Article

• The EMBO Journal (2008) 27, 1447 - 1457
• doi:10.1038/emboj.2008.78

Published online: 17 April 2008

• Subject Category:

  • Chromatin and Transcription

Cooperative activity of cdk8 and GCN5L within Mediator directs tandem phosphoacetylation of histone H3

Krista D Meyer¹, Aaron J Donner², Matthew T Knuesel¹, Autumn G York¹, Joaquín M Espinosa² and Dylan J Taatjes¹

1. Department of Chemistry and Biochemistry, Cellular and Developmental Biology, University of Colorado, Boulder, CO, USA
2. Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, CO, USA

Correspondence to:

Dylan J Taatjes, Department of Chemistry and Biochemistry, University of Colorado, Campus Box 215, Boulder, CO 80309, USA. Tel.: +1 303 492 6929; Fax: +1 303 492 5894; E-mail: taatjes@colorado.edu

Received 27 October 2007; Accepted 27 March 2008

• Keywords:

  • chromatin,
  • histone H3,
  • Mediator,
  • phosphoacetylation

Introduction

The human Mediator complex is a multisubunit cofactor that regulates RNA polymerase II-dependent transcription (Conaway et al, 2005). Although its precise biochemical activity is poorly understood, it is clear that Mediator plays multiple roles in regulating the pre-initiation complex (PIC), which minimally consists of TFIIA, IIB, IID, IIE, IIF, IIH, and RNA polymerase II (pol II) (Hahn, 2004). For example, Mediator interacts directly with pol II and helps recruit this complex to gene promoters (Davis et al, 2002; Näär et al, 2002); Mediator also stimulates the TFIIH-dependent phosphorylation of the pol II CTD, which is critical for transcription initiation (Kim et al, 1994; Akoulitchev et al, 2000). Furthermore, Mediator is a general target of DNA-binding transcription factors. Thus, Mediator is believed to function in part by communicating regulatory signals from promoter- and enhancer-bound transcription factors directly to the PIC.

One simple way in which Mediator activity is controlled is through subunit exchange: addition or subtraction of subunits to alter its biochemical function (Taatjes et al, 2004; Malik and Roeder, 2005). For instance, a cdk8 subcomplex (consisting of cdk8, cyclin C, Med12 and Med13) can reversibly associate with the 'core' Mediator to inhibit its co-activator function (Taatjes et al, 2002; Pavri et al, 2005). Notably, core Mediator (25 subunits and 1.2 MDa in size) has no known enzymatic functions; however, association with the cdk8 subcomplex bestows kinase activity (for example, cdk8) to the complex. Whereas the core Mediator complex strongly interacts with pol II and potentiates activated transcription, the cdk8-Mediator complex does not interact with pol II and is transcriptionally inert (Davis et al, 2002; Näär et al, 2002; Taatjes et al, 2002). Although the mechanism by which cdk8-Mediator inhibits activated transcription is not completely defined, its kinase activity appears to play an important role. In fact, cdk8-dependent phosphorylation of cyclin H (a subunit of TFIIH) can inhibit TFIIH-dependent phosphorylation of the pol II CTD, which facilitates initiation of transcription (Akoulitchev et al, 2000). Interestingly, this cdk8-dependent modification does not occur in yeast; rather, an alternate inhibitory mechanism appears to function in yeast whereby cdk8 itself phosphorylates the pol II CTD before PIC assembly (Hengartner et al, 1998). Apart from cyclin H and the pol II CTD, however, few cdk8 substrates have been identified in humans.

Although a vast body of literature has demonstrated that the cdk8 subcomplex can negatively regulate transcription, a number of observations suggest that the cdk8 subcomplex may play some roles in activating gene expression (Liu et al, 2004; Wang et al, 2005; Donner et al, 2007). First, activators can interact with either the core Mediator or cdk8-Mediator, suggesting that cdk8-Mediator may play some roles in the activation of transcription (Taatjes et al, 2002). Second, although the majority of activators target subunits shared between core- and cdk8-Mediator, a few are known to recruit Mediator directly via the cdk8 subcomplex (Zhou et al, 2002; Fryer et al, 2004; Kim et al, 2006; Wang et al, 2006). Third, recent work in human cells suggests that cdk8-Mediator is recruited at an early stage in the activation of some genes. For example, at genes regulated by the retinoic acid receptor (RAR) or CEBP, recruitment of cdk8-Mediator occurs before activation of transcription; moreover, only upon loss of the cdk8 subcomplex—leaving behind core Mediator—is pol II recruited and transcription activated at these genes (Mo et al, 2004; Pavri et al, 2005). These results and others (Andrau et al, 2006; Zhu et al, 2006) suggest that cdk8 recruitment is temporally regulated and can correlate with transcription. However, a biochemical mechanism for cdk8-dependent activation of transcription has remained elusive.

In addition to Mediator and the PIC, histone modifications play major roles in the regulation of transcription (Lee and Workman, 2007; Shahbazian and Grunstein, 2007). Specific histone modifications—such as acetylation and phosphorylation—work to alter chromatin structure and recruit co-activators or co-repressors to specific genomic loci. It is becoming increasingly evident that histone marks work in combinatorial fashion and that modification at one site can promote or antagonize modifications at other sites. One example is tandem S10/K14 phosphoacetylation of histone H3, which correlates strongly with active expression of at least a subset of human genes. Although the precise mechanisms by which tandem H3 phosphoacetylation affect transcription are not completely defined, it is clear that both modifications can stimulate or antagonize recruitment of co-regulatory factors such as 14–3–3 proteins or HP1 (Mateescu et al, 2004; Fischle et al, 2005; Hirota et al, 2005; Winter et al, 2008).

Over the years, the identities of many enzymes that post-translationally modify histone substrates have been determined, with most isolated as components of large, macromolecular complexes. These distinct, multi-subunit chromatin-modifying complexes possess many shared subunits. For example, GCN5 is present in multiple chromatin-modifying complexes in yeast and humans (Lee and Workman, 2007). Notably, human GCN5L interacts directly with the 430 kDa TRRAP polypeptide (McMahon et al, 2000; Park et al, 2001), which is believed to function primarily as a scaffold protein because it has no known enzymatic function but is found in a number of distinct multi-subunit complexes that play varied roles in the cell (Robert et al, 2006; Lee and Workman, 2007). Interestingly, the Nut1 subunit of the yeast Mediator complex possesses sequence similarity to GCN5, and yeast Mediator complexes display HAT activity in a Nut1-dependent fashion (Lorch et al, 2000). However, the Nut1 subunit is not conserved in higher organisms, and human Mediator is not known to interact with histones in any way.

In this study, we describe the isolation and biochemical activity of a cdk8-Mediator complex that contains the TRRAP and GCN5L polypeptides. Significantly, the cdk8 and GCN5L subunits within this 'T/G-Mediator' complex work cooperatively to catalyze tandem phosphoacetylation of H3 S10/K14, providing a mechanistic basis for GCN5L association with cdk8-Mediator. Because tandem H3 phosphoacetylation is associated with active transcription, these results identify a biochemical role for cdk8 in transcriptional activation. As expected, however, the cdk8-containing T/G-Mediator complex was unable to directly activate transcription in a highly purified, reconstituted human system, suggesting that Mediator itself performs multiple roles in the regulation of gene expression, including potential early events such as post-translational modification of chromatin.

