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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Cancer Lett. Author manuscript; available in PMC May 28, 2011.
Published in final edited form as:
PMCID: PMC2849871
NIHMSID: NIHMS156286
Histone acetyltransferase p300 is a coactivator for transcription factor REL and is C-terminally truncated in the human diffuse large B-cell lymphoma cell line RC-K8
Michael R. Garbati, Gökçen Alço, and Thomas D. Gilmore*
Department of Biology, Boston University, Boston, MA 02215, USA
* Corresponding author: T.D. Gilmore, Biology Department, Boston University, 5 Cummington Street, Boston, MA 02215, USA; 617-353-5444 (phone); 617-353-6340 (fax); gilmore/at/bu.edu
Human c-Rel (REL) is a member of the NF-κB family of transcription factors. REL’s normal physiological role is in the regulation of B-cell proliferation and survival. The REL gene is amplified in many human B-cell lymphomas and overexpression of REL can transform chicken lymphoid cells. In this report, histone acetyltransferase p300 enhanced REL-induced transactivation and interacted with REL both in vitro and in REL-transformed chicken spleen cells and the B-lymphoma cell line RC-K8, in which REL is constitutively active and required for proliferation. However, due to a deletion in the EP300 locus, only a C-terminally truncated form of p300 is expressed in RC-K8 cells. These results suggest a role for p300 in REL-mediated oncogenic activity in B-lymphoma.
Keywords: c-Rel, p300, CBP, RC-K8, Diffuse large B-cell lymphoma
The human c-rel proto-oncogene (REL) encodes a transcription factor in the NF-κB family of proteins. Expression of c-rel is important for normal and malignant B-cell proliferation and survival [1]. c-rel knockout mice have immune deficiencies because their B cells do not proliferate in response to mitogenic stimulation [2], and misregulation of REL has been implicated in B-cell malignancy in several ways. The REL gene is amplified or mutated in some types of B-cell lymphoma, including diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, and Hodgkin’s lymphoma [1,3]. Overexpression of REL can transform chicken lymphoid cells [4]. Additionally, REL is constitutively active in many B-lymphoma cell lines, including the human DLBCL cell line RC-K8 [5].
The REL protein contains a conserved N-terminal DNA-binding/dimerization domain of approximately 300 amino acids (aa) called the Rel homology domain (RHD). The C-terminal half of REL (aa 296-587) contains a transactivation domain (TAD) with two subdomains: TAD1 consists of aa 422-497 and TAD2 aa 518-587 [6,7]. Deletion of the entire C-terminal TAD in mice abolishes the ability of c-Rel to perform its normal functions in the B-cell immune response [8]. Moreover, the transforming activity of REL requires at least one of its two TAD subdomains to be present [7]. Therefore, understanding the mechanism by which REL activates transcription will help elucidate REL’s normal and oncogenic activities.
The related histone acetyltransferases CBP and p300 can act as coactivators for many transcription factors [9,10]. Several groups have shown that CBP/p300 can interact with NF- κB transcription factor RelA to enhance its transactivating activity [1119], but little is known about the effect of CBP/p300 on other NF- κB transcription factors. Co-expression of p300 has been shown to enhance the transactivaton ability of REL [20,21], and Wang et al. [19] showed a weak interaction between REL and CBP in whole cell extracts. However, a direct and functional nuclear interaction between REL and CBP/p300 has not been rigorously described. In this report, we show that both p300 and CBP can interact with REL through its C-terminal TAD sequences, and that this interaction enhances REL-dependent transactivation. Additionally, we show that the RC-K8 DLBCL cell line expresses only a C-terminally truncated form of p300, which can still interact with nuclear REL.
2.1. Plasmids
DNA manipulations were carried out by standard methods [22]. Complete details of all subclones and primers used in this study are described in supplementary information and at www.nf-kb.org.
2.2. Cell culture and transfection
A293 human embryonic kidney cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Biologos, Montgomery, IL, USA) as described [23]. RC-K8, BJAB, BL41, Daudi, IB4, and SUDHL4 human lymphoma cell lines, and REL-transformed chicken spleen cells were maintained in DMEM supplemented with 20% heat-inactivated FBS. The REL-transformed chicken spleen cell culture was generated using spleen necrosis virus vector JD-REL as described previously [4].
