All three members of the PLAG family have been shown to be involved in tumorigenesis (reviewed in (Van et al., 2007
; Abdollahi, 2007
)), however, little is known about the mechanisms that regulate their activity. In the present study, we identified PC2, a component of the ARC/Mediator complex, as a novel PLAGL2-binding protein and an important modulator of PLAGL2 transactivation.
The partial PC2434-784
clone isolated from the yeast-two hybrid screen demonstrated that the C-terminal domain of PC2 was sufficient for PLAGL2 binding. In vivo
Co-IP studies in HEK293 cells confirmed the yeast two-hybrid results and show that PC2 binds to PLAGL2 via the C-terminus of PLAGL2. This is consistent with previous reports showing a direct interaction of the Mediator subunit, PC2, with other transcriptional regulators, including the sterol regulatory element binding protein (SREBP), and Smad factors 2/3 and 4 (Yang et al., 2006
; Kato et al., 2002
). The ability of PC2 to interact with various gene-specific transcription factors, suggests that, as a component of the ARC/Mediator complex, PC2 may play a key role in targeting Mediator to a distinct set of promoters.
Full-length PLAGL2 expressed in the Co-IP studies was shown to be nuclear, as previously reported (Zheng and Yang, 2005
). We further ascertained that, like PLAG1 (Braem et al., 2002
), nuclear localization was determined by the N-terminal domain of PLAGL2. Although we did not attempt to specifically identify a nuclear localization signal, PLAGL2 does contain a similar sequence (PRPR) at the same amino acid location corresponding to the putative PLAG1 nuclear localization sequence (KRKR). Thus, it will be of interest to determine if the PRPR sequence of PLAGL2 serves a similar function.
The binding of PLAGL2 to the NCF2
TRR in previous studies was somewhat surprising, as there appeared to be no obvious binding site for PLAGL2 within this sequence. In this report, mutagenesis of the TRR showed that the nucleotide sequence required for PLAGL2 binding contained the PLAG1 consensus core sequence (GGRGGCC) but lacked the G-cluster motif. Although this binding site is located on the reverse strand, this arrangement is similar to the functional PLAG1 binding site characterized in the IGF-II
promoter (Voz et al., 2000
). Mutagenesis of PLAGL2 zinc fingers demonstrated that zinc fingers 5 and 6 were important for TRR binding, with zinc finger 2 playing much less of a role. These data are consistent with a previous report demonstrating the importance of PLAGL2 zinc fingers 5 and 6 in binding to the PLAG1 core sequence and of zinc finger 2 in binding to the G-cluster (Hensen et al., 2002
). The ability of PLAGL2 to bind relatively efficiently to the core sequence alone demonstrates that, like PLAGL1, PLAGL2 can also bind GC-rich DNA in the absence of the G-cluster. These data support the idea that these family members can recognize DNA-binding sites that are, in general, GC rich with some variability in sequence, possibly leading to functional redundancy between proteins and/or competition for similar target genes. Elucidation of the complete set of PLAG target genes and DNA-recognition sites in their respective target promoters will be important in addressing this issue.
As stated earlier, overexpression of PLAGL2 alone had little to no affect on the activity of the NCF2
promoter-reporter plasmid in HEK293 cells, although we demonstrated binding of endogenous PLAGL2 to the NCF2
promoter in TNF-α-treated MM1 cells in a recent study (Ammons et al., 2007
). The lack of NCF2
promoter-reporter activity by PLAGL2 alone, however, was not unprecedented, as similar results were reported for PLAGL2 transactivation of an IGF-II
promoter-reporter (Ning et al., 2008
). In those studies, overexpression of PLAGL2 had no significant effect on the activity of the IGF-II
promoter-reporter, however, when PLAGL2 was overexpressed with Tip60, a PLAGL2-binding protein that modulates PLAGL2 transactivation through acetylation, the activity of the IGF-II
promoter-reporter was stimulated approximately ~2 fold. In contrast, another PLAGL2 target promoter, SP-C, showed no enhanced activity in the presence of Tip60, demonstrating that the effect of Tip60 on PLAGL2 transactivation was promoter-specific. In this study, we show that PC2 enhanced PLAGL2 transactivation of the NCF2
promoter 2- to 3-fold in cooperation with PU.1. We also show the importance of endogenous PC2 in PLAGL2-induced IGF-II
expression, implying that the effect of PC2 on PLAGL2 target promoter activity may be a general mechanism of PLAGL2 transactivation. Additional studies, however, are required to determine if PC2 is indeed being targeted to the IGF-II
promoter via PLAGL2 binding. Together, the findings presented here demonstrate that PC2 is a modulator of PLAGL2 transactivation, and, it will be of interest to determine if PC2 plays a role in the regulation of all PLAGL2 responsive genes or, as suggested for Tip60 regulated PLAGL2 activation, if the effect is limited to a subset of PLAGL2 target promoters.
The data presented here support a model whereby PLAGL2, PU.1, and PC2 physically associate with the NCF2 promoter directly, or indirectly through protein-protein interactions, and that they cooperate to enhance the activity of the NCF2 promoter-reporter. The data suggest that PLAGL2 and PU.1 are required to bind to their respective sites in the promoter, and that PC2 is targeted to the promoter via the C-terminus of PLAGL2, the region required for transactivation. We demonstrate that GFP-PLAGL2 interacts with endogenous PC2 and that endogenous PC2 associates with a PLAGL2 target promoter in TNF-α-treated MM1 cells in a similar region occupied by PLAGL2. In addition, we show that PLAGL2 regulation of NCF2 in response to TNF-α is inhibited with PC2 knockdown, thus demonstrating the physiological relevance of PC2 as an effector of PLAGL2 transactivation. We cannot, however, rule out the possibility that the interaction between PLAGL2 and PC2 is indirect, and that there may be additional proteins acting as linkers between PLAGL2 and PC2, as suggested by the inconclusiveness of the sequential ChIP studies (data not shown). The role of PC2 as a subcomponent of the large multi-protein Mediator complex implies that the PC2-enhanced NCF2 promoter activity is likely the result of Mediator recruitment to the promoter via PLAGL2, allowing formation of a stable preinitiation complex. Additional studies are necessary to further characterize this model and to determine if the effect of PC2 is indeed through targeting Mediator to PLAGL2 target promoters.
Although there are many reports alluding to the role of the PLAG proteins in key physiological processes, including oncogenesis, understanding the mechanisms whereby PLAG proteins regulate these processes, including the identification of the complete set of target genes and regulatory cofactors, is far from complete. This study further contributes to our understanding of PLAG gene regulation by establishing PC2 as a novel PLAGL2-binding protein and modulator of PLAGL2 transactivation. Considering the oncogenic potential of PLAG proteins, elucidating their mechanisms of activation by identifying gene targets, signaling pathways, and the functional relationship between family members will be important in indentifying avenues for early diagnosis and novel therapeutic targets for treatment of diseases and/or disorders associated with the aberrant expression of these proteins.