TRRAP/GCN5L stably associate with human Mediator

This work initiated from an unexpected observation made upon purification of Mediator from partially purified extracts. A typical purification starts with fractionation of HeLa nuclear extract over a phosphocellulose column; Mediator will elute in the P0.5 M and the P1 M fractions. The P1 M fraction is enriched in the core Med26-containing Mediator complex, whereas the P0.5 M fraction is enriched in the larger cdk8-containing Mediator complex (Figure 1A). Further purification using a VP16 activation domain affinity resin indicated that Mediator samples derived from the P0.5 M fraction—but not the P1 M fraction—showed the presence of a polypeptide unique to Mediator that co-migrated with the complex on a glycerol gradient (Figure 1B and data not shown). This polypeptide was of high molecular weight (>300 kDa), enabling its easy detection by gel electrophoresis and silver staining. Notably, we observed similar results using different activation domains for purification from the P0.5 M fraction (data not shown).

Figure 1.

TRRAP/GCN5L associates with human Mediator. (A) TRRAP/GCN5L and cdk8-Mediator are enriched in the P0.5 M fraction; core Mediator is enriched in the P1 M fraction. Each lane (PFT, P0.3, P0.5, P1 M) contained 11 g total protein. (B) Silver-stained gel (7% acrylamide) of glycerol-gradient fractions from VP16 affinity purification. The identities of the subunits are indicated at the right; arrow indicates the obvious 'unknown' co-migrating polypeptide. (C) Mediator purified from the P0.5 M fraction (Input, lane 1) was incubated with an anti-TRRAP or anti-Med26 antibody resin. Following extensive washing with 0.5 M KCl HEGN, bound proteins were eluted with TRRAP peptide antigen. Mediator remained bound and was eluted from the anti-TRRAP antibody resin only (lanes 2 and 3). A full-colour version of this figure is available at The EMBO Journal Online.

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To identify this unknown polypeptide, glycerol gradient fractions 15–17 (Figure 1B) were pooled, concentrated by TCA precipitation and separated by SDS–PAGE. A tryptic digest of the unknown band analyzed by mass spectrometry revealed 31 peptide matches to TRRAP/PAF400, a protein found in a number of large, multi-subunit complexes (McMahon et al, 1998; Ogryzko et al, 1998; Wieczorek et al, 1998; Ikura et al, 2000). Further MS analysis of other excised bands from the P0.5M-derived Mediator sample identified predicted Mediator subunits, with the exception of one 95 kDa band that was identified as GCN5L (Table I). TRRAP interacts directly with GCN5L and both subunits are found together in other large protein complexes such as TFTC and STAGA (Martinez et al, 1998; Wieczorek et al, 1998). However, MS analysis did not identify any additional subunits from TFTC/STAGA in these samples. Thus, we suspected that TRRAP/GCN5L might associate with Mediator.

Table 1: MS results from P0.5 M-derived Mediator samples purified using affinity chromatography and glycerol gradient sedimentation

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As a first step to investigate a potential TRRAP/GCN5L interaction with Mediator, we generated antibodies against TRRAP and tested whether these antibodies would immunoprecipitate the Mediator complex from HeLa nuclear extracts. As shown in Supplementary Figure 1, Mediator subunits co-immunoprecipitated with the anti-TRRAP antibody. In a reciprocal experiment, TRRAP and GCN5L were observed to co-IP on a cdk8 antibody resin (Supplementary Figure 1). To further test this association, Mediator was affinity-purified from the P0.5 M fraction, eluted and incubated over an anti-TRRAP antibody resin or an anti-Med26 resin. Significantly, the purified Mediator complex specifically bound the anti-TRRAP antibody resin, whereas the same Mediator sample did not bind an anti-Med26 column (compare lanes 2 and 4, Figure 1C). Moreover, Mediator could be eluted from the anti-TRRAP resin with its peptide antigen (Figure 1C, lane 3). Coupled with the MS data (Table I), the results from Figure 1 suggested that TRRAP and GCN5L can stably associate with Mediator.

TRRAP/GCN5L interact with the cdk8-Mediator complex

Mediator exists in two major forms in human cells: a smaller form that interacts strongly with pol II and activates transcription, and a large form that does not interact strongly with pol II and does not directly activate transcription. Notably, the 'small' and 'large' Mediator complexes differ in their subunit composition: the Med26 subunit preferentially associates with the small, active complex, whereas cdk8, cyclin C, Med12 and Med13 associate with the large Mediator complex (Taatjes et al, 2004). Thus, with the exception of Med26, the small Mediator complex represents a core Mediator structure that is present within the large, cdk8-Mediator complex.

To determine whether TRRAP/GCN5L specifically associate with the Med26- or the cdk8-containing Mediator complex (or both), we first examined how TRRAP and GCN5L tracked with Mediator subunits over a series of ion-exchange columns. Although the P0.5 M fraction is enriched in cdk8-Mediator, a small amount of Med26 remains in this fraction (Figure 2A). We can further segregate Med26 from cdk8 on a HiTrap Q column. As shown in Figure 2A, TRRAP, GCN5L, cdk8 and other Mediator subunits co-eluted in the P0.5M/QFT fraction, whereas Med26 was no longer detectable. This suggested that TRRAP/GCN5L associates specifically with the cdk8-Mediator complex. Further purification from the P0.5M/QFT fraction using affinity chromatography followed by glycerol gradient sedimentation indicated that TRRAP and GCN5L co-purified with cdk8-Mediator using this more rigorous purification protocol (Figure 2B–D). As expected, the STAGA subunits SAP130 and SPT3 were not detected in this sample (Figure 2D). Importantly, similar results were obtained using different affinity chromatography resins (for example, SREBP-1a or p53 instead of VP16—see Supplementary Figure 2). In addition, binding assays with anti-Med26 or anti-cdk8 antibody resins indicated specific association of TRRAP/GCN5L with cdk8-Mediator but not core Mediator (Supplementary Figure 3).

Figure 2.

Purification of T/G-Mediator. (A) Western blots against partially purified column fractions. Each lane contained 24 g total protein. (B) Purification protocol used for isolation of T/G-Mediator. (C) Silver-stained gel (7% acrylamide) showing glycerol gradient fractions from the purification outlined in (B). Subunit identities are shown at the right. (D) Western blots against various subunits from the T/G-Mediator sample. Numbers in parenthesis represent the approximate molecular weight of each protein. Subunits in red are specific to STAGA, whereas Med26 is specific to core Mediator. A full-colour version of this figure is available at The EMBO Journal Online

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As a final means to confirm TRRAP/GCN5L association with the cdk8-Mediator complex, we used electron microscopy (EM). Core Mediator and cdk8-Mediator can be readily distinguished with EM by completing 2D classification of single-particle images, as shown in Figure 3A: the larger cdk8-Mediator complex has a size and shape distinct from core Mediator. As expected, analysis of the purified sample on a gradient (5–15%) polyacrylamide gel indicated the presence of polypeptides specific for cdk8-Mediator (for example, Med12 and Med13), whereas Med26, a subunit specific for core Mediator, was not observed (Figure 3B). This result was substantiated upon EM analysis of the sample: the 2D classes generated from the data revealed complexes of size and shape consistent with cdk8-Mediator, but not core Mediator (representative classes shown in Figure 3C).

Figure 3.

T/G-Mediator contains the cdk8 submodule. (A) Representative 2D classes of cdk8-Mediator and core Mediator. Scale bar: 150 Å. (B) Silver-stained gradient (5–15% acrylamide) gel showing the T/G-Mediator sample used for EM analysis. Note that only prominently staining bands are identified and smaller, poorly staining polypeptides representing other consensus Mediator subunits are not labelled (Sato et al, 2004). Asterisk: insulin (used for TCA precipitation). (C) EM analysis indicates that T/G-Mediator sample has a size and shape consistent with cdk8-Mediator. Representative 2D classes are shown; each class (1–4) was obtained by averaging 150 aligned single-particle images. Scale bar: 150 Å. A full-colour version of this figure is available at The EMBO Journal Online

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Taken together, the results summarized in Figures 1, 2 and 3 indicate that TRRAP/GCN5L stably associate with the cdk8-Mediator complex but not the core Mediator complex. For simplicity, we will refer to this complex as T/G-Mediator.