For transfections, A293 cells were seeded such that they were approximately 60% confluent on the following day when transfections were performed using polyethylenimine (PEI) (Polysciences, Warrington, PA, USA). On the day of transfection, DNA:PEI was incubated at a ratio of 1:3 in serum-free DMEM (200l for a 35-mm plate; 500 μl for a 100-mm plate) for 15 min at room temperature. The DNA/PEI mixture was then added to two (35-mm plate) or ten ml (100-mm plate) of DMEM containing 10% FBS and the final mixture was added to the cells. The next day, the transfection media was replaced with fresh DMEM containing 10% FBS. Cells were harvested and lysed 24 h later.
2.3. GST pulldown assays
GST and GST-REL fusion proteins were expressed in E. coli and purified on glutathione-agarose beads as previously described [24]. Five percent of the protein-bound beads from each sample were separated on an SDS-polyacrylamide gel and stained with Coomassie Blue to verify that approximately equal amounts of each GST-fusion protein had been purified. The remaining beads were incubated with 3 mg of A293 or RC-K8 whole cell extract for 2 h at 4°C. The beads were then washed with AT lysis buffer (20 mM HEPES pH 7.9, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 20% v/v glycerol, 1% v/v Triton X-100, 20 mM NaF, 1 mM Na4P2O7·10H2O, 1 mM DTT, 1 mM Na3VO4, 1 mM phenylmethylsulfonylfluoride [PMSF], 1 μg/ml pepstatin, 1 μg/ml leupeptin, 10 μg/ml aprotinin [Sigma, St. Louis, MO, USA]) and proteins were eluted by heating at 95°C in SDS sample buffer. Proteins were electrophoresed on a 6% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane using a large-protein transfer buffer (20 mM Tris, 150 mM glycine, 0.05% SDS, 10% methanol). One percent of the amount of extract used for one pulldown (30 μg) was included on the gel as an input lane. The membrane was then subjected to anti-p300 or anti-CBP Western blotting.
2.4. Western blotting
Western blotting was performed as described previously [7,23]. The following antisera were used: rabbit anti-REL (1:10,000 dilution; anti-C-terminal, #265, Nancy Rice, NCI, Frederick, MD, USA), rabbit anti-REL (1:2500; anti-NLS, #1206, Nancy Rice), mouse anti-CBP (1:200; sc-7300, Santa Cruz Biotechnology, Santa Cruz, CA USA), rabbit anti-CBP (1:1000; #4772, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-p300 (1:200; anti-N-terminal, sc-584, Santa Cruz Biotechnology), rabbit anti-p300 (1:200; anti-C-terminal, sc-585, Santa Cruz Biotechnology), rabbit anti-FLAG (1:1000; #2368, Cell Signaling Technology), rabbit anti-HA (1:500; sc-805, Santa Cruz Biotechnology), mouse anti-MYC (1:200; sc-40, Santa Cruz Biotechnology), rabbit anti-IκBα(1:500; sc-371, Santa Cruz Biotechnology), and rabbit β-tubulin (1:500; sc-9104, Santa Cruz Biotechnology).
2.5. Immunoprecipitation
For co-immunoprecipitation of overexpressed proteins, A293 cells in 100-mm plates were co-transfected with 5 μg HA-p300 and 5 μg FLAG-REL, FLAG-REL TAD1, FLAG-REL TAD2, FLAG-REL-RHD, or pcDNA-FLAG; or with 5 μg pCMV-CBP and 5 μg MYC-REL, MYC-REL-RHD, or pcDNA. Two days later, cells were washed in PBS and nuclear extracts were prepared as described previously [25]. For anti-FLAG immunoprecipitation, 500 μg of each nuclear extract was incubated with anti-FLAG M2 affinity gel (Sigma) in high salt buffer (20 mM HEPES pH 7.9, 400 mM NaCl, 1 mM EDTA, 1 mM EGTA) overnight at 4°C. For anti-MYC immunoprecipitations, 3 μg of anti-MYC antiserum (sc-40, Santa Cruz Biotechnology) was incubated with protein G Sepharose beads in high salt buffer overnight at 4°C. Antibody-bound beads were then washed and incubated with 500 μg of each nuclear extract in high salt buffer overnight at 4°C. Beads were then washed in high salt buffer and proteins were eluted by heating at 95oC in SDS sample buffer. Proteins were electrophoresed on a 6% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane using a large-protein transfer buffer as described above. For all samples, 1% of the amount of nuclear extract used for one pulldown (5 μg) was included on the gel as an input lane. The membranes were then subjected to anti-HA (to detect p300) or anti-CBP Western blotting.
