Genes transcribed by RNA polymerase II (Pol II) contain chromatin modifications that facilitate initiation and early elongation (Li et al., 2007
, Wang et al., 2009
, Wang et al., 2008
, Agalioti et al., 2002
, Li et al., 2008
). These modifications often bind to ATP-dependent remodeling proteins such as the bromodomain-containing SWI/SNF and the chromodomain-containing CHD1, which mobilize nucleosomes to allow binding of the Pol II machinery (Hargreaves et al., 2011
). Our groups are interested in how the assembly and function of the Pol II preinitiation complex (PIC) is coordinated with the chromatin modification and remodeling events at a promoter. To address this issue and to study its mechanism, we have recreated transcription on naked DNA and chromatin templates, and captured the resulting PICs using templates immobilized on magnetic beads (Lin et al., 2011
, Black et al., 2006
, Johnson et al., 2002
). This approach permits a detailed examination of PIC composition and function.
In our initial studies on immobilized naked DNA templates, we found that the activator GAL4-VP16 formed a complex with the TFIID and Mediator co-activators that was necessary for efficient recruitment of Pol II and the general transcription factors (GTFs) (Johnson et al., 2002
) in broad agreement with the view of these co-activators as bridging factors (D'alessio et al., 2009
, Naar et al., 2001
, Kornberg, 2005
). On chromatin, we found that the histone acetyltransferase p300 acts in concert with the Mediator co-activator very early in PIC assembly and prior to the recruitment of the GTFs (Black et al., 2006
, Black et al., 2008
). The p300-mediated acetylation of itself and chromatin led to p300 dissociation and allowed binding of the TFIID complex to Mediator, which facilitated assembly of the PIC.
Our early studies employed immunoblotting to identify factors thought to be involved in PIC assembly and function. To provide a more detailed understanding, we employed Multidimensional Protein Identification Technology (MuDPIT) to detect factors captured by the immobilized template on unmodified and H3K4-trimethylated (H3K4me3) chromatin templates (Lin et al., 2011
). Our analysis revealed that a wide range of protein complexes involved in chromatin modification and remodeling were recruited to the PIC along with Mediator, TFIID and Pol II. Importantly, SAGA, a well-studied H3 histone acetyltransferase in yeast and mammals, and a major co-activator in yeast (Baker et al., 2007
, Nagy et al., 2007
), was typically among the highest abundance factors. Moreover, numerous Pol II elongation complexes such as PAF and the CDK9-containing super-elongation complex (SEC) were detected (Smith et al., 2011
After initiation, Pol II pauses 30–50 bp downstream of the transcription startsite (TSS) through the action of DSIF and NELF. The SEC, which is recruited by Mediator (Takahashi et al., 2011
), plays a post-initiation role by phosphorylating DSIF and NELF, thereby releasing the paused Pol II. Since many genes in vivo contain paused Pol II (Nechaev et al., 2011
), our work suggests that in these cases, Mediator’s initial role may be in releasing the pause after which it establishes a PIC to allow multiple rounds of transcription.
The results of our initial proteomic study raised several important questions. First, are the chromatin modification/remodeling and Pol II elongation factors detected in our chromatin study components of PICs in vivo and in vitro? This bears on whether chromatin factors are general features of the PIC, whose recruitment is controlled by activator or a major co-activator. Second, are PICs formed in HeLa cells representative of PICs in other cell types like ES cells, which are emerging as an exciting area of biological interest. Finally, given the abundance of SAGA, what is its role in PIC assembly and function? In S. cerevisiae,
SAGA replaces TFIID at TATA box-containing promoters (Bhaumik et al., 2002
, Lee et al., 2000
, Huisinga et al., 2004
), while TFIID is employed at TATA-less promoters (Basehoar et al., 2004
). These findings were a surprise at the time because SAGA was thought to be simply a histone acetyltransferase and deubiquitinase.
To address these questions, we employed MuDPIT to determine the composition of GAL4-VP16-stimulated PICs in vitro on DNA templates using transcriptionally active extracts from HeLa and mouse ES cells. We then compared the DNA PICs with the composition of our chromatin PICs. We found them to be highly similar indicating that the chromatin modification and remodeling machinery is an inherent component of PICs. We then compared the in vitro PICs to Mediator-associated factors from HeLa and ES cell nuclei isolated at a low salt concentration, where PIC components remain associated with chromatin (Dignam et al., 1983
). The composition of the native PICs was remarkably similar to in vitro PICs. Finally, we delineated the roles of SAGA and Mediator. Our data suggest that the coordinated binding of most chromatin and Pol II elongation factors, which act near the startsite, is largely due to the Mediator co-activator. Importantly, we found that for the GAL4-VP16 activator, SAGA is not a co-activator in the traditional sense but functions independent of PIC assembly to allow transcription on chromatin templates.