T/G-Mediator cooperatively phosphoacetylates S10/K14 within histone H3

Having established that TRRAP/GCN5L can stably associate with cdk8-Mediator in human cells, we examined whether T/G-Mediator would display HAT activity. These experiments, which are described further in Supplementary data, demonstrated that T/G-Mediator, but not core Mediator, specifically acetylates lysine 14 on histone H3 (Supplementary Figure 5). Because T/G-Mediator also contains a kinase, cdk8, we tested whether T/G-Mediator might also phosphorylate histones; these experiments were prompted in part by related work in our lab that demonstrated that the cdk8 subcomplex itself was a potent histone kinase (MTK and DJT, unpublished data). As described further in Supplementary data, kinase assays showed that T/G-Mediator, but not core Mediator, specifically phosphorylates serine-10 on histone H3 (Supplementary Figure 6 and 7).

Individually, H3S10 phosphorylation and H3K14 acetylation are histone modifications linked to transcriptional activation. However, mounting evidence suggests that both modifications are important for expression of at least a subset of genes in yeast and mammalian cells (Clayton et al, 2000; Lo et al, 2000; Thomson et al, 2001). Although the data summarized in Supplementary Figures 5 and 7 indicated T/G-Mediator specifically phosphorylates H3S10 and acetylates H3K14, we could not conclude that T/G-Mediator was performing both modifications on the same H3 tail. To demonstrate this, we incubated T/G-Mediator with recombinant core histone octamers in the presence of both acetyl CoA and ATP. Significantly, immunoblotting experiments with antibodies specific for S10/K14 phospho-acetylated H3 confirmed that T/G-Mediator was catalyzing H3S10 phosphorylation and H3K14 acetylation on the same H3 tail (Figure 4A). Because H3S10 phosphorylation is known to stimulate GCN5 acetylation of H3K14 (Cheung et al, 2000), we also ran separate acetyltransferase assays with T/G-Mediator in the presence or absence of ATP. As expected, the added ATP had a notable affect on histone acetylation by T/G-Mediator (Figure 4B, compare lanes 2 and 4). In particular, the low levels of H2A, H2B and H4 acetylation by T/G-Mediator decreased to nearly undetectable levels in the presence of ATP, whereas acetylation of H3 remained high. This data is consistent with previous studies demonstrating cooperativity between H3S10 phosphorylation and H3K14 acetylation (Cheung et al, 2000; Clements et al, 2003) and offers further evidence that T/G-Mediator can perform both modifications on the same histone H3 tail. Figure 4C also demonstrates cooperativity between H3S10/K14 within T/G-Mediator, as mutation of S10 within the H3 tail significantly reduced acetylation at H3K14. Taken together, the results summarized in Figure 4 indicate that cdk8 and GCN5L work cooperatively within a single complex, T/G-Mediator, to perform tandem phosphoacetylation of histone H3.

Figure 4.

T/G-Mediator cooperatively phosphoacetylates histone H3. (A) Recombinant core histones alone (lane 1), T/G-Mediator alone (lane 2) or T/G-Mediator together with recombinant core histones (lane 3) were incubated with ATP and acetyl CoA. Reactions were then probed with an antibody specific to the doubly modified S10/K14 H3. (B) ATP enhances T/G-Mediator acetylation specificity for H3. Acetylation assay with T/G-Mediator and core histones in the absence (lane 2) and presence (lane 4) of ATP. Lane 1 and 3: loading reference for lanes 2 and 4. Quantitation of the [¹⁴C]-acetyl label is shown to the right of lanes 2 and 4. (C) T/G-Mediator-catalysed acetylation of H3K14 is reduced in H3S10A mutant. Equivalent amounts of T/G-Mediator were incubated with wild-type (WT) or mutant (S10A) H3 tails in the presence of ATP and [¹⁴C]-acetyl CoA. Representative data are shown; quantitation of the [¹⁴C]-acetyl label is shown beside each radiolabelled band, with WT normalized to 100. The value shown for S10A is the average of four independent experiments (=8.9). Representative loading controls (silver stain) are shown for each lane. A full-colour version of this figure is available at The EMBO Journal Online

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T/G-Mediator modifies H3 within chromatin templates

We also performed experiments that demonstrated that T/G-Mediator phosphoacetylates H3 within chromatin templates; these experiments are described further in Supplementary data and are shown in Supplementary Figure 8.

Co-occupancy of T/G-Mediator subunits at the CDKN1A and egr1 genes

To investigate the association of TRRAP with a cdk8-containing Mediator complex in vivo, we tested for their recruitment to human genes using ChIP assays. We focused on three well-characterized human genes whose activity could be easily manipulated in cell cultures: CDKN1A, egr1 and c-fos. CDKN1A is a canonical p53 target gene whose transcription is strongly stimulated by Nutlin-3, a pharmacological inhibitor of the p53 repressor MDM2 (Vassilev et al, 2004; Donner et al, 2007). Nutlin-3 treatment of HCT116 cells (wt p53) leads to sustained accumulation of the CDKN1A mRNA (Figure 5A). egr1 and c-fos are genes whose transcription is stimulated in quiescent cells via growth factor-mediated signalling. Consistent with their classification as immediate-early genes, egr1 and c-fos mRNAs rapidly but transiently accumulate upon serum replenishment of serum-starved HCT116 cells (Figure 5B and data not shown). ChIP analysis at the CDKN1A promoter reveals that, upon induction, pol II recruitment increases at both the transcription start site (amplicon B) and the intragenic region (amplicon C), thus revealing the presence of elongating pol II. Interestingly, significant amounts of TRRAP can be detected at the CDKN1A proximal promoter even before p53 activation, and TRRAP association increases modestly upon induction. Similarly, ChIP assays with cdk8 antibodies reveal that it is recruited to the CDKN1A locus upon p53 activation. Importantly, cdk8 is most likely recruited as part of the Mediator complex, as indicated by concomitant association of Med1, Med12 and cyclin C to the active CDKN1A locus (Donner et al, 2007). These results are consistent with a cdk8-containing Mediator complex coexisting with TRRAP at the active CDKN1A locus, suggesting the presence of T/G-Mediator. ChIP analysis of the egr1 or c-fos locus produced almost identical results (Figure 5B and D and data not shown), also consistent with T/G-Mediator occupancy at each promoter. ChIP assays using antibodies recognizing tandem phosphoacetylated H3 at the CDKN1A, egr1 and c-fos loci are described in Supplementary data.

Figure 5.

TRRAP and cdk8 are co-recruited to the promoter regions of active human genes; cdk8 knockdown depletes global levels of phosphoacetylated H3. (A) p53-dependent activation of CDKN1A in HCT116 cells. cDNA samples were prepared after addition of 10 M Nutlin-3 to the culture media. Values are expressed as fold induction over untreated cells after normalization to 18S rRNA. (B) Serum-induced activation of egr1 in HCT116 cells. cDNA samples were prepared after serum replenishment, following 24 h of serum withdrawal. (C) ChIP analysis of the CDKN1A locus. HCT116 cells were treated with vehicle or 10 M Nutlin-3 for 8 h before harvest and extract preparation. IPs were performed with antibodies against pol II, TRRAP and cdk8. ChIP-enriched DNA was analysed by Q–PCR with four different amplicons (A–D) covering different regions of the CDKN1A locus. (D) ChIP analysis of the egr1 locus. Serum-starved HCT116 cells were stimulated with serum for 30 min before harvest. ChIPs were performed as in (C). Error bars represent standard deviations on triplicate experiments. (E) HCT116 cells were transduced with control or anti-cdk8 shRNAs and analysed by Western blot. Quantitation of knockdown is reported as % reduction from control cells (^*average of two experiments; % reduction measured was 92/92 for cdk8, 86/75 for H3S10P/K14Ac, (-10)/16 for nucleolin (loading control)).

