We have characterized the altered EP300
genomic locus, mRNA and protein (p300ΔC) from the human diffuse large B-cell lymphoma cell line RC-K8. Although truncations and mutations have been described for p300 and the related co-activator CBP in several types of cancer [2
], the RC-K8 cell line is, to date, the only example of a p300/CBP C-terminal truncation occurring in a B-cell lymphoma [8
DNA sequencing of the breakpoint in the altered EP300
locus in RC-K8 cells showed that sequences in the intron after exon 17 of EP300
are fused to sequences normally found on chromosome 6, within an intron of the BCKDHB
gene. At the site of the EP300
-chromosome 6 fusion in RC-K8 cells, there is a 9-bp sequence of unknown origin (see ). The first 5 of these 9 bp are identical to the sequence on the chromosome 22 (EP300
) side of the junction, and the last 5 bp are identical to an adjacent sequence on the chromosome 6 side. We noted a similar occurrence at the rearranged REL
junction in RC-K8 cells where 12 of 26 new bases at the chromosomal fusion junction are identical to the proximal downstream NRG
]. The presence of duplications at breakpoint junctions suggests that these alterations are the result of abnormal breakage-joining such as normally occurs in V(D)J recombination. Of note, the immunoglobulin recombinase genes RAG1
have been reported to be NF-κB target genes [29
], suggesting that the high NF-κB activity in RC-K8 cells could promote aberrant recombination.
In RT-PCR reactions of RC-K8 cell mRNA using a p300 primer and a 3’ p300ΔC-specific primer, we amplified a major band of ~120 bp that corresponds to the spliced version of the p300ΔC mRNA, as well as a minor band that co-migrated with the ~220-bp band amplified by PCR of genomic DNA (). The 220-bp band seen in this RT-PCR probably arose from either contamination of the mRNA with genomic DNA or inefficient splicing of the pre-mRNA for p300ΔC. Of note, the sequence at the downstream splice site in the chromosome 6 non-EP300 sequences that is joined to exon 17 of EP300 is identical to the consensus splice acceptor sequence (AG at the 3’ end of the intron; see ).
The cloning of the p300ΔC cDNA from RC-K8 cells enabled us to carry out a functional characterization of the p300ΔC protein. Not surprisingly, given the deletion of the HAT domain, p300ΔC is defective as a transcriptional co-activator for REL in A293 cells (). It is likely that p300ΔC is generally defective for transcriptional enhancement activity, given that numerous studies have shown that HAT activity is required for p300/CBP co-activator activity [7
]. Furthermore, although both p300 and p300ΔC interact readily with REL, p300ΔC had a reduced ability to interact with transcription factor VP16 as compared to wild-type p300 (). Therefore, in RC-K8 cells, p300ΔC is likely to have a broad range of effects on the many different cellular transcription factors with which p300 normally interacts to effect target gene expression. Of note, MYC protein expression was not especially reduced in RC-K8 cells (), even though wild-type p300 has been implicated in repression of MYC
Like wild-type p300, p300ΔC localizes to discrete “speckles” in the nucleus of chicken cells () and can interact with REL in the nucleus of RC-K8 cells [8
]. Several groups have reported that p300 and CBP localize to nuclear subdomains, often called speckles, which correspond to sites of active transcription in interphase nuclei [33
]. Therefore, even though p300ΔC lacks the HAT domain, it is likely to localize normally in RC-K8 cells. However, the subnuclear localization of p300ΔC and its lack of co-activator activity remain to be confirmed in RC-K8 cells themselves.
RC-K8 cells have several chromosomal alterations ([35
) including one within the REL
locus on chromosome 2 [36
]. Consequently, a chimeric REL protein, termed REL-NRG, is expressed in RC-K8 cells [28
]. The RC-K8 cell line also contains two other mutations that impact the REL/NF-κB pathway: mutations that inactivate the NF-κB inhibitor IκBα [22
] and inactivating mutations in the A20 ubiquitin-modifying protein [37
]. RC-K8 cells contain abundant levels of nuclear REL (and REL-NRG) and express high levels of several REL/NF-κB target genes [22
]. As we argued previously [8
], the combination of REL-activating (IκBα and A20) and REL-inhibiting (REL-NRG and p300ΔC) mutations is likely to keep nuclear REL transcriptional activity within an effective range for oncogenic activity. Indeed, several studies have shown that optimal transformation of chicken lymphoid cells occurs when REL's transcriptional activation ability is at a moderate level [21
]. Given that over-expression of REL can be toxic [43
], the reduced REL activity afforded by mutations such as truncation of p300 may be better suited for sustained B-cell proliferation and oncogenicity in cases where REL (or other p300-dependent transcription factors) has unrestrained activity. Nevertheless, truncations of p300 were not detected in two B-lymphoma cell lines---KMH2 and L428---that also have inactivating mutations in IκBα and high NF-κB activity [44
] (). To date, we have detected an obvious truncation of p300 only in the RC-K8 cell line, among 14 B-lymphoma cell lines that we have screened (; and ref. 8
Consistent with p300ΔC contributing to the oncogenic state in RC-K8 cells, the full-length p300 protein is not expressed in RC-K8 cells (; [8
]), and knock down of p300ΔC reduced the growth of RC-K8 cells in both liquid medium and soft agar (). Although over-expression of p300ΔC blocked the ability of wild-type p300 to enhance REL-dependent transactivation in reporter gene assays, it did so efficiently only when p300ΔC was in substantial excess to wild-type p300 (). Thus, if p300ΔC contributes to the oncogenic state of RC-K8 cells by reducing normal p300-mediated co-activation, then reduced WT p300 expression may have been selected in these cells to maximize p300ΔC activity. As a defective co-activator bound to REL (or other transcription factors) in the nucleus, p300ΔC would likely reduce transactivation of several target genes. In addition, p300ΔC could block other co-activators from binding to REL (or other transcription factors). Consistent with this model, CBP can readily bind REL transactivation sequences in vitro, but CBP does not efficiently co-precipitate with REL from RC-K8 cells [8
]. Alternatively, the N-terminal sequences retained in p300ΔC contribute to the growth of RC-K8 cells in a way that is not related to p300-dependent transcriptional co-activation.
Previous studies have suggested that EP300
is a tumor suppressor gene [11
], primarily based on the deletion of the HAT domain in certain cancers as well as the ability of re-expressed p300 to reduce tumor cell growth in certain cells with p300 mutations. However, most of the p300 alterations in these cancer cell lines are C-terminal truncations accompanied by loss of heterozygosity (of the wild-type EP300
allele), suggesting that retention of the truncated EP300
copy is important for growth. Moreover, mice with a conditional knockout of the p300 gene in B cells do not develop lymphoma [46
]. Therefore, we suggest that EP300
is not a tumor suppressor per se, but rather that expression of truncated, defective p300 proteins actively contributes to tumorigenesis in some cases. Future experiments will be aimed at determining how p300ΔC affects gene expression in RC-K8 cells and whether p300 mutations occur in other human B-lymphoma cell lines, especially those that have high levels of REL/NF-κB activity.