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RESEARCH PAPER

RESEARCH PAPER

Transcription 4:1, 39–51; January/February 2013; © 2013 Landes Bioscience

Mediator MED23 regulates basal transcription in vivo via an interaction with P-TEFb Wei Wang,1 Xiao Yao,1 Yan Huang,1 Xiangming Hu,2 Runzhong Liu,2 Dongming Hou,1 Ruichuan Chen2 and Gang Wang1,* State Key Laboratory of Cell Biology; Institute of Biochemistry and Cell Biology; Shanghai Institutes for Biological Sciences; Chinese Academy of Sciences; Shanghai, China; 2 State Key Laboratory of Cellular Stress Biology; School of Life Sciences; Xiamen University; Xiamen, Fujian China

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The Mediator is a multi-subunit complex that transduces regulatory information from transcription regulators to the RNA polymerase II apparatus. Growing evidence suggests that Mediator plays roles in multiple stages of eukaryotic transcription, including elongation. However, the detailed mechanism by which Mediator regulates elongation remains elusive. In this study, we demonstrate that Mediator MED23 subunit controls a basal level of transcription by recruiting elongation factor P-TEFb, via an interaction with its CDK9 subunit. The mRNA level of Egr1, a MED23-controlled model gene, is reduced 4–5 fold in Med23-/- ES cells under an unstimulated condition, but Med23-deficiency does not alter the occupancies of RNAP II, GTFs, Mediator complex, or activator ELK1 at the Egr1 promoter. Instead, Med23 depletion results in a significant decrease in P-TEFb and RNAP II (Ser2P) binding at the coding region, but no changes for several other elongation regulators, such as DSIF and NELF. ChIP-seq revealed that Med23-deficiency partially reduced the P-TEFb occupancy at a set of MED23-regulated gene promoters. Further, we demonstrate that MED23 interacts with CDK9 in vivo and in vitro. Collectively, these results provide the mechanistic insight into how Mediator promotes RNAP II into transcription elongation.

Introduction The transcription cycle of RNA polymerase II (RNAP II) is divided into several distinct stages: pre-initiation, initiation, elongation, and termination. First, RNAP II and other general transcription factors are recruited to the promoter to form a preinitiation complex (PIC). RNAP II then initiates the synthesis of RNA and escapes from the promoter. After moving away from the promoter, RNAP II is able to begin productive elongation with the assistance of a number of elongation factors or complexes. Finally, when a gene is transcribed completely, transcription termination occurs, and the newly synthesized RNA is released from the elongation complex. Among these steps, recruitment of the RNAP II transcriptional machinery to the promoters has been considered to be the principal regulatory step for most genes.1,2 However, increasing evidence suggests that post-recruitment steps are also important for regulating gene expression.3,4 One of these post-recruitment steps is promoter-proximal pausing. A classic example is that of RNAP II paused at the promoter region of the Hsp70 gene in uninduced cells, and can be released to synthesize full-length Hsp70 transcripts upon heat induction.5,6 Promoter-proximal pausing has also been detected in human genes, including c-Myc and the human immunodeficiency virus (HIV).7,8 Recent genome-wide mapping of the distributions of RNAP II in the genomes of human and Drosophila cells has revealed that RNAP II is concentrated not only at the

promoter region of active genes but also at many quiescent genes, suggesting that promoter-proximal pausing is a prevalent regulatory step in metazoans.9-11 Although it is not fully understood how paused RNAP II is controlled, some of the key factors have been studied. DRB sensitivity-inducing factor (DSIF) and the negative elongation factor (NELF) associate with the elongation complex and cause RNAP II to pause closely downstream of the transcription start site (TSS).12,13 The release of paused RNAP II depends on positive transcription elongation factor b (P-TEFb), which phosphorylates DSIF, NELF, and the CTD of RNAP II.14,15 Evidence suggests that several transcription factors are involved in the recruitment of P-TEFb, such as c-Myc,16 NF-kappaB,17 and Brd4.18-20 Moreover, recent studies suggest that the Mediator complex also contributes to the release of paused RNAP II.21,22 The Mediator complex is an evolutionarily conserved multisubunit complex that functions as a molecular bridge linking regulatory signals from transcription factors to the RNAP II transcription apparatus by direct interactions with RNAP II, GTFs and diverse transcription factors.23,24 Through these direct interactions, Mediator is believed to play important roles at multiple stages of transcription, from pre-initiation to termination.25-27 The Mediator MED23 subunit controls the transcriptional activation of Egr1 in mouse embryonic stem (ES) cells.28,29 Egr1 is an early response gene encoding a zinc finger transcription factor that is important for cell growth, cell differentiation,

*Correspondence to: Gang Wang; Email: [email protected] Submitted: 11/09/12; Accepted: 11/12/12 http://dx.doi.org/10.4161/trns.22874 www.landesbioscience.com Transcription

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Keywords: basal transcription, Egr1, elongation, Mediator MED23, P-TEFb

Results Med23 modulates Egr1 basal transcription in vivo without changing RNAP II recruitment. We previously showed that an approximately 3-fold increase in RNAP II binding generated an approximately 13-fold increase in Egr1 mRNA under serum stimulation.29 However, this modest increase in RNAP II binding leading to a dramatic increase in transcription was not easily explained because Med23-dependent recruitment and