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Knockdown of cdk8 dramatically reduces phosphoacetylated H3 levels in human cells

Because of the inherent difficulty in identifying and proving that a human gene is specifically dependent upon the chromatin-modifying activity of T/G-Mediator (and not some other recruited kinase or acetyltransferase), we instead examined how shRNA-mediated knockdown of cdk8 affects global levels of phosphoacetylated H3. As shown in Figure 5E, knocking down cdk8 reduced the level of phosphoacetylated H3 by 80%. This corroborates our in vitro data and clearly demonstrates that cdk8 acts as a histone kinase in human cells. Furthermore, because cdk8 knockdown impacts the dual H3 S10P/K14Ac state, this suggests that cdk8 activity within T/G-Mediator plays a significant role in the tandem modification of histone H3.

T/G-Mediator does not directly activate transcription

Subunit exchange within the human Mediator complex can dramatically impact its function as a transcriptional co-activator. For example, the core Mediator complex interacts directly with RNA polymerase II and is a potent co-activator of transcription. By contrast, when associated with the 4-subunit cdk8 subcomplex (that is, cdk8, cyclin C, Med12, Med13), Mediator co-activator function is repressed: this distinct 'cdk8-Mediator' complex is unable to activate transcription (Taatjes et al, 2002). Notably, T/G-Mediator most closely resembles cdk8-Mediator in that it contains the cdk8 submodule and lacks Med26; however, T/G-Mediator also contains a known co-activator, the GCN5L acetyltransferase.

To determine whether T/G-Mediator possesses direct co-activator function, we tested its activity in our in vitro transcription system. This reconstituted human transcription system is similar to one described previously (Taatjes and Tjian, 2004) and uses chromatin templates with highly purified and recombinant general transcription factors (TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, RNA pol II) together with purified activators and Mediator. Additional details of this reconstituted human system will be provided elsewhere (KDM, MTK and DJT, unpublished data). As shown in Figure 6A, core Mediator displays robust, activator-dependent co-activator function in this assay (compare lanes 1–4), as observed previously (Taatjes and Tjian, 2004). T/G-Mediator, by contrast, was unable to activate transcription in this highly purified system, even when titrated to a concentration threefold higher than core Mediator (Figure 6A, lanes 8–11). In fact, if added together with core Mediator, T/G-Mediator can block transcription in a dose-dependent manner (Figure 6A, lanes 4–7). These results suggest that T/G-Mediator is transcriptionally inert and that repression results from competition between T/G-Mediator and core Mediator for activator (Sp1 in this case) binding/recruitment to the promoter. To ensure the observed activity was not simply a characteristic of the Sp1 promoter template, we also tested T/G-Mediator function on chromatin templates responsive to SREBP-1a/Sp1, VDR/RXR or gal4-VP16. T/G-Mediator displayed similar activity in these assays (Supplementary Figure 9).

Figure 6.

In vitro transcription on chromatin templates. (A) Unlike core Mediator, T/G-Mediator does not directly activate transcription. Core Mediator is required to reconstitute activator-dependent transcription in vitro on chromatin templates (compare lanes 1–4); T/G-Mediator blocks core Mediator-dependent activation in a dose-dependent manner (compare lanes 4–7); T/G-Mediator is unable to activate transcription even in the presence of activator (lanes 8–11). (B) PARP-1 does not activate T/G-Mediator. Titration of PARP-1 (20, 50 or 120 ng) in the presence of T/G-Mediator and Sp1 (lanes 6–8). Quantitation of RNA transcripts is shown in the bar graph below each lane.

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Given that T/G-Mediator contains the cdk8 subcomplex, we did not expect it to display co-activator function (Taatjes et al, 2002). Yet because T/G-Mediator possesses histone kinase and acetyltransferase activity, we hypothesized that it may possess latent co-activator function. Previous work by the Reinberg lab indicated that, at least at promoters responsive to RAR, the cdk8-Mediator complex could be 'activated' by PARP-1: PARP-1 was shown to bind RAR and dissociate the cdk8 subcomplex, leaving behind core Mediator at the promoter, which then worked to activate transcription (Pavri et al, 2005). Although clearly distinct from cdk8-Mediator, T/G-Mediator does contain the cdk8 submodule, and therefore we surmised that PARP-1 might dissociate the cdk8 subcomplex from T/G-Mediator (and perhaps TRRAP/GCN5L) to enable transcription activation in vitro. However, as shown in Figure 6B, PARP-1 was unable to activate T/G-Mediator in this way, at least at the promoters tested. (Note that similar results were obtained with LDLR-derived promoters or a VDR/RXR promoter, as shown in Supplementary Figure 9.) This supports the conclusions of Pavri et al, (2005) that PARP-1 acts in a promoter-specific manner.

Mediator is a primary regulator of PIC function: Mediator physically or functionally interacts with most components of the PIC and also regulates post-recruitment events in transcription initiation (Malik and Roeder, 2005; Wang et al, 2005). The results from this study suggest that human Mediator plays an expanded role in transcriptional regulation that involves post-translational modification of histone H3. Given the link between H3 acetylation and activation of transcription, it is somewhat counter-intuitive that TRRAP/GCN5L associates not with core Mediator, which possesses co-activator function, but rather with cdk8-Mediator, which does not directly activate transcription. However, our histone modification assays reveal that within T/G-Mediator, cdk8 phosphorylates H3S10, providing a biochemical rationale for the selective interaction of TRRAP/GCN5L with cdk8-Mediator instead of core Mediator. Indeed, numerous studies describe a functional cooperativity between H3S10 phosphorylation and H3K14 acetylation, at least at a subset of genes (Clayton et al, 2000; Lo et al, 2000; Anest et al, 2003; Yamamoto et al, 2003). This functional coordination, coupled with the ability of phosphorylated H3S10 to stimulate H3K14 acetylation by GCN5, led to the hypothesis that an H3S10 kinase/H3K14 acetyltransferase might reside within the same multi-subunit complex (Cheung et al, 2000). To our knowledge, T/G-Mediator represents the first demonstration that an H3S10 kinase (cdk8) and an H3K14 acetyltransferase (GCN5L) can in fact function within a single stable, multi-subunit complex.

By coordinating post-translational modifications within histone H3, T/G-Mediator may work to direct early events in the activation of gene expression (that is, before full PIC assembly). Importantly, the observation that cdk8 phosphorylates H3S10 offers a biochemical rationale for past studies showing a correlation between cdk8 recruitment and transcription activation. Paradoxically, this positive role for cdk8 remains consistent with its well-established function as a negative regulator of transcription, provided the biochemical function of cdk8 is considered at different stages of initiation. As a histone kinase within T/G-Mediator, cdk8 may work together with other chromatin-modifying/remodelling factors to establish a chromatin environment favorable for transcription. However, by blocking stable pol II assembly within the PIC and inhibiting TFIIH (Akoulitchev et al, 2000; Näär et al, 2002; Elmlund et al, 2006), cdk8 would concomitantly inhibit transcription initiation at this stage. Actual transcription initiation likely occurs only upon dissociation of the cdk8 subcomplex (and perhaps TRRAP/GCN5L) from Mediator, which would enable pol II recruitment via its stable association with core Mediator and other PIC components (for example, TFIIB). This model is consistent with our in vitro transcription data, which clearly shows that T/G-Mediator, in contrast to core Mediator, is unable to activate transcription. Furthermore, previous studies in human cells demonstrate that dissociation of cdk8 subcomplex components—but not core Mediator itself—occurs immediately before transcriptional activation at RAR- and CEBP-regulated genes (Mo et al, 2004; Pavri et al, 2005).

