We have shown that the highly related DEAD box RNA helicases p68 and p72 act as repressors of transcription in a promoter-context manner. When targeted to the TK-CAT promoter-reporter construct they both strongly repress transcription (Figure 1). Furthermore, this transcriptional repression does not appear to be due to either squelching or physical blocking of the transcription apparatus (Figure 2), implying an active transcriptional mechanism. Moreover repression of TK-CAT was observed in several cell lines (U2OS, 293, MCF-7) suggesting that it is not cell line dependent. In order to determine whether this repression activity exhibited any promoter specificity we also tested the ability of p68 and p72 to repress transcription of other constitutively active promoter-reporter constructs with high basal levels of transcription, namely MLP-CAT and SV40-CAT. Interestingly MLP-CAT revealed a difference in the ability of p68 and p72 to repress transcription, with p72 strongly repressing transcription of this promoter-reporter and p68 having no effect (Figure 1). This observation suggests that, although highly homologous (70% overall identity at the amino acid level ) p68 and p72 proteins may act differently in some contexts, perhaps through the association with different protein partners. Neither p68 nor p72 repressed transcription of SV40-CAT (Figure 1) suggesting that the repression by p68 and p72 is promoter context-dependent, an observation that has been reported for other transcription factors . These findings thus are consistent with the observed repression activity of p68/p72 being an active process. Interestingly, in this context, another DEAD box protein DP103 (Ddx20) has been found to act as a co-repressor of the Ets repressor METS/PE1 .
Using a series of deletion derivatives of p68 and p72 we identified three domains, within the core conserved among the DEAD box family of proteins, which can independently repress transcription (Figure 3c). Moreover regions encompassing residues 1–478 of p68 and 1–474 of p72, which contain the complete conserved core (Figure 3c), can repress transcription as well as the respective full-length proteins (Figure 3a,3b). In contrast, the C-terminal extension of both proteins acts as a transcriptional activator in this context (Figure 3a,5) consistent with earlier reports of these proteins acting as transcriptional co-activators [24, 25]. Thus, using this system, we have shown that there are separable transcriptional repression and activation domains within p68 and p72.
Since HDAC proteins have been extensively implicated in the repression of transcription, it was important to examine whether these proteins are likely to play a role in transcriptional repression by p68 and/or p72. Firstly, the ability of the HDAC inhibitor, TSA, to relieve repression was examined. No effect was observed on repression of TK-CAT (data not shown) implying the involvement of a HDAC-independent mechanism in repression of the TK promoter. In contrast, repression of MLP-CAT by p72 was relieved two-fold compared with the vector control (Figure 4a) suggesting the involvement of HDACs in this process. (Since p68 did not repress MLP-CAT transcription, the lack of effect by TSA is not surprising.) Supporting these data, p72 was found to co-immunoprecipitate a HDAC activity which was abolished by the addition of TSA, while p68 did not (Figure 4b). We chose to investigate whether p68 and/or p72 associate with HDAC1, since it is a well-studied example of Class I HDACs. Both p68 and p72 co-immunoprecipitate with HDAC1 (Figures 6 and 7); furthermore HDAC1, p68 and p72 co-elute in similar sized complexes by gel-filtration, which are of an appropriate size (Figure 5) supporting the idea of interactions between p68/p72 and HDAC1 in cells. Moreover, the finding that these proteins co-immunoprecipitate and co-elute from extracts which had been treated with DNase/RNase suggest that these represent protein-protein interactions rather than merely interactions via nucleic acid. While an interaction between HDAC1 and p68 is not supported by the results of the HDAC assay, or the TSA experiment, it is possible that, in some instances, p68 does recruit HDAC1 and that this mechanism is not being triggered in the MLP-CAT assay or HDAC assay. Alternatively, it is possible that the observed co-immunoprecipitation of p68 and HDAC1 is occurring through the interaction between p68 and p72  or that HDAC1 associated with p68 may have other, possibly non-transcriptional, roles [35, 36]. However, the data are entirely consistent with p72 recruiting HDAC1 to achieve active repression of transcription. Future investigations should also reveal whether the differential ability of p68 and p72 to recruit active HDAC proteins is responsible for the difference observed upon the MLP promoter.
Our attempts at correlating the different repressive functions of p68 and p72 to specific domains of the respective proteins, using deletion derivatives, were unsuccessful, as the equivalent regions of both either caused transcriptional repression or activation. While this might suggest that both helicases repress transcription in the same manner, it is more likely that the HDAC recruitment by p72 may be an additional mechanism of repression, used at specific promoters. We also cannot rule out the recruitment of other repression complexes at this stage. Our findings that p68 and p72 differ in their ability to repress the MLP promoter and to recruit HDAC activity suggest that, at least in some contexts, p68 and p72 repress transcription by different mechanisms.
In summary, it is clear that p68 and p72 act to repress transcription in a differential manner dependent upon promoter context. It will be important to determine which endogenous promoters are subject to repression by p68/p72 in a physiological context. However, until the signal transduction pathways, which target these proteins to the appropriate promoters, are elucidated it will be necessary to use a targeting system (such as GAL4) to undertake a molecular analysis of transcriptional repression by p68 and p72. Since it is now clear that p68/p72 can act both to activate and repress transcription future work will involve dissection of the transcriptional activation/repression complexes in which p68 and p72 are involved, as well as characterisation of the molecular 'switch' which determines whether these proteins will be part of transcriptional activation or repression complexes.