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post-recruitment mechanisms were combined together to impact on transcription. To determine the mechanism by which Med23 controls Egr1, we treated WT and KO ES cells with serum for different times after 12-h of depletion of serum from the medium. The Egr1 mRNA level was analyzed by quantitative real-time PCR (Q-RT-PCR), and a constitutively expressed translation elongation factor 2 (EF2) gene was used as an internal control. Consistent with our earlier work,29 Egr1 mRNA level in WT cells increased rapidly after adding serum to the medium, reached a maximum at approximately 30 min, and decreased after 60 min. In KO cells, Egr1 transcriptional level was largely reduced during the course of serum induction (Fig. 1A). Interestingly, before or several hours after serum induction, the “basal” level of Egr1 expression seemed to be reduced several-fold in KO cells to a lower, “sub-basal” level. Here, the term “basal” refers to the basic gene expression in cells under an unstimulated physiological condition, including the steady-state and the serum starvation. In vitro “basal transcription” refers to transcription from a minimal promoter without transcription factor binding sites, which does not exist in the in vivo environment. We then carefully examined mRNA levels of Egr1 gene in both steady-state and serum starvation. Again, we found that Egr1 mRNA level was 5-fold higher in WT than in KO ES cells under both conditions (Fig. 1B). WT and KO mouse embryonic fibroblast (MEF) cells showed similar results (data not shown). Therefore, the presence or absence of Med23 seems to correlate with a higher or lower basal level of Egr1 expression, respectively, in vivo. To test whether the binding of RNAP II to the Egr1 promoter correlates with transcription, we performed ChIP experiments using an antibody against total RNAP II followed by Q-RTPCR. Correlated with the Egr1 expression level, serum addition rapidly increased RNAP II recruitment at the Egr1 promoter in WT cells, whereas the increase in RNAP II binding at the Egr1 promoter in KO cells was greatly attenuated. Surprisingly, the RNAP II occupancy was equal between WT and KO cells before serum stimulation (Fig. 1C) even though the transcription level between the two types of cells exhibited a 5-fold difference (Fig. 1A, 1B). To map the sites of RNAP II and other binding factors more accurately, we scanned the Egr1 promoter region with 5 sets of primers that amplified fragments centered at -836 (amplicon A), -598 (amplicon B), -62 (amplicon C), +366bp (amplicon D), and +674 (amplicon E) relative to the Egr1 transcription start site (TSS). As shown in Figure 1D, RNAP II is primarily enriched at the TSS (amplicon C), and, as shown in Figure 1C, RNAP II occupancy at the Egr1 promoter was not significantly different between WT and KO cells under the unstimulated condition. Thus, we observed that the equal RNAP II binding at the Egr1 promoter in WT and KO cells gave rise to 5-fold different levels of transcription. Med23 deficiency does not alter the occupancies of GTFs at the Egr1 promoter. To understand the discrepancy between RNAP II binding and mRNA levels, we investigated a series of events that occur at different stages of transcription. First, we examined whether PIC assembly at the Egr1 promoter differs between WT and KO cells. Using ChIP assays, we examined different GTFs binding at the Egr1 promoter under the

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and apoptosis.30 The serum-responsive transcription of Egr1 is regulated by serum response factor (SRF) and ELK1, which cooperatively bind to the serum response elements (SREs) in its upstream promoter region.30 MAPK-signaling-activated phosphorylation of ELK1 stimulates Egr1 transcription by recruiting the Mediator complex to the Egr1 promoter through an interaction with the MED23 subunit.28,29 Previously, we found that upon serum induction, Med23 knockout resulted in an approximately 3-fold reduction in the recruitment of preinitiation complexes to the Egr1 promoter. If the relationship between RNAP II recruitment and mRNA synthesis were linear, there would be an approximately 3-fold reduction of Egr1 transcriptional activation. However, the level of Egr1 transcription was actually attenuated approximately 13-fold in Med23 -/- (KO) cells compared with wild-type (WT) cells after serum addition. The observation that the presence of Med23 dramatically enhanced transcription but only modestly increased the recruitment of RNAP II and GTFs led to our proposal of the post-recruitment model: i.e., the Mediator complex stimulates RNAP II activity in addition to its function in recruiting RNAP II machinery. An important criticism of this model is that even a modest enhancement in PIC formation by the Mediator complex may account for a drastic increase in transcription given the possibility that the relationship between RNAP II occupancy and transcription is nonlinear. Therefore, the post-recruitment model needs to be re-examined, and the molecular mechanisms by which the Mediator complex functions in post-recruitment steps remain to be further elucidated. In this study, we observed that under the unstimulated condition, Egr1 basal transcription was reduced 5-fold in KO cells compared with WT cells. However, pre-bound RNAP II, GTFs, ELK1, and Mediator complex occupy the Egr1 promoter equally in both WT and KO ES cells. This result unequivocally demonstrates that PIC formation can be uncoupled from the level of transcription, strongly supporting the post-recruitment function of the Mediator complex in stimulating RNAP II activity. Furthermore, close examination revealed that the binding of the elongating RNAP II at the Egr1 coding region is 50% lower in KO cells than in WT cells, suggesting that the defects resulting from Med23-deficiency likely occur at the elongation stage. CDK9 was the only elongation regulator found to be reduced by 50% at the Egr1 locus, and further investigations revealed that Mediator MED23 interacts with CDK9 in vivo and in vitro. Collectively, our results provide in-depth mechanistic insight for the post-recruitment model; specifically, MED23 interacts with P-TEFb to regulate elongation.