Notably, dissociation of the cdk8 submodule was shown to be dependent upon PARP-1 at RAR/RXR-regulated genes in vitro and in vivo; however, PARP-1 was unable to similarly 'activate' T/G-Mediator. This suggests that PARP-1 dissociation of the cdk8 subcomplex may be promoter specific, as proposed previously (Pavri et al, 2005). Indeed, PARP-1 was shown to interact directly with RAR, and similar interactions may not occur with the activators tested here (Sp1, SREBP-1a, VDR/RXR). Alternately, the presence of TRRAP/GCN5L may block PARP-1-dependent dissociation of the cdk8 subcomplex from T/G-Mediator to prevent transcription activation. In either case, it is likely that an activity distinct from PARP-1 is required to dissociate the cdk8 submodule within T/G-Mediator. Future work will be directed towards identification of such factors.

The regulatory role of combined H3S10 phosphorylation and H3K14 acetylation appears to vary in different promoter contexts, and it is not established whether these tandem modifications are a general phenomenon of active gene expression. Furthermore, it is likely that tandem H3 S10/K14 phosphoacetylation does not occur cooperatively at all genes that require this modification (Thomson et al, 2001). Yet it is clear that each modification can facilitate transcription activation via recruitment of co-activators (for example, TAF1 or GCN5L itself) and/or loss of co-repressors (for example, HP1) (Kasten et al, 2004; Fischle et al, 2005; Hirota et al, 2005; Hassan et al, 2007). In mammalian cells, phosphorylation of H3S10 has been linked to activation of specific genes in response to growth factors or inflammation (Sassone-Corsi et al, 1999; Thomson et al, 1999; Anest et al, 2003; Yamamoto et al, 2003); moreover, H3S10 phosphorylation correlates with expression of at least a subset of RAR and myc target genes (Lefebvre et al, 2002; Zippo et al, 2007). In several of these cases, distinct kinases were directly or at least indirectly responsible for H3 phosphorylation (for example, IKK, MSK2/1, PIM1), suggesting that each may play gene-specific roles in transcriptional activation (Soloaga et al, 2003).

Our examination of modified H3 in human cells reveals that knockdown of cdk8 has a major impact on global levels of phosphoacetylated H3. Although these effects could be indirect, our in vitro results overwhelmingly support a direct role for cdk8 in the phosphoacetylation of H3. In fact, the striking correlation between the levels of cdk8 and phosphoacetylated H3 suggests that T/G-Mediator is a prominent regulator of this H3 modification. Further studies involving genome-wide approaches in human cells will be required to assess which genes may be specifically dependent upon the chromatin-modifying function of T/G-Mediator. Although cdk8 and Mediator itself appear to be globally recruited to promoter and enhancer regions in yeast (Andrau et al, 2006; Zhu et al, 2006), we predict that tandem phosphoacetylation by T/G-Mediator will be critical for expressing only a subset of human genes due to redundant histone kinase and acetyltransferase activities; furthermore, it is not evident that tandem H3 phosphoacetylation is generally required for expression of all protein-coding genes. Indeed, like cdk8, GCN5 is also globally recruited to gene promoters in yeast (Robert et al, 2004; Pokholok et al, 2005), yet in higher organisms, changes in histone acetylation are not uniformly observed for all genes upon activation (Nowak and Corces, 2000; Lefebvre et al, 2002). Notably, cdk8 can also negatively regulate transcription via phosphorylation of TFIIH (Akoulitchev et al, 2000); thus, precise control of cdk8 substrate specificity may be critical for regulating transcription initiation in humans.

Interestingly, TRRAP and GCN5L are also subunits of the STAGA/TFTC complex in humans, which is biochemically similar to yeast SAGA (Baker and Grant, 2007; Nagy and Tora, 2007). Because human TRRAP/GCN5L are shared between STAGA/TFTC and T/G-Mediator, these subunits (particularly TRRAP) may help orchestrate recruitment of STAGA/TFTC and Mediator at human genes. Indeed, ChIP studies in yeast and human cells have shown that recruitment of SAGA and Mediator can occur simultaneously or sequentially (Bhoite et al, 2001; Cosma et al, 2001; Bryant and Ptashne, 2003; Govind et al, 2005), and that SAGA may aid in Mediator recruitment, at least at some genes (Bhaumik et al, 2004; Qiu et al, 2005; Liu et al, 2008). Genetic studies in yeast also suggest functional interactions between SAGA and Mediator (Roberts and Winston, 1997), specifically with Mediator containing the cdk8 submodule (Larschan and Winston, 2005). Although a potential mechanism for this interdependent recruitment is not established, the results shown here suggest that STAGA/TFTC and Mediator co-association or exchange may be dependent upon the 430 kDa TRRAP polypeptide. Notably, this is consistent with the perceived biochemical role of TRRAP as a molecular scaffold/architectural factor within the PIC (Murr et al, 2007).

Purification of T/G-Mediator

A typical purification starts with nuclei isolated from approximately 100 l of HeLa cells, as outlined in Figure 2B. For the affinity purification step, after binding QFT, the resin was washed with 50 column volumes (CV) 0.5 M KCl HEGN (pH 7.9, 20 mM HEPES, 0.1 mM EDTA, 10% glycerol, 0.1% NP-40) and 10 CV 0.15 M KCl HEGN (0.02% NP-40). T/G-Mediator was then eluted from the GSH resin with 2 CV buffer containing 30 mM glutathione (pH 7.6, 20 mM Tris, 0.1 mM EDTA, 10% glycerol, 0.15 M KCl). This material was then loaded onto a 2 ml glycerol gradient (15–40% glycerol in 0.15 M KCl HEGN) and centrifuged at 55K r.p.m. for 6 h at 4°C. Fractions were collected from the top of the gradient in 100 l aliquots.

LC MS/MS analysis

Mass spectrometry experiments were completed on a PE Sciex API QSTAR Pulsar MS with capillary electrospray interface, essentially as described (Knuesel et al, 2005). In-gel trypsin digestion of proteins was completed following reduction (DTT) and alkylation (iodoacetamide) using standard techniques.

Antibodies

Santa Cruz Biotechnology: Med1, GCN5L, cdk8, pol II; Bethyl Inc.: Med23; Upstate: phospho-H3T3, -H3S10, -H3S28, phosphoacetyl-H3S10/K14; Lab stocks: TRRAP, Med15, Med26, Med14.

Core histone purification

Purification of core histones was completed with 140 g of 12–24 h Drosophila embryos, essentially as described (Laybourn and Kadonaga, 1991). Expression and purification of recombinant core histones was carried out as described (Luger et al, 1999).

