In this study, we have uncovered a novel function of the human STAGA coactivator complex in physical recruitment of Mediator and stimulation of MYC-dependent transcription and cell proliferation, which requires the integrity of the complex or minimally its STAF65γ-dependent SPT-TAF module (which includes SPT3, TAF9, TAF10, TAF12, and associated histone fold partners; Fig. ). We have shown that STAF65γ is required for activation of TERT transcription by MYC and for the association of both SPT3 (STAGA) and MYC with Mediator in human cells. Consistent with this, we found that STAGA and Mediator complexes directly interact and bind in a partially interdependent manner to the TAD of MYC in vitro. Collectively, our results suggest a model in which MYC interacts with the STAGA complex via its TRRAP and/or GCN5 subunits, while the STAF65γ-dependent SPT-TAF components physically recruit a core Mediator complex (i.e., devoid of MED12 and MED13) to the TERT promoter and direct transcription activation at a step(s) mostly distinct from, and likely subsequent to, the loading of TFIID and Pol II (Fig. ).
FIG. 7. Model for STAGA-dependent transcription activation of TERT by MYC involving recruitment and/or stabilization of a core Mediator complex at the promoter. Double arrows indicate direct interactions between MYC, STAGA, and Mediator described here and previously (more ...)
Similar to the nonessential gene-specific function of Spt7 (SAGA) in yeast, we have shown that STAF65γ plays a structural role within the human STAGA complex and is not essential for viability of U2OS and HeLa cells or for normal growth of U2OS cells. However, STAF65γ is important for proliferation of HeLa cells, which depend on MYC for growth, suggesting the intriguing possibility that STAF65γ (or an associated SPT-TAF component) may have MYC- and/or cell type-specific functions. The exact nature of this cell-type specificity, however, remains to be elucidated, and it will be important to determine whether STAF65γ is required specifically for MYC-induced cell proliferation and transformation. Our results demonstrate that STAF65γ is required for MYC-dependent activation of TERT
transcription (as well as for transcription of several other MYC target genes) and for the association of SPT3, TAF9, and probably other components of the SPT-TAF group with the TRRAP and GCN5 subunits. Thus, TRRAP and GCN5 may belong to a separate module(s) in the complex (e.g., a “TRRAP/domain I” module and a “GCN5/ADA2/ADA3” module) (49
). Consistent with this, MYC directly interacts with both TRRAP (60
) and GCN5 (N. Zhang, S. Lo and E. Martinez, unpublished results) and recruits TRRAP to various promoters (23
) and GCN5 (as shown here) to the TERT
promoter independently of STAF65γ. It is possible that, in addition to MYC, other promoter-associated factors, perhaps including acetylated histones, might contribute to the stable binding of TRRAP and/or GCN5 to the TERT
promoter. However, MYC is essential for the stable recruitment of all STAGA components analyzed and interacts directly with a purified STAGA complex in vitro (44
). Our results do not exclude the possibility that the STAF65γ-independent binding of TRRAP and GCN5 to the TERT
promoter might instead (or in addition) reflect the recruitment by MYC of other complexes, i.e., distinct from STAGA-type complexes.
In contrast to the STAF65γ-independent recruitment of TRRAP and GCN5, the MYC-directed recruitment of SPT3 and TAF9 to the TERT
promoter is strictly dependent on STAF65γ. While STAF65γ and SPT3 are specific to STAGA-type complexes, TAF9 is shared by STAGA-type and TFIID complexes: note that the polyclonal TAF9 antibodies used also recognize the highly similar TAF9b (formerly TAF9L) paralog that coexists with TAF9 in both TFIID and STAGA/TFTC (25
). Since we have shown that STAF65γ depletion selectively affects the association of TAF9 with STAGA but not with TFIID (Fig. ), these results suggest the intriguing possibility that TAF9 is recruited to the TERT
promoter as part of the SPT-TAF module of STAGA. This would be consistent with increasing evidence indicating the existence of structurally and functionally distinct TFIID complexes, including TFIID complexes that lack TAF9 (reviewed in reference 55
). Note, however, that we cannot formally exclude the possibility that TAF9 is associated with the TERT
promoter (as part of TFIID) in STAF65γ-knockdown cells but cannot be detected by ChIP.
