, and known to interact with CBP/p300, they do not directly interact with STAT6 (17). However, p/CIP mRNA and protein levels are up-regulated by IL-4, and p/CIP enhances IL-4 transcriptional responses through still an unknown mechanism that involves interaction with p300 (20). Thus, STAT6 employs several coactivator proteins through both direct and indirect interactions in recruiting CBP/p300 and basal transcriptional machinery to IL-4-responsive promoters. The results from this and earlier studies allow us now to make the first outline of the IL-4 responsive enhanceosome (Fig. 10). STAT6 interacts directly with p100 and SRC-1 to recruit CBP/p300 to the promoter. p/CIP in turn binds to p300 and facilitates the transcriptional function of STAT6 by a mechanism that may involve induction of structural alterations in CBP/p300 that modulate the duration and level of transcriptional activation. Furthermore, our results also indicate that various STATs are utilizing different mechanisms to recruit CBP and HAT activity. While STAT6 connects to CBP through bridging factors like p100, STAT1 was found to recruit CBP and HAT activity directly and p100 did not affect the recruitment of HAT activity (Fig. 8B and data not shown).



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  		FIG. 10.
Schematic model of IL-4-responsive enhanceosome.

 
SN-like domains of p100 structurally resemble staphylococcal nucleases, small calcium-dependent enzymes, that hydrolyze both DNA and RNA (42). The SN-domains consist of two domains, an oligonucleotide/oligosaccharide-binding (OB) domain with {beta}-sheet topology, and the second domain containing two independently folded {alpha}-helices. However, SN domains of p100 lack the catalytic amino acids present in nucleases, which suggests that these domains may only function as interaction domains. It is currently not known, whether p100 can directly bind to DNA. However, there is more evidence on SN-like domains functioning in protein binding: p100 has been reported to interact with c-Myb and Pim-1 serine/threonine kinase through sequences located in SN-like domains (15, 46). The SN domains also mediate the interaction between STAT6-TAD and CBP. Individual SN-domains failed to mediate the interaction and tandem SN-domains showed diminished binding compared with the entire domain suggesting that the four SN-like domains of p100 are required for high affinity associations and can create a contact area for several simultaneous interactions.

Transcriptional activation of cytokine-responsive genes requires the cooperation of several proteins. The interaction between p100, CBP, STAT6, and RNA pol II provide insight into the mechanisms underlying the activation of STAT6-mediated transcriptional activation. Our findings support a hypothesis, that p100 brings CBP to STAT6 response elements causing the nucleosomes to unfold and facilitating the access of STAT6-p100 protein complex to the basal transcription machinery and formation of the preinitiation complex.


   FOOTNOTES
 
* This work was supported by the Medical Research Council of Academy of Finland, Medical Research Fund of Tampere University Hospital, the Finnish Foundation for Cancer Research, the Sigrid Juselius Foundation, the National Natural Science Foundation of China No. 30300070, and the Tuberculosis Foundation of Tampere. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Both authors contributed equally to this work. Back

** To whom correspondence should be addressed: Institute of Medical Technology, University of Tampere, Biokatu 8, FIN-33014 Tampere, Finland. Tel.: 358-3-215-7845; Fax: 358-3-215-7332; E-mail: olli.silvennoinen{at}uta.fi.

1 The abbreviations used are: STAT, signal transducer and activator of transcription; HAT, histone acetyltransferase; IL, interleukin; CBP, CREB-binding protein; CREB, cAMP-response element-binding protein; HA, hemagglutinin; PIPES, 1,4-piperazinediethanesulfonic acid; GST, glutathione S-transferase; siRNA, small interfering RNA; TAD, transactivation domain; SN, staphylococcal nuclease-like domain; TD, tudor domain. Back


   ACKNOWLEDGMENTS
 
We thank Merja Lehtinen and Paula Kosonen for technical assistance, Dr. Anna Polesskaya for providing the biotinylated H4 peptide, and Dr. Elliot Kieff for providing the anti-p100 antibody.



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