Co-immunoprecipitations of endogenous REL with p300 or CBP were performed using the Nuclear Complex Co-IP Kit essentially as described by the manufacturer (Active Motif, Carlsbad, CA, USA). Three μg of normal rabbit IgG (sc-2027, Santa Cruz Biotechnology), anti-p300 antiserum (sc-584, Santa Cruz Biotechnology) or anti-CBP antiserum (#4774, Cell Signaling Technology) was incubated with protein A Sepharose beads (Amersham Biosciences, Piscataway, NJ, USA) in IP Low Buffer (Active Motif) overnight at 4°C. Antibody-bound beads were then washed with IP Low Buffer and incubated with 250 μg of nuclear extract from RC-K8 cells or REL-transformed chicken spleen cells in IP Low Buffer overnight at 4°C. Beads were washed with IP Low Buffer and proteins were eluted by heating at 95°C in SDS sample buffer. To detect REL and p300ΔC, proteins were electrophoresed on a 7.5% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane. To detect p300 or CBP, proteins were electrophoresed on a 6% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane using a large-protein transfer buffer containing 0.05% SDS and 10% MeOH. One percent (for anti-REL) or 10% (for anti-p300 or anti-CBP) input lanes were included. The membranes were then subjected to anti-REL, anti-p300, or anti-CBP Western blotting.
2.6. Luciferase reporter assays
Luciferase reporter assays were performed as described previously [24]. A293 cells in 35-mm plates were transfected with 0.5 μg of reporter plasmid pGL2-3x-κB-luciferase and 0.5 μg of normalization plasmid RSV-βgal. Cells were co-transfected with 0.5 μg of pcDNA-REL or vector alone, along with 0.5 μg of HA-p300, pCMV-CBP, or vector alone. Cells were harvested in Reporter Lysis Buffer (Promega, Madison, WI, USA) and luciferase activity was measured using the Luciferase Assay System (Promega). Luciferase and β-galactosidase activites were determined, and values were normalized to the relevant vector control (1.0). Values are the averages of three experiments performed with triplicate samples.
2.7. Northern blotting
Northern blotting was performed essentially as described previously [22]. Total RNA was isolated from A293 and RC-K8 cells using TRIzol Reagent according to the manufacturer’s protocol (Invitrogen, Carlsbad, CA, USA). RNA was separated by agarose gel electrophoresis and then transferred by capillary action to a Zeta-Probe GT membrane (Bio-Rad, Hercules, CA, USA). RNA was UV-crosslinked to the membrane with a Stratalinker 2400 (Stratagene, La Jolla, CA, USA). Probes were produced using PCR products generated from the 5′ approximately 500 bases of EP300 (using primers p300-5′probe-For and p300-5′probe-Rev) and from approximately 600 bases of exon 31 of EP300 (using primers p300-exon31-For2 and p300-exon31-Rev2) (see Fig. S1 and supplementary information). These PCR products were used as templates to generate random-primed 32P-labeled DNA probes as previously described [26]. Membranes were prehybridized for 1 h in Amersham Rapid-hyb buffer (GE Healthcare, Little Chalfont, UK) at 65°C with rotation in a Robbins Model 2000 Micro Hybridization Incubator (Robbins Scientific, Sunnyvale, CA, USA). The denatured probe was then added to the hybridization buffer and incubated with the membrane overnight at 65°C with rotation. Membranes were washed twice with 50 ml of 2X SSC/0.1% SDS for 20 min each at 25°C, twice with 50 ml of 0.2X SSC/0.1% SDS for 15 min each at 65°C, and twice with 50 ml of 0.1X SSC/0.1% SDS for 15 min each at 65°C. Membranes were then exposed to X-ray film with intensifying screens for 2–7 days at −80°C.