In addition to SPT3 and TAF9 components of STAGA, STAF65γ is also strictly required for MYC-dependent recruitment of core Mediator subunits (MED1 and MED17) to the TERT
promoter. This supports observations in yeast indicating at least a partial dependency on SAGA for Mediator recruitment to certain yeast promoters (6
). However, neither MYC nor STAF65γ is essential for TFIID or Pol II binding to the human TERT
promoter in HeLa cells (although we note a moderate decrease in the association of TBP and Pol II). Moreover, STAF65γ is also dispensable for histone H3 and H4 acetylation. Altogether, these results suggest a role of the STAF65γ-dependent SPT-TAF components of STAGA in MYC-dependent TERT
transcription by (at least in part) enhancing core Mediator association with the promoter and facilitating a transcription step(s) postrecruitment of TFIID and Pol II. This model is in accord with previous reports indicating that MYC activates transcription of TERT
and other target genes after Pol II engagement, possibly by stimulating Pol II C-terminal domain phosphorylation and/or promoter clearance or elongation (8
) and with recent studies indicating a role of yeast SAGA in transcription elongation (12
). Similarly, Mediator has also been shown to play a role postrecruitment of Pol II during the initiation phase of transcription in mouse cells (74
), stimulates TFIIB recruitment and initiation/reinitiation in vitro (2
), and physically and/or functionally associates with various elongation factors, including TFIIS (76
), Brd4/P-TEFb (84
), and DSIF (46
), and with coding regions of yeast genes (1
). Clearly, it will be important to identify the steps downstream of TFIID/Pol II loading that are controlled by STAGA and core Mediator on the TERT
promoter and the detailed activation mechanisms involved. The results presented here nonetheless provide the first evidence for a concerted function of STAGA and core Mediator in MYC-dependent transcription activation in human cells at a step mostly distinct from histone acetylation and binding of TFIID and Pol II to the promoter. Importantly, our results also indicate a partial dependency on MYC (but not STAF65γ) for histone H3 acetylation on the endogenous TERT
promoter in HeLa cells, which is consistent with previous findings in different cells/systems (8
). These results suggest that MYC-dependent histone H3 acetylation does not require the STAF65γ-dependent SPT-TAF components of STAGA (or Mediator) but most likely depends on GCN5, p300/CBP, and/or other recruited HATs (8
). Thus, we propose that the STAGA complex may contribute, perhaps in a cell context-dependent manner, to the two distinct transactivating functions of MYC (i) at the level of chromatin acetylation and (ii) at subsequent steps after Pol II engagement, via separate TRRAP-GCN5 and SPT-TAF components, respectively.
We have demonstrated that MYC and STAF65γ (STAGA) are required for the recruitment of core Mediator components (i.e., MED1, MED16, and MED17) but not MED12 or MED13 to the endogenous TERT
promoter in HeLa cells. This is consistent with our in vitro interaction assays indicating that, in contrast to core MED subunits, MED12 and MED13 in nuclear extracts and CDK8 in our affinity-purified Flag-Nut2 Mediator complexes interact poorly with MYC TAD. In addition, the interaction in vitro of MYC TAD with MED1 but not MED16, in purified Flag-Nut2/Mediator complexes, is enhanced by STAGA. This supports the reported heterogeneity of purified Flag-Nut2 MED10-containing Mediator complexes (86
) and suggests that STAGA may facilitate the interaction of MYC TAD with selectively MED1-containing core complexes—e.g., those lacking the CDK8 module (which comprises MED12, MED13, cyclin C, and CDK8). Consistent with this, the preferential interaction of MYC TAD with core MED subunits in nuclear extracts was both activation domain specific (i.e., not observed with GST-VP16) and at least partially STAF65γ dependent. Note that, in contrast to MED12 and MED13, CDK8 in nuclear extracts interacts efficiently with MYC TAD, but this association is independent of STAF65γ (our data not shown) and of cyclin C (20
) and thus might not reflect an interaction with a bona fide Mediator complex.
Our ChIP analyses have further uncovered a differential association of MED12 and MED13 (relative to core MED components) on the TERT
promoters. Note that this differential association does not indicate the levels of the different MED subunits on either promoter. This contrasts with the reported uniform binding of Mediator components including subunits of the CDK8 module in genomewide location analyses in yeast (1
). However, this differential interaction of MED subunits is in agreement with two previous reports describing the recruitment to native human promoters of CDK8-containing and CDK8-lacking Mediator complexes by, respectively, repressive and active forms of C/EBPβ (54
) and MED1-containing and MED1-deficient Mediator complexes by the estrogen receptor and p53, respectively (86
). Thus, at least in human cells, the association of Mediator components with promoters appears to vary in a promoter-specific and regulated manner, consistent with the original biochemical characterization of different forms of human Mediator (see references 15
, and 71
and references therein).
We have shown that purified STAGA and Mediator complexes directly interact and bind to MYC TAD both independently and, at least for specific components (i.e., MED1, GCN5, and SPT3), also in an interdependent manner. Thus, it is possible that MYC might also recruit specific Mediator components to promoters in vivo independently of STAGA and Mediator might help STAGA recruitment to certain human promoters. The latter possibility would be consistent with the observed dependency on Mediator for SAGA recruitment to certain promoters in yeast (63
). Moreover, the interdependent recruitment of SAGA and Mediator to specific yeast promoters (6
) strongly suggests analogous physical interactions of SAGA and Mediator complexes in yeast and highly conserved mechanisms for SAGA/STAGA-dependent transcription activation from yeasts to humans.
In addition to STAGA and core Mediator, MYC also recruits p300 to the TERT
) and this is independent of STAF65γ/STAGA and core Mediator (as shown here). How MYC coordinates the recruitment of p300, STAGA, and core Mediator remains to be determined. Beyond cooperative interactions, coactivator exchange mechanisms may be required as proposed for the concerted interactions of p300 and Mediator with PGC1-α and Gal4-VP16 (7
). In summary, our results support and extend the notion that at least certain HATs such as p300 (7
) and HAT complexes such as STAGA (this report) can directly communicate with Mediator via physical interactions and suggest that eukaryotic SAGA/STAGA-type complexes may coordinate different steps in transcription activation: from chromatin modification and facilitated recruitment of TBP/TFIID and Pol II to subsequent steps in transcription that require components of the SAGA/STAGA-specific SPT-TAF module and interactions with a specific core Mediator complex.