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unstimulated condition. Most of the ChIP signals from GTFs could be detected and peaked near the TSS (amplicon C; Figure 2A-E). Similar to the RNAP II binding, the binding levels of TFIID (indicated by TBP), TFIIA, and TFIIB at the Egr1 promoter in WT cells were equal to those in KO cells (Fig. 2A-C). However, there was no detectable peak for TFIIE and TFIIH binding at the Egr1 promoter in either WT or KO cells (Fig. 2D-E), which may indicate that the binding is below the level of detection or that the binding does not occur under the unstimulated condition. It is unlikely that the antibodies against TFIIE and TFIIH are not appropriate for ChIP, because both factors could be detected after serum addition, and the binding signals peaked at region C after serum addition (Fig. 2D-E). To explore whether TFIIE/TFIIH is required for basal transcription in mouse ES cells, we knocked down TFIIE or TFIIH in the WT and KO mES cells using retroviral RNAi (Fig. 2F-G). We found that Figure 1. Med23 modulates Egr1 basal transcription in vivo without changing RNAP II recruitment. Egr1 mRNA level was largely reduced in (A) Time-course analysis of Egr1 expression after serum addition to serum-starved WT and KO ES both the siTFIIE and siTFIIH WT cells, cells. The expression was normalized to EF2 mRNAs, and the normalized value in serum-starved WT which demonstrates that TFIIE and cells at time 0 was defined as 1. (B) Real-time PCR analysis of the Egr1 expression at steady-state and TFIIH are required for basal Egr1 tranunder serum starvation. The expression was normalized to EF2 mRNA expression, and the normalscription, though their binding to Egr1 ized value in WT cells at steady-state was defined as 1. In this and subsequent figures, results are expressed as the mean ± s.d. of n = 3 independent experiments. (C) Time-course ChIP analysis of are undetectable under the unstimulated RNAP II binding to the Egr1 promoter after serum addition. Values from real-time PCR were normalcondition (Fig. 2F-G). This observation ized to the percentage of input chromatin. (D) ChIP analysis of RNAP II binding to the Egr1 gene is also consistent with early findings that using primers for the indicated fragments. The scale is in base pairs. TFIIE and TFIIH play important roles in promoter clearance and elongation.31,32 However, in the KO cells, when TFIIH or TFIIE was knocked SREs in the Egr1 promoter (Fig. 3A-B). Deletion of Med23 did down, Egr1 mRNA level did not go down much further under not affect the level of ELK1 binding at region B of the Egr1 prothe basal condition (Fig. 2F-G), suggesting that Med23 may be moter between WT and KO cells (Fig. 3A). Likewise, we did also required for the minimal amount of TFIIE/TFIIH to be not observe any difference in phospho-ELK1 binding (Fig. 3B). functional. Based on these results, we conclude that although Mediator complex recruitment was Med23-dependent at the Egr1 there was an approximately 5-fold difference in the unstimulated promoter after serum induction.29 To determine whether the transcription of the Egr1 gene between WT and KO cells, exami- Mediator complex is also Med23-dependent under the unstimunation of the GTFs binding at the Egr1 promoter revealed no lated condition, we performed ChIP assays using two antibodies significant difference. against Mediator subunits MED1 and MED17. Surprisingly, the ELK1 and Mediator occupancies at the Egr1 promoter are not Mediator complex also bound equally to the Egr1 promoter in affected by the presence or absence of Med23. Comparable num- WT cells as in KO cells (Fig. 3C). bers of PICs at the Egr1 promoter between WT and KO cells were Distinct forms of the Mediator complex can be characterable to produce different levels of mRNA under an unstimulated ized by the presence or absence of the CDK8 kinase module.33-35 condition. To better understand this paradox, we investigated CDK8 has been thought to be a transcriptional repressor.36 We whether Med23 depletion affected the binding and the activity previously observed that CDK8 associated with the activated of transcription factor ELK1 to the Egr1 promoter. To address Egr1 promoter,29 which was supported by a recent finding that this question, we performed ChIP experiments using antibod- CDK8 indeed acted as a positive regulator for gene activity.21 ies against ELK1 or its phosphorylation. Unlike RNAP II and Because CDK8 can be either negative or positive for transcripGTFs, which mainly bound to region C at the Egr1 promoter, tion, we investigated whether the binding of CDK8 at the Egr1 ELK1 bound most often to region B, spanning the four clustered promoter may account for the different levels of Egr1 basal

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Because all of these factors exhibited similar binding at the Egr1 promoter, we investigate whether Med23 was relevant to the difference in Egr1 basal transcription. To verify the effects of Med23 on Egr1 basal transcription, the human Med23 gene was reintroduced into KO MEF cells. A stable cell line was selected with hygromycin, and western blot confirmed the re-expression of Med23 in KO cells (Fig. 3E). Indeed, ectopic expression of Med23 rescued Egr1 transcription under both unstimulated and stimulated conditions (Fig. 3F), reinforcing the idea that the presence or absence of MED23 dictates the basal and sub-basal level of Egr1 expression in WT and KO cells, respectively. The interaction between ELK1 and MED23 is necessary for maintaining Egr1 basal transcription. MAPK-phosphorylated ELK1 interacts with MED23 to recruit RNAP II and GTFs for Egr1 activation.28,29 To determine whether phosphorylation of ELK1 is necessary for the basal level of Egr1 transcription, we used an inhibitor of MEK1/2, U0126, to inhibit the phosphorylation of ELK1. After the addition of U0126 to WT cells, the basal level of Egr1 transcription was reduced to a level that was equivalent to the sub-basal level in KO cells (Fig. 4A). Importantly, the sub-basal level of Egr1 transcription did not decrease further when U0126 was added to KO cells (Fig. 4A) because Med23 deletion had already disrupted the transmission of MAPK Figure 2. Med23 deficiency does not alter the occupancies of GTFs at the Egr1 promoter under an signaling to the Egr1 gene transcripunstimulated condition. ChIP experiments were performed using antibodies against TBP (A), TFIIA tion. This observation also suggests (B), TFIIB (C), TFIIE (D) and TFIIH (E). The precipitated DNA was analyzed by real-time PCR with primers that MAPK signaling and Med23 described in Figure 1D. WT+S/KO+S: 30 min after serum addition. (F) and (G), Egr1 mRNA levels in siCtrl are both required and in the same and siTFIIEa/siCdk7 cells. The expression was normalized to EF2 mRNA expression, and the normalized value in siCtrl WT cells was defined as 1. genetic pathway for maintaining the basal level of Egr1 transcription. Surprisingly, we found no obvious transcription between WT and KO cells. Again, we found that difference in RNAP II recruitment to the Egr1 promoter between there was no difference in CDK8 occupancy between the two cell WT and KO cells with or without U0126 addition (Fig. 4B). types (Fig. 3D). These results indicate that under the unstimu- These results suggest that the disruption of the MAPK-ELK1lated condition, activator ELK1, its phosphorylated form, and MED23 axis does not affect RNAP II recruitment; instead, it Mediator complex (including CDK8) at the Egr1 promoter, are may affect other events downstream of PIC formation to control Med23 independent. Thus, the recruitment of RNAP II, GTFs, the basal level of Egr1 transcription. ELK1, and the Mediator complex, including CDK8, may not be To further characterize the interface between MED23 and the reason for the reduced Egr1 expression in KO cells. ELK1 that controls Egr1 basal transcription, two phosphorylated