2.8. Real-time PCR
For reverse transcription real-time quantitative PCR, total RNA was obtained from A293 and RC-K8 cells using TRIzol Reagent according to the manufacturer’s protocol (Invitrogen). RNA was reverse transcribed into cDNA using M-MLV reverse transcriptase (Promega) and random primers (Promega). One thirtieth of the synthesized cDNA was combined with EP300 primers (see Fig. S1) or GAPDH primers and Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA). PCRs were performed in the ABI Prism 7900HT Sequence Detection System (Applied Biosystems) using 40 cycles of 94°C for 15 sec and 60°C for 1 min. Cycle threshold (Ct) levels were obtained for each sample and the average of the three wells was calculated. To normalize the EP300 samples, the Ct value for GAPDH cDNA amplification was subtracted from each value (Δ Ct). Then, the ΔCt value for A293-cell samples was subtracted from each value for RC-K8-cell samples (ΔΔCt). Relative EP300 mRNA expression levels were then calculated from the ΔΔCt values (2−ΔΔCt) as described previously [27]. Normalized relative expression values are the averages of three experiments and standard error was calculated.
For genomic qPCR, genomic DNA was isolated from BJAB and RC-K8 cells. Fifty ng of DNA was combined with EP300 exon-specific forward and reverse primers and Power SYBR Green PCR Master Mix (Applied Biosystems). PCR was then performed as described above. The Ct value for EP300 exon 2 amplification was subtracted from each value (ΔCt). Next, the ΔCt value for each BJAB cell sample was subtracted from the value for the corresponding EP300 exon from the RC-K8 cell sample (ΔΔCt). The relative abundance of each exon was then calculated from the ΔΔCt values (2−ΔΔCt) as described previously [27]. Normalized relative abundance values are the averages of three experiments and standard error was calculated. The sequences of all primers are listed in supplementary information.
2.9. Semi-quantitative RT-PCR
Total RNA was isolated from A293 and RC-K8 cells using TRIzol Reagent and reverse transcribed into cDNA as described above. Twenty-seven cycles of PCR was performed on 1/15 of the synthesized cDNA using EP300 primers (see Fig. S1). PCR products were separated by agaorse gel electrophoresis and visualized by ethidium bromide staining. ImageJ software (National Institutes of Health, Bethesda, MD, USA) was used to measure band densities. The value from the no-template control (NTC) lane was subtracted from the value for each band and then values were normalized to those of amplicons from cDNA obtained from A293 cells (1.0).
3.1. CBP and p300 interacted with REL transactivation domain sequences in vitro
To determine whether CBP and p300 can interact with REL, pulldown experiments were performed using bacterially-expresed GST-fusion proteins containing subdomains of REL and A293 human embryonic kidney whole cell extracts (Fig. 1A). First, GST fusion proteins on beads were incubated with equal portions of an A293 cell extract, and the presence of CBP in the pulldowns was assessed by Western blotting. Fig. 1B shows that the REL transactivation domain (GST-REL-TAD) interacted strongly with CBP, the RHD (GST-REL-RHD) interacted weakly with CBP, and GST alone did not interact with CBP. To define the C-terminal REL sequences that interacted with CBP, GST-REL fusions containing deletions of one (GST-RELΔTAD1; GST-RELΔTAD2) or both (GST-RELΔTAD1,2) of the C-terminal TAD subdomains were used in a similar pulldown experiment. GST-RELΔTAD1 and GST-RELΔTAD2 pulled down CBP, albeit less efficiently than the complete TAD in GST-REL-TAD (Fig. 1C). GST-RELΔTAD1,2 and GST alone did not pull down REL. Taken together, these results indicate that each TAD subdomain contributed to REL’s interaction with CBP.
Fig. 1
Fig. 1
CBP and p300 interact with REL transactivation domain sequences in vitro. (A) Structures of full-length REL (top) and GST-REL fusion proteins used in this study. RHD, Rel homology domain; TAD, transactivation domain; GST, glutathione-S-transferase. (B (more ...)