QRT–PCR and ChIP assays

HCT116 were maintained in McCoys 5A media (Gibco-Invitrogen) supplemented with 10% fetal bovine serum and antibiotic/antimycotic mix. For CDKN1A induction, cells were treated with 10 M Nutlin-3 (Cayman Biochemicals). For egr1 and c-fos induction, cells were serum-starved for 24 h before serum replenishment. QRT–PCRs and ChIPs assays were performed essentially as described (Gomes et al, 2006). Briefly, for RT–PCR, total RNA was isolated using the RNeasy Mini Kit (Qiagen), and cDNA was generated with the SuperScriptase II (Invitrogen). For ChIP assays, cells were fixed with 1% formaldehyde and harvested for whole cell lysate preparation. A quantity of 1 mg of protein lysate was used per ChIP with antibodies against pol II, TRRAP and cdk8. ChIP-enriched DNA was analysed by Q-PCR as described (Gomes et al, 2006). See Supplementary Table 1 and 2 for primer sequences.

shRNA knockdown of cdk8

Control or anti-cdk8 shRNA sequences were cloned into a derivative of the pLL3.7 vector containing a G418 resistance cassette. After packaging of lentiviral particles in HEK293FT cells, HCT116 cells were transduced, selected in G418 and individual stable clones were grown and tested for cdk8 knockdown by Western blot.

Chromatin assembly

For kinase or acetyltransferase assays, chromatin was assembled using purified Drosophila core histones and a DNA template (G5E4) derived from the sea urchin 5S rRNA gene, essentially as described (Ikeda et al, 1999). The quality of the assembled chromatin was determined by micrococcal nuclease digestion. For in vitro transcription, supercoiled template plasmids were assembled into chromatin using Drosophila embryo cytosolic extract (S-190), purified Drosophila core histones and an ATP-regenerating system as described (Näär et al, 1998). Chromatin assembly was carried out at 27°C for 5 h.

In vitro transcription

Chromatin templates were incubated with activators (Sp1, SREBP-1a or gal4-VP16: 4 nM each; or VDR/RXR: 10 nM each plus ligand) for 30 min, followed by addition of purified and recombinant general transcription factors (40 nM TFIIA, 10 nM TFIIB, 0.8 nM TFIID, 10 nM TFIIE, 10 nM TFIIF, 0.5 nM TFIIH and 2 nM pol II) with/without non-limiting amounts of Mediator and/or T/G-Mediator (0–15 nM). Note that acetyl CoA (5 M) was also added in some experiments and had no effect (not shown). After allowing 15 min for PIC assembly, NTPs (0.5 nM final concentration) were added; 30 min later, transcription was terminated by adding three volumes of stop solution (20 mM EDTA, 0.2 M NaCl, 1% SDS). RNA was then isolated and analysed by primer extension.

We thank J Goodrich and X Liu for helpful comments on the manuscript; S Zhou for initial MS experiments and S Zheng and DS King for assistance with antibody production and design and synthesis of TRRAP-derived peptides. We also thank K Luger for providing plasmids for expression of recombinant core histones and J Workman for the 5S G5E4 template. This work was supported by grants from the Colorado Cancer League, the NCI (P20-CA103680) and the Ellison Medical Foundation to DJT and the NCI (R01-CA117907) to JME.

Akoulitchev S, Chuikov S, Reinberg D (2000) TFIIH is negatively regulated by cdk8-containing mediator complexes. Nature 407: 102–106 | Article | PubMed | ISI | ChemPort |

Andrau J, van de Pasch L, Lijnzaad P, Bijma T, Koerkamp MG, van de Peppel J, Werner M, Holstege FCP (2006) Genome-wide location of the coactivator Mediator: binding without activation and transient cdk8 interaction on DNA. Mol Cell 22: 179–192 | Article | PubMed | ChemPort |

Anest V, Hanson JL, Cogswell PC, Steinbrecher KA, Strahl BD, Baldwin AS (2003) A nucleosomal function for IB kinase- in NFB-dependent gene expression. Nature 423: 659–663 | Article | PubMed | ISI | ChemPort |

Baker SP, Grant PA (2007) The SAGA continues: expanding the cellular role of a transcriptional co-activator complex. Oncogene 26: 5329–5340 | Article | PubMed | ChemPort |

Bhaumik SR, Raha T, Aiello DP, Green MR (2004) In vivo target of a transcriptional activator revealed by fluorescence resonance energy transfer. Genes Dev 18: 333–343 | Article | PubMed | ISI | ChemPort |

Bhoite LT, Yu Y, Stillman DJ (2001) The Swi5 activator recruits the Mediator complex to the HO promoter without RNA polymerase II. Genes Dev 15: 2457–2469 | Article | PubMed | ISI | ChemPort |

Bryant GO, Ptashne M (2003) Independent recruitment in vivo by Gal4 of two complexes required for transcription. Mol Cell 11: 1301–1309 | Article | PubMed | ISI | ChemPort |

Cheung P, Tanner KG, Cheung WL, Sassone-Corsi P, Denu JM, Allis CD (2000) Synergistic coupling of histone H3 phosphorylation and acetylation in response to epidermal growth factor stimulation. Mol Cell 5: 905–915 | Article | PubMed | ISI | ChemPort |

Clayton AL, Rose S, Barratt MJ, Mahadevan LC (2000) Phosphoacetylation of histone H3 on c-fos- and c-jun-associated nucleosomes upon gene activation. EMBO J 19: 3714–3726 | Article | PubMed | ISI | ChemPort |

Clements A, Poux AN, Lo W, Pillus L, Berger SL, Marmorstein R (2003) Structural basis for histone and phosphohistone binding by the GCN5 histone acetyltransferase. Mol Cell 12: 461–473 | Article | PubMed | ISI | ChemPort |

Conaway RC, Sato S, Tomomori-Sato C, Yao T, Conaway JW (2005) The mammalian Mediator complex and its role in transcriptional regulation. TIBS 30: 250–255 | PubMed | ChemPort |

Cosma MP, Panizza S, Nasmyth K (2001) Cdk1 triggers association of RNA polymerase to cell cycle promoters only after recruitment of the mediator by SBF. Mol Cell 7: 1213–1220 | Article | PubMed | ISI | ChemPort |

Davis JA, Takagi Y, Kornberg RD, Asturias FA (2002) Structure of the yeast RNA polymerase II holoenzyme: Mediator conformation and polymerase interaction. Mol Cell 10: 409–415 | Article | PubMed | ISI | ChemPort |

Donner AJ, Szostek S, Hoover JM, Espinosa JM (2007) CDK8 is a stimulus-specific positive coregulator of p53 target genes. Mol Cell 27: 121–133 | Article | PubMed | ChemPort |

Elmlund H, Baraznenok V, Lindahl M, Samuelsen CO, Koeck PJB, Holmberg S, Hebert H, Gustafsson CM (2006) The cyclin-dependent kinase 8 module sterically blocks Mediator interactions with RNA polymerase II. Proc Natl Acad Sci USA 103: 15788–15793 | Article | PubMed | ChemPort |

Fischle W, Tseng BS, Dormann HL, Ueberheide BM, Garcia BA, Shabanowitz J, Hunt DF, Funabiki H, Allis CD (2005) Regulation of HP1-chromatin binding by histone H3 methylation and phosphorylation. Nature 438: 1116–1122 | Article | PubMed | ISI | ChemPort |

Fryer CJ, White JB, Jones KA (2004) Mastermind recruits CycC:Cdk8 to phosphorylate the notch ICD and coordinate activation with turnover. Mol Cell 16: 509–520 | Article | PubMed | ISI | ChemPort |

Gomes NP, Bjerke G, Llorente B, Szostek SA, Emerson BM, Espinosa JM (2006) Gene-specific requirement for P-TEFb activity and RNA polymerase II phosphorylation within the p53 transcriptional program. Genes Dev 20: 601–612 | Article | PubMed | ISI | ChemPort |

Govind CK, Yoon S, Qiu H, Govind S, Hinnebusch AG (2005) Simultaneous recruitment of coactivators by Gcn4p stimulates multiple steps of transcription in vivo. Mol Cell Biol 25: 5626–5638 | Article | PubMed | ChemPort |

