The DEAD/H box family of RNA helicases has been demonstrated to be involved in virtually all processes that require manipulation of RNA including transcription, pre-mRNA and pre-rRNA processing, RNA export, ribosome assembly and translation [1
]. Although, in vitro
, several members of this family have been shown to unwind RNA duplexes, relatively few appear to be true processive helicases and it is clear that, in the cell, many are likely to be involved in unwinding of short base paired regions of RNA or in the modulation of RNA-protein interactions.
DNA helicases belong to a superfamily of proteins that is distantly related to DEAD/H box RNA helicases and includes the Werner syndrome protein (WRN) [2
] and the Xeroderma pigmentosum XPB and XPD proteins [3
], which have well established roles in transcription. Although the functions of DEAD/H box RNA helicases in other cellular processes, such as pre-mRNA processing and translation have been well studied, their role in transcriptional regulation is only now emerging. Examples of DEAD/H box RNA helicases involved in transcription include RNA helicase II (RHII/Gu) and RNA helicase A (RHA/NDHII). RHII/Gu was demonstrated to be a cofactor for c-Jun-activated transcription [4
] and was shown to translocate from the nucleolus to the nucleoplasm after UV or anisomycin treatment (which activates JNK signalling). Although RHII/Gu was found to associate with phosphorylated c-Jun in a non-phosphorylated state, this association was observed to increase after anisomycin treatment, implying a stronger interaction when c-Jun is phosphorylated [4
]. RHA is a homologue of the Drosophila
maleless (MLE) gene product [5
] and is thought to be important for gene dosage compensation on the X-chromosome [6
]. RHA has been shown to be required for complex formation between the transcriptional co-activator, CREB binding protein (CBP), and RNA polymerase II [7
]. Furthermore, different regions of the RNA helicase protein were found to interact with both CBP and RNA polymerase II. The association of RHA with RNA polymerase II was further investigated, and narrowed down to a 50 amino acid stretch, outwith the conserved helicase motifs [7
]; this study also showed that RHA could regulate CREB-dependent transcription either through recruitment of Pol II or by ATP-dependent mechanisms. A later study reported that RHA acts as a bridging molecule between the breast tumour specific transcriptional activator, BRCA1 and the RNA polymerase II holoenzyme complex [8
]. These reports thus provide clear evidence of a role for RNA helicases as transcription factors.
p68 is a prototypic member of the DEAD box family of proteins [9
] and an established RNA helicase [10
]. The subsequent discovery of p72 [11
] and the finding that p68 and p72 share remarkable homology (90% over the central conserved core and 60% and 30% at the N- and C-terminal extensions respectively) suggests that these proteins may form a specific sub-group of DEAD box proteins and may have similar, but perhaps subtly different, functions in the cell, perhaps through interaction with different RNA substrates or proteins. In vitro
, both proteins exhibit the RNA-dependent ATPase and RNA helicase activities characteristic of members of the DEAD box family [10
] and have also been reported to catalyse rearrangement of RNA structure via branch migration [13
]. Moreover p68 and p72 can interact with each other, as well as self-associate, and appear to preferentially form heterodimers in cells [15
]. This provides the potential for a wide range of functions for p68 and p72 with the possibility of their co-operation in some contexts.
More recently p68 and p72 have been shown to be involved in a range of processes in the cell, including pre-mRNA and pre-rRNA processing and alternative splicing [16
]. p68 and p72 have also been shown to be growth- and developmentally-regulated [19
] and, furthermore, p68 appears to be over-expressed and poly-ubiquitylated in colorectal tumours [23
]. Interestingly p68 has been shown to act as a transcriptional co-activator, specific for the activation function 1 (AF-1) domain of estrogen receptor alpha (ERα) [24
]. This interaction was dependent upon phosphorylation of AF-1 at serine118
, a residue phosphorylated by mitogen-activated protein kinase (MAPK). Interestingly the RNA helicase function of p68 appeared to be dispensable for this activity as a mutant p68 (Lys144
to Arg) in the ATP binding site (conserved motif I) retained the ability to co-activate ERα, although in a later study RNA binding appeared to be required [25
]. p72 was subsequently shown to share this property and both proteins were shown to interact with the activation domain 2 (AD2) of p160 co-activators [25
]. Furthermore p68 was also found to interact with the CBP co-activator and RNA polymerase II [26
]. More recently p68 has been shown to be recruited to the promoter of the ERα target gene pS2 [27
], suggesting a direct involvement in transcriptional regulation.
In this report we explore further potential mechanisms through which p68 and p72 may contribute to transcriptional regulation. We find that, in some contexts, p68 and p72 can also act as transcriptional repressors and that these proteins exhibit clear promoter specificity in this function. By directing GAL4-tagged p68/p72 (GAL4 DNA binding domain aa1-147) to promoters containing GAL4 binding sites, we show that both p68 and p72 can repress transcription from the herpes virus thymidine kinase (TK) promoter but not the simian virus 40 promoter/enhancer. Moreover, while p72 can repress transcription from the Adenovirus major late promoter, p68 appears to have no effect, suggesting that these proteins do not behave in an identical way in all contexts. Furthermore we show, by co-immunoprecipitation, that both p68 and p72 interact with histone deacetylase 1 (HDAC1) and that HDAC1/p68/p72 co-elute by gel filtration, indicating that they exist in the same complex in the cell and suggesting a possible mechanism by which these proteins may exert their repressive effect.