TIP60's role as a co-activator of gene expression is well documented and this activity is primarily through its recruitment to the promoter and acetylation of histones. TIP60 is also a haplo-insufficient tumor suppressor (Gorrini et al., 2007
). Here we have identified (i) that the TIP60 tumor suppressor is destabilized by E6, a viral oncogene, (ii) TIP60 destabilization is targeted by E6 of both high- and low-risk HPV, (iii) YY1 facilitates binding of TIP60 to the promoter, (iv) TIP60 acts as a repressor of the major viral promoter, (v) this repression is through TIP60's ability to regulate acetylation of histone H4 to facilitate Brd4 recruitment to the HPV promoter, (vi) TIP60 represses cellular genes and, (vii) even low-risk HPV E6 attenuates p53-dependent activation of apoptotic pathways by destabilizing TIP60. Collectively, these findings provide new insight into the roles of the E6 proteins during the viral life cycle common to both high- and low-risk HPV types.
The mechanism of HPV E6-mediated destabilization of TIP60 is independent of E6AP, a cellular ubiquitin ligase known to be involved in E6-mediated degradation of the tumor suppressor p53. Mdm2 and Cul3 ubiquitin ligases are known to be involved in degradation of TIP60 (Bhoumik et al., 2008
; Legube et al., 2002
). However, depletion of E6AP, Mdm2 and Cul3 did not stabilize TIP60 in HeLa cells suggesting that these ligases do not co-operate with HPV E6 for destabilizing TIP60 (data not shown). HPV E6 destabilizes its targets by acting as an adaptor and facilitates interaction of the target protein with cellular ubiquitin ligases. Previous studies suggest that HIV-tat could act as an adaptor protein and facilitate destabilization of TIP60 through p300 and Mdm2 (Col et al., 2005
). However, we did not see any stabilization of TIP60 in HPV positive cells after depleting Mdm2 or p300 (data not shown). This could be because we failed to deplete these proteins sufficiently, or because a novel E3 ligase is utilized by E6 to target TIP60 to proteasomes. Another possibility is that TIP60 is destabilized by proteasomes in an ubiquitin independent pathway as reported for several other proteins (Mao et al., 2008
The inability of p53 to induce expression of PUMA
in cells expressing the low-risk HPV E6, or N-terminal (1-43) region of E6 highlights the significance of TIP60 in the activation of apoptotic pathway (). TIP60 acetylates p53 on lysine 120 and targets p53 to promoters of pro-apoptotic genes (Sykes et al., 2006
; Tang et al., 2006
). An alternate hypothesis suggests that binding of TIP60 to p53 is sufficient to recruit HIPK2, a homeodomain-interacting protein kinase-2 which targets p53 to pro-apoptotic genes by phosphorylating Ser46 (Li et al., 2009
). We expect both these pathways to be abrogated by TIP60 degradation. In addition, TIP60 is also involved in activation of the checkpoint pathway through acetylation of ATM (Sun et al., 2005
), so that destabilizing TIP60 would also attenuate the checkpoint pathway response. Our results suggest that simply stabilizing p53 in HPV-infected cancers by inhibiting E6AP may not be sufficient to cure cancers without concordantly restoring TIP60.
Although TIP60 and hisone H4 acetylation is normally thought of as an activator of gene expression, we provide evidence that TIP60 can also repress viral and cellular promoters. For the viral promoter, this represson is a direct effect of TIP60 binding and activity with the acetylated H4 recruiting a bromodomain containing cellular repressor, Brd4. While this paper was under review, a genome wide siRNA screen reported that four subunits of the TIP60\NuA4 complex (EP400, EPC1, Brd8 and YEATS\GAS41), along with Brd4, are required for repressing the HPV promoter by HPV E2 (Smith et al.
). Thus mechanisms similar to that shown here for the E2-independent repression of the HPV promoter, may also be at play in the E2-dependent repression pathways.
The mechanisms underlying the few occasions when TIP60 represses a cellular promoter have been studied (reviewed in (Sapountzi et al., 2006
)). We speculate that the Bromodomain-dependent mechanism reported here may also explain the repression of some cellular promoters by TIP60 and the de-repression by E6. Involvement of TIP60 in regulating transcription of cellular genes suggests that by degrading TIP60, E6 could alter the differentiation program of keratinocytes. Inactivation of the p130, a pRB-related pocket protein, by the high- and low-risk HPV E7 proteins promote S phase re-entry by the differentiated keratinocytes to facilitate viral DNA amplification (Genovese et al., 2008
; Zhang et al., 2006
) while the viral E6 proteins of high- and low-risk HPV inactivate the transcriptional activity of the major tumor suppressor p53 by interfering with its acetylation by p300 (Thomas and Chiang, 2005
). This is the first instance of a cellular protein being targeted for degradation by both low- and high-risk HPV E6, raising the possibility that degradation of the tumor suppressor TIP60 is equally important for the life cycle and pathogenesis of the virus and in the cellular deregulation seen in HPV-associated cancers and benign papillomas.