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sites (S383,389) within the ELK1 activation domain fused to the GAL4 DNA binding domain were mutated to alanine (Fig. 4C). Phosphorylation of the serine 383/389 of the ELK1 activation domain was shown to be required for binding to MED23 and for transcription activation.29,37 We compared the transcriptional activities of wildtype GAL4-ELK1 and GAL4-ELK1(S383,389A) (GAL4-AA) in a transient transfection luciferase assay. Under unstimulated steady-state, the transcriptional activity of GAL4-AA was approximately 12-fold lower than that of GAL4-ELK1 in WT MEF cells (Fig. 4D). However, in KO MEF cells, the transcriptional activities of GAL4-ELK1 and GAL4-AA were comparably low (Fig. 4D), indicating that the activity of ELK1 is dependent on MED23 for sensing its signaling or phosphorylation. Collectively, these data suggest that ELK1 activation and its interaction with MED23 are prerequisites for maintaining basal Egr1 transcription. MED23 is required for RNAP II CTD phosphorylation and RNAP II elongation but not for RNAP II recruitment. Although RNAP II bound equally to the Egr1 promoter, its binding at the Egr1 coding region was lower in KO cells (Fig. 5A), which correlated with the lower transcription level of Egr1. RNAP II CTD plays multiple critical roles in controlling transcription initiation and elongation via its different phosphorylation patterns. For example, Ser5 phosphorylation accompanies the transition from pre-initiation to elongation and helps recruit the 5' capping enzymes.38 Ser2 phosphorylation is implicated in facilitating the transition Figure 3. ELK1 and Mediator occupancies at the Egr1 promoter are not affected by the presence or absence of Med23. ChIP experiments were performed using antibodies of initiating RNAP II into a productive elonagainst ELK1 (A), p-ELK1 (B), the Mediator Complex (Med1 and Med17) (C), and CDK8 (D). gating form in addition to 3' cleavage and poly(E) KO MEF cells were infected with retroviruses encoding hMED23 and subsequently se14,39,40 adenylation. Ser5 phosphorylation peaks lected for hygromycin resistance. Western blotting was used to detect the protein levels near the 5' end of genes, and Ser2 phosphorylaof MED23 in WT, KO, and KO+Med23 cells. The anti-TBP blot was included as an internal tion increases toward the 3' end of genes.41,42 To control. (F) Real-time PCR analysis of the Egr1 expression in states of serum starvation and serum induction (30 min). The expression was normalized to EF2 mRNA expression, further analyze the events downstream of PIC and the normalized value in serum-starved WT cells was defined as 1. formation that are affected by Med23 status, we monitored CTD phosphorylation throughout the Egr1 gene in WT and KO cells under the unstimulated con- elongation, such as DSIF and NELF. Although Ser5-P at the dition. With ChIP assays, we found that depletion of Med23 Egr1 promoter was reduced in KO cells compared with WT cells, clearly reduced the level of Ser5 phosphorylation (Ser5P) at we failed to detect any enriched signal of CDK7 binding at the the Egr1 gene, especially at the transcription initiation region Egr1 promoter under the unstimulated condition (Fig. 2E). By (Fig. 5B). Similarly, Med23 knockout strongly reduces the level examining the occupancies of DSIF and NELF with antibodies of Ser2 phosphorylation (Ser2P) throughout the Egr1 coding against Spt5 and NELF-E, we observed that the binding of DSIF region (Fig. 5C), which explains the lower transcriptional level and NELF to the Egr1 promoter was not affected by MED23 in KO cells. depletion under the unstimulated condition (Fig. 6A and B). In MED23 is partially required for the recruitment of CDK9 contrast, the binding of CDK9 to the Egr1 promoter decreased to the Egr1 promoter. Given the effects of Med23 knockout on by roughly 50% in KO cells, which is consistent with reduced RNAP II CTD phosphorylation, we next analyzed the CTD Ser2 phosphorylation of CTD at the Egr1 coding region, sugkinases CDK7 and CDK9 under the unstimulated condition gesting that Med23 may help recruit CDK9 to the Egr1 promoter in addition to other factors regulating RNAP II pausing and (Fig. 6C). These results suggest that MED23 may target CDK9