Hahn S (2004) Structure and mechanism of the RNA polymerase II transcription machinery. Nat Struct Mol Biol 11: 394–403 | Article | PubMed | ISI | ChemPort |

Hassan AH, Awad S, Al-Natour Z, Othman S, Mustafa F, Rizvi TA (2007) Selective recognition of acetylated histones by bromodomains in transcriptional co-activators. Biochem J 402: 125–133 | Article | PubMed | ISI | ChemPort |

Hengartner CJ, Myer VE, Liao S, Wilson CJ, Koh SS, Young RA (1998) Temporal regulation of RNA polymerase II by Srb10 and Kin28 cyclin-dependent kinases. Mol Cell 2: 43–53 | Article | PubMed | ISI | ChemPort |

Hirota T, Lipp JJ, Toh B, Peters J (2005) Histone H3 serine 10 phosphorylation by Aurora B causes HP1 dissociation from heterochromatin. Nature 438: 1176–1180 | Article | PubMed | ISI | ChemPort |

Ikeda K, Steger DJ, Eberharter A, Workman JL (1999) Activation domain-specific and general transcription stimulation by native histone acetyltransferase complexes. Mol Cell Biol 19: 855–863 | PubMed | ISI | ChemPort |

Ikura T, Ogryzko VV, Grigoriev M, Groisman R, Wang J, Horikoshi M, Scully R, Qin J, Nakatani Y (2000) Involvement of the TIP60 histone acetylase complex in DNA repair and apoptosis. Cell 102: 463–473 | Article | PubMed | ISI | ChemPort |

Kasten M, Szerlong H, Erdjument-Bromage H, Tempst P, Werner M, Cairns BR (2004) Tandem bromodomains in the chromatin remodeler RSC recognize acetylated histone H3 lys14. EMBO J 23: 1348–1359 | Article | PubMed | ISI | ChemPort |

Kim S, Xu X, Hecht A, Boyer TG (2006) Mediator is a transducer of Wnt/beta-catenin signaling. J Biol Chem 281: 14066–14075 | Article | PubMed | ISI | ChemPort |

Kim Y, Bjorklund S, Li Y, Sayre MH, Kornberg RD (1994) A multiprotein mediator of transcriptional activation and its interaction with the C-terminal repeat domain of RNA polymerase II. Cell 77: 599–608 | Article | PubMed | ISI | ChemPort |

Knuesel M, Cheung HT, Hamady M, Barthel KK, Liu X (2005) A method of mapping protein sumoylation sites by mass spectrometry using a modified small ubiquitin-like modifier 1 (SUMO-1) and a computational program. Mol Cell Proteomics 4: 1626–1636 | Article | PubMed | ISI | ChemPort |

Larschan E, Winston F (2005) The Saccharomyces cerevisiae Srb8-Srb11 complex functions with the SAGA complex during Gal4-activated transcription. Mol Cell Biol 25: 114–123 | Article | PubMed | ISI | ChemPort |

Laybourn PJ, Kadonaga JT (1991) Role of nucleosomal cores and histone H1 in regulation of transcription by RNA polymerase II. Science 254: 238–245 | Article | PubMed | ISI | ChemPort |

Lee KK, Workman JL (2007) Histone acetyltransferase complexes: one size doesn't fit all. Nature Rev Mol Cell Biol 8: 284–295 | Article

Lefebvre B, Ozato K, Lefebvre P (2002) Phosphorylation of histone H3 is functionally linked to retinoic acid receptor beta promoter activation. EMBO Rep 3: 335–340 | Article | PubMed | ISI | ChemPort |

Liu X, Vorontchikhina M, Wang Y, Faiola F, Martinez E (2008) STAGA recruits Mediator to the MYC oncoprotein to stimulate transcription and cell proliferation. Mol Cell Biol 28: 108–121 | Article | PubMed | ChemPort |

Liu Y, Kung C, Fishburn J, Ansari AZ, Shokat KM, Hahn S (2004) Two cyclin-dependent kinases promote RNA polymerase II transcription and formation of the scaffold complex. Mol Cell Biol 24: 1721–1735 | Article | PubMed | ISI | ChemPort |

Lo WS, Trievel RC, Rojas JR, Duggan L, Hsu JY, Allis CD, Marmorstein R, Berger SL (2000) Phosphorylation of serine 10 in histone H3 is functionally linked in vitro and in vivo to Gcn5-mediated acetylation at lysine 14. Mol Cell 5: 917–926 | Article | PubMed | ISI | ChemPort |

Lorch Y, Beve J, Gustafsson CM, Myers LC, Kornberg RD (2000) Mediator-nucleosome interaction. Mol Cell 6: 197–201 | Article | PubMed | ChemPort |

Luger K, Rechsteiner TJ, Richmond TJ (1999) Expression and purification of recombinant histones and nucleosome reconstitution. Methods Mol Biol 119: 1–16 | Article | PubMed | ChemPort |

Malik S, Roeder RG (2005) Dynamic regulation of pol II transcription by the mammalian Mediator complex. TIBS 30: 256–263 | PubMed | ChemPort |

Martinez E, Kundu TK, Fu J, Roeder RG (1998) A human SPT3-TAFII31-GCN5-L acetylase complex distinct from transcription factor IID. J Biol Chem 273: 23781–23785 | Article | PubMed | ISI | ChemPort |

Mateescu B, England P, Halgand F, Yaniv M, Muchardt C (2004) Tethering of HP1 proteins to chromatin is relieved by phosphoacetylation of histone H3. EMBO Rep 5: 490–496 | Article | PubMed | ISI | ChemPort |

McMahon SB, Van Buskirk HA, Dugan KA, Copeland TD, Cole MD (1998) The novel ATM-related protein TRRAP is an essential cofactor for the c-Myc and E2F oncoproteins. Cell 94: 363–374 | Article | PubMed | ISI | ChemPort |

McMahon SB, Wood MA, Cole MD (2000) The essential cofactor TRRAP recruits the histone acetyltransferase hGCN5 to c-Myc. Mol Cell Biol 20: 556–562 | Article | PubMed | ISI | ChemPort |

Mo X, Kowenz-Leutz E, Xu H, Leutz A (2004) Ras induces mediator complex exchange on C/EBP. Mol Cell 13: 241–250 | Article | PubMed | ISI | ChemPort |

Murr R, Vaissiere T, Sawan C, Shukla V, Herceg Z (2007) Orchestration of chromatin-based processes: mind the TRRAP. Oncogene 26: 5358–5372 | Article | PubMed | ChemPort |

Näär AM, Beaurang PA, Robinson KM, Oliner JD, Avizonis D, Scheek S, Zwicker J, Kadonaga JT, Tjian R (1998) Chromatin, TAFs, and a novel multiprotein coactivator are required for synergistic activation by Sp1 and SREBP-1a in vitro. Genes Dev 12: 3020–3031 | Article | PubMed | ISI | ChemPort |

Näär AM, Taatjes DJ, Zhai W, Nogales E, Tjian R (2002) Human CRSP interacts with RNA polymerase II CTD and adopts a specific CTD-bound conformation. Genes Dev 16: 1339–1344 | Article | PubMed | ISI | ChemPort |

Nagy Z, Tora L (2007) Distinct GCN5/PCAF-containing complexes function as co-activators and are involved in transcription factor and global histone acetylation. Oncogene 26: 5341–5357 | Article | PubMed | ChemPort |

Nowak SJ, Corces VG (2000) Phosphorylation of histone H3 correlates with transcriptionally active loci. Genes Dev 14: 3003–3013 | Article | PubMed | ISI | ChemPort |