To determine whether REL can also interact with p300, a similar pulldown experiment was performed. GST-REL-TAD interacted strongly with p300, GST-REL-RHD interacted weakly with p300, whereas GST-RELΔTAD1,2 and GST alone did not interact with p300 (Fig. 1D). This indicates that p300, like CBP, bound to the REL TAD.
3.2. p300 and CBP interacted with REL in vivo and acted as coactivators for REL-induced transactivation
To determine whether REL interacts with p300 in vivo, FLAG-REL expression constructs for full-length REL, REL-RHD, RELΔTAD1, and RELΔTAD2 or vector alone were co-transfected with an HA-p300 expression plasmid into A293 cells. Nuclear extracts were later immunoprecipitated with anti-FLAG antiserum and immune complexes were subjected to anti-HA Western blotting. HA-p300 co-precipitated with full-length FLAG-REL, FLAG-RELΔTAD1, and FLAG-RELΔTAD2, but HA-p300 was not detected in immunoprecipitates from cells transfected with FLAG-REL-RHD or vector alone (Fig. 2A). These results suggest that p300 interacted with REL in vivo primarily through the C-terminal TAD of REL. To determine whether REL interacts with CBP in vivo, expression vectors for MYC-tagged versions of full-length REL, REL-RHD (lacking the entire REL TAD), or vector alone were co-transfected into A293 cells with a CBP expression vector. Nuclear extracts were immunoprecipitated with anti-MYC antiserum, and immune complexes were subjected to anti-CBP Western blotting. CBP interacted with full-length MYC-REL, but interacted only weakly with MYC-REL-RHD (Fig. 2B). Therefore, similar to p300, CBP interacted with REL primarily through the C-terminal TAD of REL.
Fig. 2
Fig. 2
p300 and CBP co-immunoprecipitate with REL and act as REL coactivators. (A) Anti-FLAG immunoprecipitation (IP: FLAG) was performed on nuclear extracts of A293 cells that had been transfected with 5 μg of expression plasmids for the indicated FLAG-REL (more ...)
To determine whether p300 can affect REL-dependent transactivation, A293 cells were transfected with expression vectors for REL plus p300 or vector alone and a luciferase reporter plasmid with a promoter containing three upstream κB sites. p300 enhanced REL-mediated transactivation by approximately two-fold (Fig. 2C, left panel). A similar experiment showed that CBP also enhanced REL activity by approximately two-fold (Fig. 2C, right panel).
3.3. RC-K8 cells express a C-terminally truncated p300 protein
In the course of screening human B-lymphoma cells for p300 expression, we discovered that the RC-K8 cell line expressed a smaller version of p300. That is, Western blotting of RC-K8 cell extracts using an antibody directed against the N terminus of p300 detected only one anti-p300-reactive protein which migrated at about 160 kDa (Fig. 3A). This smaller anti-p300-reactive protein in RC-K8 cells was expressed approximately ten-fold less than wild-type p300 in A293 cells (Fig. 3A).
Fig. 3
Fig. 3
RC-K8 cells express a C-terminally truncated p300 protein. (A) Anti-p300 Western blotting of A293 and RC-K8 whole cell extracts. Twenty-five μg of A293 extract and 25, 50, 100, and 200 μg of RC-K8 extract were loaded. (B) The six indicated (more ...)
Western blotting with the N-terminal p300 antibody showed that p300 is expressed as an apparently full-length protein in the BJAB, BL41, Daudi, IB4, and SUDHL4 human B-lymphoma cell lines (Fig. 3B). As controls, extracts from A293 cells and REL-transformed chicken spleen cells, both of which express full-length p300, were also analyzed. As before, the only p300-reactive protein in RC-K8 cell extracts was the 160-kDa protein, which was less abundant than full-length p300 expressed in other cell lines (Fig. 3B, top panel). The RC-K8-cell 160-kDa protein was not detected by Western blotting with a C-terminal p300 antibody (Fig. 3B, 2nd panel from top). Therefore, this RC-K8-specific protein is likely to be a C-terminally truncated form of p300, herein called p300ΔC.
The RC-K8 cell line expresses a mutant REL fusion protein (REL-NRG) but no IκBα protein, due to inactivating mutations [5,28]. To confirm that the cell line used in the current studies was indeed RC-K8, we showed that REL-NRG, but not IκBα, was expressed in these cells (Fig. 3C).