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the increase in Egr1 expression. Importantly, CDK9 occupancy at the Egr1 promoter in KO cells was still significantly lower than that in WT cells (Fig. 6G), even when CDK9 was equally overexpressed in both WT and KO cells (Fig. 6E). These results suggest that Med23 is, at least in part, critical for CDK9 recruitment, and the decreased P-TEFb activity at the Egr1 gene upon Med23 depletion may explain the post-recruitment defects in KO cells. Mediator associates with CDK9 via the MED23 subunit in vivo and in vitro. So far, our data suggest that Med23 is important for elongating RNAP II, CTD phosphorylation, and CDK9 recruitment at the Egr1 locus. These observations are consistent with a recent study indicating that the Mediator complex plays a role in RNAP II elongation via Figure 4. The interaction between ELK1 and MED23 is necessary for maintaining Egr1 basal transcription. an interaction with P-TEFb by the (A) The MEK1/2 inhibitor U0126 was added to the culture medium for 30 min, and total RNA samples were CDK8 submodule in an in vitro harvested and analyzed by real-time PCR. (B) WT and KO ES cells were treated or not treated by U0126 for co-IP experiment.21 However, it is 30 min. ChIP assays were performed using RNAP II antibody. (C) The structures of Gal4-Elk1 and Gal4-AA not clear which subunit of this sub(S383,389A) are schematically depicted. The sequences are shown below. D: D-box domain; C: C-box domodule or whether other submodmain. (D) 293T cells were transfected with a 5 × Gal-E1B-TATA-luciferase reporter construct and a plasmid encoding the Gal4-Elk1 activation domain or Gal4-Elk1 mutant, with a MEKK expression plasmid. Firefly ule/subunits directly interact with luciferase activity was normalized to Renilla luciferase activity. P-TEFb. Because the presence or absence of Med23 impacts P-TEFb recruitment and CTD phosphorylation to impact Egr1 basal recruitment, we tested whether MED23 could associate with transcription. CDK9 directly. First, we expressed and purified a double-tag To further verify whether the effects of MED23 depletion soluble His-FLAG-MED23 protein, which was historically difon RNAP II activity at post-recruitment steps are the result of ficult to purify as a solubilized form, from baculovirus-infected decreased P-TEFb binding, we treated cells with the CDK9 insect cells.25,37 Utilizing this soluble recombinant protein, we inhibitor flavopiridol. RNA samples were collected from 90% identified MED23-interacting proteins from HeLa nuclear confluent WT and KO ES cells after 1 h treatment with flavo- extracts by sequential affinity purification (using Ni-NTA beads piridol (250 nM) and analyzed by Q-RT-PCR. The expression and then anti-FLAG M2 beads, respectively; Figure 7A). The of Egr1 was inhibited in WT cells (Fig. 6D). However, the sub- proteins eluted from the anti-FLAG M2 beads were visualized basal level of Egr1 expression in KO cells was reduced to an even by silver staining (Fig. 7B), followed by shotgun mass speclower level, which may have been a response to the inhibition of trometry. Mass spectrometry analysis revealed that CDK9 was the 50% remaining CDK9 at the Egr1 promoter (Fig. 6D). among the MED23-associating proteins. Western blot confirmed Having observed that CDK9 is an effector for Med23- that CDK9 and its partner Cyclin T1 were among the eluted controlled basal Egr1 expression, we overexpressed CDK9 to a proteins associated with MED23, but not with another subunit similar level in both WT and KO cells using retroviral trans- of Mediator MED29 (Fig. 7C). Consistent with the previous duction, and stable cells were selected with puromycin (Fig. 6E). study,21 we also found that CDK9 may also associate with the The expression of Egr1 increased in both WT and KO cells when CDK8 subunit, though whether this association is direct or indiCDK9 was overexpressed. However, CDK9 overexpression could rect was not determined. not overcome the effects of loss of MED23, as the fold differTo further verify the interaction between MED23 and CDK9, ence in Egr1 expression remained the same between WT and KO we went on to perform GST pull down, co-transfection and cells (Fig. 6F). ChIP experiments revealed that CDK9 overex- endogenous immunoprecipitation experiments. GST and GSTpression also increased the occupancy of CDK9 at the Egr1 pro- CDK9 were purified from baculovirus infected insect cells and moter of both WT and KO cells (Fig. 6G), which correlated with immobilized on glutathione agarose to test their interaction with

Discussion Our previous work suggested that Mediator Med23 regulates RNAP II activity at both recruitment and post-recruitment steps upon serum stimulation,29 but the mechanism by which Med23 regulates transcription at a post-recruitment step remains elusive. In the present report, we took a more effective and clearer approach to characterize the function of the Mediator complex in post-recruitment steps. We found that, under the unstimulated condition, Egr1 mRNA level was largely reduced in KO ES cells.

Figure 5. MED23 is required for RNAP II CTD phosphorylation and RNAP II elongation but not RNAP II recruitment. ChIP experiments were performed using antibodies against total RNAP II (A), phosphor-Ser5 CTD (Ser5P) (B), and phospho-Ser2 (Ser2P) (C) at the Egr1 locus using primers as indicated below.

Nevertheless, the amount of basal transcription machinery was not dependent on the recruitment function of Mediator, because the occupancy of RNAP II, TFIIA, TFIIB, TBP, TFIIE, and TFIIH at the Egr1 promoter showed no significant difference between WT and KO cells. Moreover, we did not detect any differences in the occupancy of Mediator complex, activator ELK1 or ELK1 phosphorylation. These data clearly indicate that the