Ogryzko VV, Kotani T, Zhang X, Schlitz RL, Howard T, Yang XJ, Howard BH, Qin J, Nakatani Y (1998) Histone-like TAFs within the PCAF histone acetylase complex. Cell 94: 35–44 | Article | PubMed | ISI | ChemPort |

Park J, Kunjibettu S, McMahon SB, Cole MD (2001) The ATM-related domain of TRRAP is required for histone acetyltransferase recruitment and myc-dependent oncogenesis. Genes Dev 15: 1619–1624 | Article | PubMed | ISI | ChemPort |

Pavri R, Lewis B, Kim TK, Dilworth FJ, Erdjument-Bromage H, Tempst P, de Murcia G, Evans R, Chambon P, Reinberg D (2005) PARP-1 determines specificity in a retinoid signaling pathway via direct modulation of mediator. Mol Cell 18: 83–96 | Article | PubMed | ISI | ChemPort |

Pokholok DK, Harbison CT, Levine S, Cole M, Hannett NM, Lee TI, Bell GW, Walker K, Rolfe PA, Herbolsheimer E, Zeitlinger J, Lewitter F, Gifford DK, Young RA (2005) Genome-wide map of nucleosome acetylation and methylation in yeast. Cell 122: 517–527 | Article | PubMed | ISI | ChemPort |

Qiu H, Hu C, Zhang F, Hwang GJ, Swanson MJ, Boonchird C, Hinnebusch AG (2005) Interdependent recruitment of SAGA and Srb mediator by transcriptional activator Gcn4p. Mol Cell Biol 25: 3461–3474 | Article | PubMed | ChemPort |

Robert F, Hardy S, Nagy Z, Baldeyron C, Murr R, Dery U, Masson JY, Papadopoulo D, Herceg Z, Tora L (2006) The transcriptional histone acetyltransferase cofactor TRRAP associates with the MRN repair complex and plays a role in DNA double-strand break repair. Mol Cell Biol 26: 402–412 | Article | PubMed | ISI | ChemPort |

Robert F, Pokholok DK, Hannett NM, Rinaldi NJ, Chandry M, Rolfe A, Workman JL, Gifford DK, Young RA (2004) Global position and recruitment of HATs and HDACs in the yeast genome. Mol Cell 16: 199–209 | Article | PubMed | ISI | ChemPort |

Roberts SM, Winston F (1997) Essential functional interactions of SAGA, a Saccharomyces cerevisiae complex of Spt, Ada, and Gcn5 proteins, with the Snf/Swi and Srb/mediator complexes. Genetics 147: 451–465 | PubMed | ISI | ChemPort |

Sassone-Corsi P, Mizzen CA, Cheung P, Crosio C, Monaco L, Jacquot S, Hanauer A, Allis CD (1999) Requirement of Rsk-2 for epidermal growth factor-activated phosphorylation of histone H3. Science 285: 886–891 | Article | PubMed | ISI | ChemPort |

Sato S, Tomomori-Sato C, Parmely TJ, Florens L, Zybailov B, Swanson SK, Banks CAS, Jin J, Cai Y, Washburn MP, Conaway JW, Conaway RC (2004) A set of consensus mammalian mediator subunits identified by multidimensional protein identification technology. Mol Cell 14: 685–691 | Article | PubMed | ISI | ChemPort |

Shahbazian MD, Grunstein M (2007) Functions of site-specific histone acetylation and deacetylation. Annu Rev Biochem 76: 1–26 | Article | PubMed | ChemPort |

Soloaga A, Thomson S, Wiggin GR, Rampersaud N, Dyson MH, Hazzalin CA, Mahadevan LC, Arthur JSC (2003) MSK2 and MSK1 mediate the mitogen- and stress-induced phosphorylation of histone H3 and HMG-14. EMBO J 22: 2788–2797 | Article | PubMed | ISI | ChemPort |

Taatjes DJ, Marr MT, Tjian R (2004) Regulatory diversity among metazoan co-activator complexes. Nature Rev Mol Cell Biol 5: 403–410 | Article

Taatjes DJ, Näär AM, Andel F, Nogales E, Tjian R (2002) Structure, function, and activator-induced conformations of the CRSP coactivator. Science 295: 1058–1062 | Article | PubMed | ISI | ChemPort |

Taatjes DJ, Tjian R (2004) Structure and Function of CRSP/Med2: a promoter-selective transcriptional co-activator complex. Mol Cell 14: 675–683 | Article | PubMed | ChemPort |

Thomson S, Clayton AL, Hazzalin CA, Rose S, Barratt MJ, Mahadevan LC (1999) The nucleosomal response associated with immediate-early gene induction is mediated via alternative MAP kinase cascades: MSK1 as a potential histone H3/HMG-14 kinase. EMBO J 18: 4779–4793 | Article | PubMed | ISI | ChemPort |

Thomson S, Clayton AL, Mahadevan LC (2001) Independent dynamic regulation of histone phosphorylation and acetylation during immediate-early gene induction. Mol Cell 8: 1231–1241 | Article | PubMed | ISI | ChemPort |

Vassilev LT, Vu BT, Graves B, Carvajal D, Podlaski F, Filipovic Z, Kong N, Kammlott U, Lukacs C, Klein C, Fotouhi N, Liu EA (2004) In vivo activation of the p53 pathway by small-molecule antagonists of MDM2. Science 303: 844–848 | Article | PubMed | ISI | ChemPort |

Wang G, Balamotis MA, Stevens JL, Yamaguchi Y, Handa H, Berk AJ (2005) Mediator requirement for both recruitment and postrecruitment steps in transcription initiation. Mol Cell 17: 683–694 | Article | PubMed | ISI | ChemPort |

Wang X, Yang N, Uno E, Roeder RG, Guo S (2006) A subunit of the mediator complex regulates vertebrate neuronal development. Proc Natl Acad Sci USA 103: 17284–17289 | Article | PubMed | ChemPort |

Wieczorek E, Brand M, Jacq X, Tora L (1998) Function of TAF(II)-containing complex without TBP in transcription by RNA polymerase II. Nature 393: 187–191 | Article | PubMed | ISI | ChemPort |

Winter S, Simboeck E, Fischle W, Zupkovitz G, Dohnal I, Mechtler K, Ammerer G, Seiser C (2008) 14-3-3 Proteins recognize a histone code at histone H3 and are required for transcriptional activation. EMBO J 27: 88–99 | Article | PubMed | ChemPort |

Yamamoto Y, Verma UN, Pajapati S, Kwak Y, Gaynor RB (2003) Histone H3 phosphorylation by IKK-alpha is critical for cytokine-induced gene expression. Nature 423: 655–659 | Article | PubMed | ISI | ChemPort |

Zhou R, Bonneaud N, Yuan CX, de Santa Barbara P, Boizet B, Schomber T, Scherer G, Roeder RG, Poulat F, Berta P, Tibor S (2002) SOX9 interacts with a component of the human thyroid hormone receptor-associated protein complex. Nucleic Acids Res 30: 3245–3452 | Article | PubMed | ISI | ChemPort |

Zhu X, Wiren M, Sinha I, Rasmussen NN, Linder T, Holmberg S, Ekwall K, Gustafsson CM (2006) Genome-wide occupancy profile of Mediator and the srb8-11 submodule reveals interactions with coding regions. Mol Cell 22: 169–178 | Article | PubMed | ChemPort |

Zippo A, De Robertis A, Serafini R, Oliviero S (2007) PIM1-dependent phosphorylation of histone H3 at serine 10 is required for MYC-dependent transcriptional activation and oncogenic transformation. Nat Cell Biol 9: 932–944 | Article | PubMed | ChemPort |

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