3.4. p300ΔC interacted with REL in RC-K8 cells and with REL TAD sequences in vitro
To determine whether p300ΔC retains the ability to interact with REL, we performed an anti-p300 immunoprecipitation on nuclear extracts from RC-K8 cells. Anti-REL Western blotting showed that REL specifically co-immunoprecipitated with p300ΔC (Fig. 4A). REL did not, however, interact strongly with CBP in RC-K8 cells, as little REL was detected in anti-CBP immunoprecipitates (Fig. 4B). REL also co-immunoprecipitated with full-length p300 from nuclear extracts of REL-transformed chicken spleen cells (Fig. 4A). These results show that REL interacts with p300 (or p300ΔC) under conditions where REL has oncogenic activity.
Fig. 4
Fig. 4
p300ΔC interacts with REL in RC-K8 cells and with REL TAD sequences in vitro. (A) RC-K8 (top) and REL-transformed chicken spleen (bottom) cell nuclear extracts were immunoprecipitated (IP) with normal rabbit IgG or anti-p300 antiserum. Immunoprecipitates (more ...)
To determine whether p300ΔC also interacted with REL through the C-terminal TAD, a GST pulldown assay was performed using RC-K8 whole cell extracts. Anti-p300 Western blotting showed that GST-REL-TAD pulled down p300ΔC, whereas GST-REL-RHD, GST-REL-TADΔ1,2, and GST did not (Fig. 4C). These results indicate that, similar to full-length p300, p300ΔC interacted with REL through the TAD of REL.
3.5. RC-K8 cells expressed an EP300 mRNA truncated at its 3′ end
We next performed Northern blotting to determine whether there was an alteration in the size of the normal 9.5-kb p300 mRNA (EP300) in RC-K8 cells. A probe complementary to 5′ sequences of EP300 detected predominantly an EP300 mRNA from RC-K8 cells that migrated at about 8 kb, whereas wild-type EP300 mRNA from A293 cells migrated at the expected 9.5 kb (Fig. 5A, left panel). A probe complementary to sequences encoded by exon 31, the last exon of EP300, detected the wild-type 9.5 kb EP300 mRNA from A293 cells, but detected little, if any, EP300 mRNA in RC-K8 cells after an exposure similar to that of the 5′ EP300 probing (Fig. 5A, right panel). These results indicate that most EP300 mRNA in RC-K8 cells lacked the wild-type 3′ sequences, and strongly suggest that this alteration was responsible for the expression of p300ΔC.
Fig. 5
Fig. 5
RC-K8 cells express a 3′ truncated EP300 mRNA. (A) Northern blotting using probes from 5′ (exons 1–2) and 3′ (exon 31) sequences of EP300 was performed. Fifty, 50, and 20 μg of A293 RNA and 80, 50, and 20 μg (more ...)
As a second means of looking at EP300 mRNA in RC-K8 cells, we performed real-time PCR analysis of cDNA using 5′ (exon 2) and 3′ (exon 31) primer sets. As compared to A293 cells, most of the EP300 transcripts expressed in RC-K8 cells lacked exon 31 sequences (Fig. 5B). That is, while EP300 expression in RC-K8 cells was 77% of that in A293 cells as judged by amplification with exon 2 primers, EP300 expression in RC-K8 cells was only 16% of that in A293 cells when exon 31 primers were used. This suggests that ~20% (0.16/0.77) of the EP300 transcripts expressed in RC-K8 were full-length, and that ~80% lacked exon 31 sequences. Similar results were obtained by semi-quantitative RT-PCR comparing amplification of EP300 cDNA sequences encoded by exons 3–12 to those encoded by exon 31 in A293 vs RC-K8 cells (Fig. 5C).