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MED23. As shown in Figure 7D, MED23 was capable of binding to GST-CDK9 (lane 2) but not to GST (lane 1). We further performed a co-immunoprecipitation (co-IP) experiment by cotransfecting tagged Med23 and Cdk9 or its mutant T186A-Cdk9 into 293T cells. Immunoprecipitated of FLAG-MED23 from transfected 293T nuclear extracts revealed that CDK9-HA but little T186A-CDK9-HA could bind to MED23 (Fig. 7E). This indicates that T186 of CDK9 may be important for the interaction between MED23 and CDK9. As T186 of CDK9 is important for its binding to 7SK snRNP and its enzymatic activity,43 we are not certain whether the enzymatic activity of CDK9 is required for its ability in binding to Mediator. Finally, we performed a co-IP experiment to detect the interaction between endogenous proteins. Extracts prepared from WT or KO ES cells were subjected to immunoprecipitation (IP) with anti-MED1 and anti-MED17 antibodies, followed by western blotting. The protein levels of several Mediator subunits, MED1, MED26, CDK8, and MED6, were not altered in input and IP samples from KO ES cells compared with WT ES cells, suggesting that Med23 depletion does not affect most of Mediator integrity. Importantly, endogenous CDK9 and Cyclin T1 were pulled down together with Mediator complex from WT cell extracts, but not from KO cell extracts under various stringent wash conditions (Fig. 7F). Earlier studies found Brd4 associates with Mediator complex,18-21 we found that Brd4 was co-immunoprecipitated with the Mediator complex in WT cell extracts but not in KO cell extracts, suggesting that, similar to CDK9/Cyclin T1, the association of Brd4 with Mediator is also Med23-dependent. Taken together, these data suggest that the Mediator complex may directly interact with P-TEFb via its MED23 subunit to regulate transcriptional elongation, specifically for a basal level of Egr1 expression. To further address the question of whether MED23 affects the recruitment of CDK9 in a genomic scale, we performed a ChIP-seq experiment to compare the CDK9 binding profile in WT and KO ES cells. We first performed a microarray analysis between WT and KO cells under the unstimulated condition and identified 548 genes whose basal level of transcription is reduced at least 1.5-fold in KO cells. ChIP-seq revealed that CDK9 are enriched near the transcription start site (TSS) regions of these genes in WT cells, whereas Med23 depletion largely reduced CDK9 occupancy at the promoter region. CDK9 binding intensity in the gene body was also slightly reduced (Figs. 7G and 7H). Therefore, MED23 affects the basal transcription of a subset of genes by regulating CDK9 occupancy at the promoter region.

amount of pre-poised PIC could be uncoupled from the amount of transcribed mRNA, which highlights the post-recruitment function of Mediator complex. These results further suggest that Med23 depletion may affect transcription beyond PIC formation. In fact, we observed a significant decrease in RNAP II

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and Ser2P occupancy at the coding region and an approximately 50% reduction in CDK9 binding at the Egr1 promoter in KO cells, which may account for the defects in Egr1 transcription. Consistent with these observations, we have demonstrated that MED23 can directly interact with elongation factor CDK9 in

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Figure 6. MED23 is partially required for the recruitment of CDK9 to the Egr1 promoter. ChIP experiments were performed using antibodies against NELF-E (A), Spt5 (B), and CDK9 (C) at the Egr1 locus, as described in Figure 1. (D) Egr1 mRNA levels were measured by real-time PCR in WT and KO ES cells with or without 1 h of flavopiridol (250 nM) pre-treatment. The expression was normalized to the EF2 mRNA expression. (E) WT and KO ES cells were infected with retroviruses encoding CDK9. Stable cell lines were selected for hygromycin resistance, and the CDK9 protein level was analyzed by western blotting. β-actin was used as an internal control. (F) Egr1 mRNA levels were measured by real-time PCR in control- and CDK9-overexpressed WT and KO ES cells. (G) ChIP analysis using an antibody against CDK9 at the Egr1 locus in control- and CDK9-overexpressed WT and KO ES cells. The mean of at least three separate experiments is shown, and the standard deviation is indicated. Student’s t test, **p < 0.001.

©2012 Landes Bioscience. Do not distribute Figure 7. For figure legend, see page 10.

vitro and in vivo. These results provide a mechanistic explanation for how MED23 regulates transcription elongation.

The Mediator complex is crucial for basal transcription in vitro23 but, in vivo, evidence has been absent. Here, we provide

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evidence that in vivo Egr1 basal transcription is dependent on Med23. When Med23 is absent, transcription of Egr1 is reduced to a sub-basal level. The human Mediator complex may enhance basal transcription by facilitating recruitment of TFIIB during PIC assembly.44 However, in the present study, we did not observe any defects in the recruitment of TFIIB, TFIIE or TFIIH in KO cells, which may reflect a discrepancy between in vivo and in vitro. In contrast, we found that depletion of Med23 affected the elongation stage of transcription. The Mediator complex has been suggested to play key roles at multiple stages of eukaryotic transcription,23,24,45 including the elongation stage. Using Med23-/- cells, we discovered that the Mediator complex regulates Egr1 transcription at both recruitment and post-recruitment steps under serum induction.29 The Mediator CDK8 kinase module has also been implicated in recruiting P-TEFb to facilitate transcriptional activation of serum response genes21 and the thyroid hormone receptor target gene DioI.46 Consistent with the findings of the Espinosa’s lab,21 we also found that CDK9 associates with His-FLAG-CDK8 (Fig. 7A). Another subunit, MED26, has been shown to function as a docking site for super-elongation complexes containing ELL/EAF and P-TEFb and, therefore, to regulate a small set of MED26-controlled gene elongation.22 Now, we show that Mediator subunit MED23 recruits P-TEFb to promote the transition of RNAP II into the elongation stage by directly interacting with CDK9. These results are not necessarily conflicting with each other as Mediator may make multiple contacts with any given factor including RNAP II and P-TEFb, and multiple mechanisms could be utilized in regulating transcription elongation. Considering the multiple components and the huge size of the Mediator complex, it is conceivable that the Mediator complex could be involved in diverse protein interactions at the distinct stages of the transcriptional process. Promoter-proximal pausing is a general feature of transcription by RNAP II in metazoan cells.9-11,16 DSIF and NELF function together to cause pausing.47 Release of the paused RNAP II is mediated by P-TEFb, which overcomes the negative effects of NELF and DSIF.14 Multiple mechanisms are involved in regulation of P-TEFb. In mammals, P-TEFb is negatively regulated by 7SK RNA and HEXIM1, which form a complex to inhibit P-TEFb kinase activity.43,48,49 In contrast, 7SK/HEXIM1-bound P-TEFb is converted into an active form through its association with bromodomain protein Brd4.18,19 In addition to Brd4, other activators are responsible for recruiting P-TEFb to target genes,

