The work presented here provides mechanistic insights into the coordinated transcriptional activation of multiple histone subtypes by NPAT at the G1
/S-phase transition. Our data indicate that the TRRAP-Tip60 complex interacts with NPAT and becomes associated with histone promoters at the G1
/S-phase boundary in an NPAT-dependent manner. Moreover, consistent with the presence of the TRRAP-Tip60 HAT complex at histone promoters, histone H4 acetylation, which is associated with transcriptional activation (25
), increases at histone promoters during the G1
/S-phase transition. This increase in histone H4 acetylation also depends on NPAT. Suppression of TRRAP or Tip60 expression through RNA interference leads to the inhibition of histone gene transcriptional activation. These results, together with the previous observations that the association of NPAT with histone promoters as well as NPAT-mediated histone promoter activation is regulated by cyclin E-Cdk2 phosphorylation (34
), suggest there is a mechanism underlying the coordinated transcription of histone subtypes during the G1
/S-phase transition (Fig. ): NPAT becomes phosphorylated by cyclin E-Cdk2 at the G1
/S-phase boundary, and this phosphorylation promotes the association of NPAT with histone promoters, likely through its interactions with the subtype-specific factors, such as Oct1/OCA-S and HiNF-P, which bind to specific DNA sequences (e.g., SSREs) in the promoters of the different histone gene subtypes. As a result, a TRRAP-containing HAT complex(s), for example, the Tip60 HAT complex, is recruited to the promoters of multiple histone genes. This HAT complex recruitment in turn leads to histone acetylation and, subsequently, transcriptional activation of multiple histone promoters.
FIG. 8. A model for coordinated transcriptional activation of histone subtypes by cyclin E-Cdk2 substrate NPAT. SSRE, subtype-specific regulator element; SSBP, proteins, such as Oct-1 and HiNF-P that directly bind SSRE elements within the promoters of a histone (more ...)
It was previously shown that the phosphorylation of NPAT by cyclin E-Cdk2 regulates its activity in histone gene activation (34
). It appears that cyclin E-Cdk2 modulates NPAT function by regulating the localization of NPAT at histone gene clusters (53
). It is not clear whether cyclin E-Cdk2 regulates NPAT through additional mechanisms. Our preliminary results indicate that NPAT interacts with TRRAP throughout the cell cycle, suggesting that the interaction of NPAT with TRRAP may be independent of cyclin E-Cdk2 activity.
The results shown in Fig. and suggest that the majority of TRRAP/Tip60 recruitment to histone gene promoters occurs immediately prior to S-phase entry. It is possible that TRRAP/Tip60 recruitment may involve a two-step mechanism in which additional recruitment of these factors takes place in S phase, as intermediate levels of association of NPAT and TRRAP with histone gene promoters were observed in the presence of aphidicolin. In addition to NPAT, the recruitment of the TRRAP/Tip60 complex to histone gene promoters may also involve other histone gene transcription factors.
According to the model proposed in Fig. , one might expect the NPAT (the LFD-to-AAA) mutant to be dominant negative. Our results, however, indicate that this mutant apparently has no inhibitory activity on histone promoter activation (Fig. ). The exact reason why the NPAT (AAA) mutant protein fails to function as a dominant-negative mutant is not clear. One possible explanation is that one or more of the proteins that interact with the NPAT transactivation domain may be involved in stabilizing the interaction of NPAT with histone promoters. Without this stabilizing interaction, the presence of the mutant at the promoter may be merely transient, thus resulting in a failure of the NPAT (AAA) mutant to be dominant negative.
In this study, we have identified a domain in NPAT that possesses intrinsic transactivation potential. Interestingly, this domain, referred to as the transactivation domain of NPAT, contains a DLFD motif that is required for NPAT-mediated transcriptional activation and is functionally conserved in E2F and adenovirus E1A proteins. Our results clearly demonstrate that the DLFD motif is crucial for the interaction of NPAT with TRRAP, as well as for NPAT-mediated transcriptional activation (Fig. and ). The DLFD motif also appears to be crucial for the interaction of several E2F proteins with TRRAP and for their transcriptional activation function. It was previously observed that deletion of the DLFD sequence in a transactivation domain of E2F1 (residues 389 to 422) fused to the GAL4 DNA-binding domain resulted in an almost complete loss of its transcriptional activation capability (13
). Consistent with this observation, the LFD-to-AAA mutation in the transcriptional activation domain of E2F3 (residues 391 to 465) results in the loss of transactivation when the mutant domain is fused to the GAL4 DBD (our unpublished observation). Moreover, the replacement of the LFD sequence with AAA in the transactivation domain abolishes the interaction of E2F3 with TRRAP (our unpublished observation). It was reported that the last seven amino acids of E2F4, which include the second aspartic acid residue in the DLFD motif, are critical for its interaction with TRRAP and E2F4-mediated reporter activation (30
). E1A may also utilize the DLFD motif to interact with TRRAP. It was shown that the deletion of E1A from the CR1 region, which includes the DLFD motif, abolishes both TRRAP binding and transformation (7
). Thus, the DLFD motif functions as a TRRAP-interacting module that is conserved in NPAT, E2F, and E1A proteins. Several other TRRAP-interacting proteins, such as c-Myc, p53, and BRCA1 (2
), apparently lack the DLFD motif and therefore likely interact with TRRAP through a different sequence motif(s). The existence of multiple TRRAP-interacting motifs may allow the recruitment of TRRAP-containing complexes by distinct factors to be differentially regulated. It is interesting to note that the DLFD motif is also part of the sequences in E2F proteins shown to interact with the retinoblastoma protein pRB (13
). Hence, pRB may inhibit E2F function by preventing the association of E2F with TRRAP-containing HAT complexes.
TRRAP has been shown to be a component of a number of HAT complexes, including the GCN5/PCAF and Tip60 complexes (6
). We focused on the TRRAP-Tip60 HAT complex in this study because we observed an interaction between the NPAT transactivation domain and two other components of the Tip60 HAT complex, Tip48 and Tip49, in our initial mass spectrometric analysis (Fig. ). It is possible that, similar to E2F and c-Myc, which recruit Tip60 as well as GCN5 complexes to their target promoters, NPAT may interact with and recruit additional TRRAP-containing HAT complexes to histone promoters in vivo. Compared with the NPATflox/−
cells infected with Ad-LacZ, the Ad-Cre-infected NPATflox/−
cells showed only a moderate (30 to 55%) reduction in histone H4 acetylation at histone promoters at the G1
/S-phase boundary. This might be due to the fact that some residual NPAT protein remains in these cells and can still recruit the TRRAP-Tip60 complex to the histone promoters (Fig. , , and ). Alternatively, other protein factors might also recruit a HAT complex (or complexes) to the histone gene promoters to induce histone acetylation in concert with, but independent of, NPAT. Since proteins other than histones can also be the substrates of HATs (17
), the NPAT-recruited HAT(s) may also play a role in histone gene transcription by acetylating nonhistone proteins at histone promoters.
In addition to components of the Tip60 complex (Fig. ), NPAT appears to interact with YY1, BZAP45, and Hsp70, which have been implicated in histone gene transcription (12
). Although an in vivo interaction of NPAT with these proteins remains to be determined, the observation raises the possibility that these proteins may participate in regulation of histone gene transcription through their cooperation with NPAT. The transactivation domain of NPAT apparently interacts with a number of additional proteins (Fig. ), which have not been shown to be involved in histone gene transcription. Further studies are needed to determine their interactions with NPAT in vivo, as well as their roles in transcriptional activation of histone genes. Such studies may shed new light on the coordinated regulation of histone gene transcription.