3.6. Genomic EP300 sequences were deleted after exon 17 in one allele in RC-K8 cells
To determine whether one of the EP300 genomic loci in RC-K8 cells contained a deletion, we performed real-time PCR analysis of individual exons (exons 13 through 27) of EP300 from RC-K8 cell genomic DNA. As controls, we amplified EP300 exon 2 from RC-K8 cells (which we knew was present in EP300 mRNA from RC-K8 cells), and we compared the values for each exon amplification from RC-K8 cells to a parallel amplification of EP300 DNA from BJAB cells (which are not known to contain any EP300 alteration). The same relative amounts of product were obtained for EP300 exons 13–17 from BJAB and RC-K8 cell DNA, whereas amplifications from exons 18–27 yielded approximately half the amount of product from RC-K8 cell DNA (Fig. 6A). These results suggest that RC-K8 cells have lost EP300 sequences after exon 17 on one allele.
Fig. 6
Fig. 6
EP300 sequences are deleted after exon 17 on one allele in RC-K8 cells. (A) Real-time PCR for individual exons of EP300 was performed using BJAB and RC-K8 genomic DNA. Specific primer sets for exons 2, 13–25 and 27 were used for PCR amplification (more ...)
This study reports that the histone acetyltransferases p300 and CBP directly interacted with and acted as transcriptional coactivators for transcription factor REL. Also, p300 and REL were found in a complex in nuclear extracts from both REL-transformed chicken spleen cells and the DLBCL cell line RC-K8, which has constitutively nuclear REL activity. Given that REL’s oncogenic activity depends on its ability to activate transcription, these results suggest that the REL-p300 interaction plays a role in REL-dependent transformation. Furthermore, we show that the RC-K8 cell line exclusively expressed a C-terminally truncated version of p300, due to a genomic loss of 3′ sequences from one allele of EP300. These results represent the first identification of a p300 truncation in a B-lymphoma cell line.
The RC-K8 DLBCL cell line is noteworthy because of the presence of mutations in genes encoding four components of the NF-κB/REL pathway: REL, IκBα, A20, and p300. We know of no other example of a tumor cell line that has four mutations which impact a single signaling pathway. First, RC-K8 cells express a C-terminally truncated REL protein, REL-NRG, in which sequences of the RHD are fused to non-REL gene sequences [28,29]; because the TAD sequences are missing in REL-NRG, the protein can no longer activate transcription [5]. Second, RC-K8 cells contain inactivating mutations in the REL inhibitor IκBα and no IκBα protein can be detected in these cells; ectopic expression of functional IκBα inhibits the proliferation of RC-K8 cells [5]. Third, there are inactivating mutations in the gene encoding A20, an upstream negative regulator of NF-κB, and expression of wild-type A20 in RC-K8 cells induces apoptosis [30]. Finally, as we show herein, RC-K8 cells express a truncated version of p300, a coactivator for REL-dependent transactivation. Given that chronic, low level REL transactivation is optimal for avian B-cell transformation [7,31], it is likely that RC-K8 cells have fine-tuned their level of REL-dependent target gene transactivation by balancing mutations that positively influence REL activity (mutations in IκBα and A20) with ones that reduce REL’s transactivation ability (REL-NRG and p300ΔC). In this model, inactivation of IκBα provides the major impetus for constitutively nuclear REL activity. However, because high level expression of REL is toxic in certain settings [32], mutations that created the DNA-binding competent but transcriptionally inactive REL-NRG protein and the p300ΔC mutant protein may have been selected to dampen REL-driven transactivation of target genes. In addition, the interaction of p300ΔC with REL may block the ability of CBP to compensate for the loss of normal p300 co-activating function in RC-K8 cells. No RelA-containing nuclear complexes are detected in RC-K8 cells, suggesting that the constitutive NF-κB activity is exclusively due to REL complexes [5]. p300ΔC was also expressed in RC-K8 cells at a much lower level than wild-type p300 in both A293 cells and five B-lymphoma cell lines (Fig. 3A and B).
A20 acts as a negative upstream regulator of NF-κB activity by deubiquinating TRAF6 and modifying the ubiquitination of RIP1 [33,34]. Whether A20 mutations contribute to the oncogenic state of RC-K8 cells by promoting unrestrained NF-κB activity is not clear. That is, expression of wild-type A20 was shown to induce apoptosis in RC-K8 cells [30]; however, it was conspicuously not shown that expression of A20 reduces nuclear NF-κB complexes in these cells. Indeed, given that there is no IκBα protein in RC-K8 cells, the reconstitution of normal A20 upstream activity would not be expected to reduce the amount of nuclear REL protein. Thus, at least in RC-K8 cells, A20 mutations may either contribute to oncogenesis by affecting NF-κB/REL at a downstream step or by affecting other pathways. Indeed, A20, through TRAF6 deubiquitination, also negatively regulates MAPK activity [35].