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such as DNA-bound activator NF-kappaB,17 c-Myc,16,50,51 and RNA-bound activator HIV Tat.52 Recently, P-TEFb has been shown to associate with Mediator complex via CDK8 sub-module21 or MED26.22 However, these studies failed to demonstrate which subunit of the Mediator complex interacts with P-TEFb directly or via other co-factors indirectly. We performed both in vitro and in vivo experiments in this study to demonstrate the interaction between the Mediator complex and P-TEFb and revealed a physical interaction between MED23 and CDK9. Importantly, we also noticed that the MED23-CDK9 interaction may not be solely responsible for P-TEFb recruitment or its function in elongation, because deletion of Med23 resulted in only a 50% reduction in CDK9 binding at the Egr1 promoter and modestly decreased Egr1 transcription to the sub-basal level. Consistent with the idea that CDK9 could have a Med23independent function, inhibition of CDK9 by flavopiridol in KO cells can further reduce Egr1 transcription to a much lower level than sub-basal transcription, presumably because flavopiridol directly inhibits the kinase activity of P-TEFb. Therefore, there may be other Med23-independent mechanisms by which the Mediator complex recruits P-TEFb via other subunits or factors, such as MED26, CDK8, and Brd4. Collectively, the Mediator complex may utilize multiple mechanisms to regulate P-TEFb for paused RNAP II release. Materials and Methods Cell lines and reagents. WT and KO28 mouse ES cells were cultured in Knockout DMEM (Invitrogen) with 15% fetal bovine serum (Hyclone), 2 mM L-glutamine (Invitrogen), 50 μg/ml pen/strep (Invitrogen), 0.1 mM non-essential amino acids (Invitrogen), 10-4 M β-mercaptoethanol, 1000 U/ml LIF (Chemicon). Cells were grown on plates treated with 0.2% gelatin. WT and KO37 MEFs were cultured in DMEM (Hyclone) with 10% fetal bovine serum (Hyclone). 293T cells were grown in the same medium. Plasmids, retroviral infection, and luciferase assay. The pMSCV-hMed23 plasmid has been described previously.37 The mouse CDK9 cDNA was amplified by PCR from mouse ES cDNA, subcloned into the pMSCV-puro and pGEX-4T-1 vectors, and verified by sequencing. Ser-383 and -389 within the Elk1 activation domain of Gal4-Elk1 (307–428) were mutated by site-directed mutagenesis to alanine residues using the KOD Hot Start DNA Polymerase kit (TOYOBO).

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Figure 7. MED23 interacts with CDK9 in vitro and in vivo. (A) Scheme for tandem affinity purification and MS/MS to identify Mediator subunit binding proteins. (B) Baculovirus-expressed His-Flag-MED23 (M23), His-Flag-CDK8 (CDK8) and His-Flag-MED29 (M29) were purified by Ni-NTA beads and then FLAG M2 beads sequentially, as described in (A). The proteins eluted from FLAG M2 beads were analyzed by SDS- PAGE followed by silver staining. (C) The proteins eluted from FLAG M2 beads were analyzed by SDS- PAGE followed by immunoblotting with antibodies against CDK9 and Cyclin T1. (D) Soluble His-Flag-MED23 was incubated at 4 degree overnight with immobilized GST or GST-CDK9. After washing, the bound proteins were eluted by boiling and immunoblotted with the indicated antibodies. (E) Flag-Med23 plasmid was co-transfected with HA-cdk9 or its mutant HA-cdk9 (T186A) into 293T cells. Whole-cell extracts were used for immunoprecipitation with the anti-FLAG M2 beads, followed by immunoblotting using antibodies against Tubulin, FLAG or HA. (F) Nuclear extracts prepared from wild type (WT) or Med23-/- (KO) mouse embryonic stem cells were subjected to co-IP with MED1 and MED17 antibodies under various stringent wash conditions as indicated. The immunoprecipitated proteins were detected with indicated antibodies by immunoblotting. (G) ChIP-seq analysis of CDK9 enrichment on the set of genes whose expression level are regulated by MED23, in WT (black) and KO (gray) ES cells. All these genes were normalized to 3 kb for Mata-gene, with 1 kb extended upstream from TSS, and 1 kb downstream from TTS for analyzing average profile in 50 bp bins. (H) Quantitation of CDK9 binding intensity on promoter region (1 kb upstream from TSS) and gene body region (from TSS to TTS). Both are normalized to the values derived from the WT ES cells.

