Enhancer-binding activators such as MyoD and related basic helix-loop-helix (bHLH) transcription factors are well- established drivers of skeletal muscle differentiation (Tapscott, 2005
). Although binding of MyoD to its cognate recognition sites at muscle genes is a required step, occupancy at target promoters is apparently not sufficient to mediate transcriptional activation. Instead, this key regulator of myogenesis along with some of its associated proteins are subject to post-translational modifications that can lead to either activation (promote differentiation) or repression (block differentiation) of MyoD dependent promoters (Sartorelli and Caretti, 2005
; Tapscott, 2005
). However, dramatic change in the composition of the core transcription apparatus was not a mechanism anticipated for directing muscle differentiation. The recent in vivo
finding that holo-TFIID is functionally replaced by a TAF3/TRF3 complex during myoblast to myotube differentiation highlights an unexpected and possibly integral regulatory function of the PIC during cell lineage specific transcription (Deato and Tjian, 2007
). Here we report direct biochemical evidence for the differential utilization of diversified core promoter recognition complexes to accommodate tissue-specific and gene-selective regulation during metazoan development.
We have used in vitro reconstituted assays to determine the biochemical properties of the TAF3/TRF3 complex that direct muscle-specific promoter recognition and coactivator selectivity. We establish that TAF3/TRF3 is a bona fide core promoter recognition complex capable of initiating both basal and activator-dependent transcription at the Myogenin promoter. In stark contrast, the canonical TFIID can mediate basal levels of transcription but failed to support MyoD dependent transcription. These in vitro studies have also allowed us to manipulate and dissect the cofactor requirements needed to mediate activator-specific transcription initiation at the Myogenin promoter. Importantly, these in vitro studies provide direct evidence for TAF3 as a “muscle-gene” coactivator directly targeted by the myogenic activator, MyoD. Thus, TAF3/TRF3 can serve as a bona fide alternative core promoter recognition complex that operates both in vitro and in vivo to drive cell-type selective gene regulation.
Our studies indicate that like the prototypic TAF subunits of TFIID, TAF3 mediates its coactivator functions, at least in part, by interacting directly with a muscle specific activator (MyoD). Intriguingly, although a sub-stoichiometric amount of TAF3 can at times be found associated with purified TFIID, only TAF3/TRF3 was able to support MyoD-dependent activation of Myogenin in our in vitro reconstituted reactions. This inability of TAF3-containing TFIID to potentiate activator–dependent transcription from the Myogenin promoter suggests that the larger TFIID complex may arrange the target protein-protein interface in a manner that is incompatible for productive transactions with MyoD. By contrast, MyoD-TAF3 interactions appear well suited to direct specific recruitment of the TAF3/TRF3 complex at the Myogenin promoter. Thus, the TAF3/TRF3 complex behaves as a bona fide coactivator wherein an activator signal is transmitted by direct interaction with components of the promoter recognition complex, underscoring the role of TAFs as molecular adaptors that connect activators to the core transcription machinery.
The coactivator domain of TAF3 that interacts with MyoD maps to a N-terminal region that also contains the histone fold domain implicated in various TAF-TAF and TAF-partner interactions (Gangloff et al., 2001
). In myotubes and in vitro
, this TAF3 domain is required to mediate MyoD-dependent transcriptional activation of Myogenin. In contrast, the C-terminal region of TAF3 interacts with TRF3 and does not appear to mediate interactions with MyoD (data not shown). Interestingly, this C-terminal region contains a Plant Homeobox Domain (PHD) commonly found in chromatin modifying cofactors that was recently shown to bind trimethylated lysine 4 on histone H3, a modification that marks active promoters in human cells (Bienz, 2006
; Vermeulen et al., 2007
). One study also suggested that when TAF3 is a component of TFIID it mediates its coactivation function through the PHD domain (Vermeulen et al., 2007
). Here we find that in the context of myogenesis the coactivation domain of TAF3 targeted by MyoD is independent of the PHD domain. However, it is possible, even likely, that TAF3 contains multiple coactivator domains that can operate in different contexts depending on the composition of its other partner subunits (i.e. TAF3/TRF3 versus TFIID) in the complex. Thus, the N-terminal coactivator domain of TAF3 might direct interactions with MyoD while the PHD domain could serve to read nucleosome modifications on target gene promoters. Since our in vitro
reconstituted reactions were reformed with naked DNA template, this latter function of TAF3 would not be manifested in our system but may be important for transcription from chromatin templates. Indeed, both coactivation functions may be important to distinguish transcriptional programs in differentiated cells.
Like TFIID, the CRSP/MED coactivator complex is another key component of the core transcription machinery that can be targeted by activators to form the pre-initiation complex (PIC). A recent in vivo
study of Drosophila transcription initiation uncovered an unexpected functional crosstalk between TFIID and CRSP/MED in regulating the transcriptional output of inducible genes (Marr et al., 2006
). In addition, genetic loss of function analyses have suggested the differential requirements for CRSP/MED subunits during cellular differentiation (Ge et al., 2002
; Urahama et al., 2005
). In conventional in vitro
transcription reactions, TFIID and CRSP/MED are thought to perform non-overlapping functions in order to potentiate activator-dependent transcription (Taatjes et al., 2004
). We were therefore curious to determine the functional requirements for CRSP/MED in myoblast to myotube differentiation wherein TFIID has been replaced by TAF3/TRF3. Remarkably, the absence of CRSP/MED had no measurable effect in our TAF3/TRF3 reconstituted system that supports MyoD-dependent activation of Myogenin template. Although, we cannot rule out the possibility that other muscle specific promoters or chromatinized Myogenin template may require a Mediator-like activity, it appears that at least for the non-chromatinized Myogenin promoter, CRSP/MED appears largely dispensable for MyoD-dependent transcription activation in vitro
. Consistent with this in vitro
result, we found that many of the CRSP/MED subunits are indeed severely reduced or absent from myotubes while most subunits were abundantly present in myoblasts with the exception of MED 1. This intriguing finding raises the possibility that the canonical CRSP/MED complex may also be dramatically altered and possibly disposable during terminal differentiation. It remains possible that other Mediator-like complexes or sub-complexes may substitute for the canonical complex in differentiated skeletal muscle. Paradoxically, myoblast to myotube differentiation involves the activation of genes by nuclear receptors (VDR, RAR and TR), a class of activators thought to target CRSP/MED (Halevy and Lerman, 1993
). It will be of interest for future studies to further explore the existence and function of CRSP/MED or other muscle-specific Mediator-like complexes in myotubes.
The replacement of TFIID with a TAF3/TRF3 complex in differentiated skeletal muscles has revealed a potentially new transcriptional mechanism that involves diversified core promoter recognition complexes interacting with muscle-specific activators to orchestrate spatial and temporal patterns of gene expression. Such a more equitable parsing of duties between muscle specific activators working in concert with cell selective coactivators may represent an important mechanism evolved to regulate metazoan gene expression; one that allows for a simple yet efficient way to permanently turn off a majority of genes while selectively turning on transcription patterns in differentiated cells.