Based on Northern blotting and quantitative RT-PCR of mRNA and genomic DNA, sequences from exons 18–31 appear to be missing from one of the EP300 loci in RC-K8 cells. These results indicate that p300ΔC contains only those amino acid residues encoded by EP300 exons 1–17 (aa 1-1047); therefore, p300ΔC would be missing the HAT domain (aa 1224-1669) [36] and the entire C-terminal transactivation domain (Fig. 6A and B). As such, p300ΔC would be expected to be unable to enhance transcriptional activation by REL because other studies have shown that the HAT domain is required for p300′s transcriptional enhancement ability [10,20,36,37]. Also, the N- and C-terminal regions of p300 form a bipartite transctivation domain that recruits TBP, TFIIB, and RNA polymerase [10], and this function would also be abolished in p300ΔC. Based on the size of the p300ΔC protein on SDS-polyacrylamide gels (~160 kDa), we suggest that p300ΔC is fused to approximately 45 kDa of non-p300 sequences, because exons 1–17 contain 1047 codons, which would yield approximately 115-kDa of p300 polypeptide.
Although our RT-PCR experiments indicate that some wild-type p300 mRNA is expressed in RC-K8 cells (Fig. 5), we have not been able to detect any wild-type p300 protein in these cells. Of note, both wild-type and mutant IκBα mRNAs are also expressed in RC-K8 cells, but no wild-type IκBα protein could be detected [5]. These results raise the intriguing possibility that the expression of certain mutant mRNAs (e.g., for p300 and IκBα) in RC-K8 cells can suppress translation from the corresponding wild-type mRNAs.
To our knowledge, this report provides the first evidence of a p300 truncation in a B-cell lymphoma. Nevertheless, inactivating mutations in p300, many of which result in C-terminal truncations, have been identified in other types of human tumors and tumor cell lines. Previous reports of p300 truncating mutations have been mostly in cancers of epithelial origin, including colorectal, gastric, breast, pancreatic, cervical, and ovarian [9,10,38,39]. Most cell lines containing p300 truncations have deleted or silenced second EP300 alleles, and the colorectal cancer cell line HCT116 contains different mutations in its two EP300 alleles [9,10,39]. Expression of wild-type p300 slows the growth of two cancer cell lines with biallelic inactivating mutations in EP300 [40]. Moreover, p300 knockout mice develop histiocytic sarcomas [41]. Based on these types of data, p300 has been suggested to be a tumor suppressor. Alterations of p300 have been less frequently found in hematopoietic malignancies. Translocations resulting in fusions of p300 to monocytic leukemia zinc-finger protein (MOZ) in acute myeloid leukemia patients and to mixed lineage leukemia (MLL) in myelodysplastic syndrome patients have been reported [4244].
Future work is needed to identify the precise genetic alteration that results in expression of the p300ΔC protein in RC-K8 cells, to determine whether p300ΔC contains non-p300 sequences, and to determine whether the p300ΔC protein contributes to the growth, survival, or malignant phenotype of RC-K8 cells. Along these lines, it will be interesting to determine whether overexpression of wild-type p300 or knockdown of p300ΔC can affect the growth or survival of RC-K8 cells or the interaction of REL with CBP.
Supplementary Material
Acknowledgments
This work was supported by NIH grant CA047763 and ARRA supplement CA047763-21S3 (to T.D.G.). G.A. was supported by NSF-REU CHE-0649114. We thank Moritz Brüggemann for help with this work, and Josh Leeman, Dan Starczynowski, and Melanie Herscovitch for helpful discussions.
Abbreviations
aaamino acid(s)
DLBCLdiffuse large B-cell lymphoma
FLAGflu antigen
NLSnuclear localization signal
NRGnon-REL gene
RHDRel homology domain
qPCRquantitative polymerase chain reaction
RT-PCRreverse transcriptase-PCR
TADtransactivation domain

Footnotes
Conflict of interest statement
No conflict of interest is declared.
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