For genome-wide gene expression analysis, microarray was performed using Affymetrix Mouse 430 2.0 array. Six arrays were probed with cDNA prepared from total RNAs isolated from WT and KO ES cells under the normal growth condition without stimulation. Co-immunoprecipitation (co-IP). WT and KO ES cells grew to 95–100% confluence, were harvested and washed by cold PBS, and then lysed in 1 mL lysis buffer (1 × PBS, 0.1% NP-40 (v/v), 5 mM EDTA, 5 mM EGTA, and freshly added proteinase inhibitor from Roche). After a brief sonication, the lysates were centrifuged at 12,000 rpm for 10 min. The supernatant was added with antibodies (2 μg each), and incubated at 4°C overnight. 40 μl Protein G beads were added, and after 2 h incubation, the beads were washed 3 times with lysis buffer, and then boiled in SDS loading buffer and analyzed by western blot with the indicated antibodies. Production of soluble recombinant H-F-MED23, H-FMED29, and H-F-CDK8 and tandem affinity purification. DNA sequences encoding Med23, Med29 and Cdk8 were amplified by PCR and cloned into pFASTBac-HTc, and the Flag sequence was added to the N terminus to generate the N-terminal 6xHis-Flag-tagged fusion. The baculovirus expressing His-FlagMED23, as well as His-Flag-MED29 and His-Flag-CDK8, was generated using the Bac-to-Bac baculovirus expression system (Invitrogen) as described.37 For tandem affinity purification, 1 ml undialyzed HeLa nuclear extract (4 mg/ml) prepared using standard procedures33 was diluted with an equal volume of 300 mM KCl D buffer (20 mM Hepes, pH 7.9, 300 mM KCl, 20% (v/v) glycerol, 1 mM PMSF, and 10 mM β-mercaptoethanol) plus 0.1% NP-40 and incubated with the Ni-MED23, Ni-MED29 or Ni-CDK8 column (200 μl beads each) in low flow speed for five times at 4°C. The columns were washed twice with 1ml 300 mM KCl D buffer (20 mM Hepes, pH 7.9, 300 mM KCl, 20% (v/v) glycerol, 1 mM PMSF, and 10 mM β-mercaptoethanol) plus 0.1% NP-40 and twice with 1 ml 500 mM KCl D buffer (20 mM Hepes, pH 7.9, 500 mM KCl, 20% (v/v) glycerol, 1 mM PMSF, and 10 mM β-mercaptoethanol) plus 0.1% NP-40. Then the resins were washed in 1 ml of 20 mM imidazole washing buffer (50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole) and eluted with 3 ml of 250 mM imidazole elution buffer (50 mM NaH2PO4, 300mM NaCl, 250mM imidazole) as E1 fraction. E1 fraction plus protease inhibitor (Roche) was then incubated with 100 μl Anti-Flag M2 Affinity Gel (Sigma-Aldrich) overnight at 4°C. The resins were washed with TBS three times and eluted with with 3 × Flag peptide (Sigma-Aldrich, 150 ng/μl final concentration) in 100 μl TBS with gentle shaking for 30 min at 4°C. The eluted proteins were boiled in SDS loading buffer and analyzed by western blotting with the indicated antibodies. Production of GST fusion proteins and GST pull down. DNA sequence encoding cdk9 was amplified by PCR and cloned into pGEX-4T-1. E. coli BL21 cells were transformed with plasmids encoding the glutathione S-transferase (GST) fusion protein or GST alone, grown at 30°C until the optical density at 600 nm reached a value of 0.6 to 0.8, and induced with 100 μM isopropyl-β-d-thiogalactosidase for 2 h. Bacteria were collected

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Establishing stable cell lines to overexpress a gene of interest was previously described.37 Twenty-four hours after infection, MEFs were selected with 50 μg/ml puromycin, and ES cells were selected with 5 μg /ml puromycin. WT and KO MEF cells at 90% confluency (in 12-well plates) were transfected with a 5 × Gal-E1B-TATA-luciferase reporter construct (250 ng per well) and the Gal4-Elk1 or Gal4-AA mutant (50 ng per well). Additionally, a plasmid expressing Renilla luciferase was cotransfected (50 ng per well). Transfections were performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s recommendations. At 48 h post-transfection, the cells were lysed, and the luciferase expression was quantified using the dual luciferase assay system (Promega). The Firefly luciferase activity was normalized to Renilla luciferase activity. Serum induction and signaling inhibition. WT and KO ES cells were plated on gelatinized plates. After the cells reached to 85–95% confluence, media was changed to 0% serum for more than 12 h. 30% serum was added for the indicated times before RNA preparation or ChIP assay. For signaling inhibition, the cells grew to 85–95% confluence, and were treated with U0126 for 30 min or P-TEFb inhibitor flavopiridol for 60 min before RNA preparation or ChIP assay. The final concentration of inhibitors in the medium are as follows: U0126 10 μM, flavopiridol 250nM. RNA extraction, Real-Time PCR, and western blot. Total RNA was isolated from cells using TRIZOL (Invitrogen). The first-strand cDNA was generated using MMLV transcriptase (Promega), and real-time PCR was performed using a SYBR Green PCR master mix (Takara) in an Eppendorf Mastercycler. All values were normalized to the level of EF2 mRNA, which is constitutively expressed and not changed during the time course of the experiments. Primer sequences used in the experiments are available on request. The western blot was performed using an ECL kit (Pierce) based on the manufacturer’s recommendations. HRP-conjugated secondary antibodies were purchased from the Jackson Laboratory. Chromatin Immumoprecipitation (ChIP) and ChIP-seq analysis. ChIP assays were performed as described previously (Wang et al., 2005). The immunoprecipitated DNA was quantified using real-time PCR. All values were normalized to the input. Primer sequences used in the ChIP experiments are available upon request. For ChIP-seq, ChIP DNA was quantified by Qubit Fluorometer (Invitrogen). DNA was purified using ChIP-seq sample prep kit (Illumina) and subjected to 76 bases of sequencing on Genome Analyzer IIx (Illumina). For ChIP-seq analysis, all single-read sequencing reads were aligned by Bowtie program (http://bowtie-bio.sourceforge.net/index.shtml) and the parameters are: -v1,–best,–strata, -k2, -m1. Peak calling was performed using MACS,53 with the following settings: bw = 300, tsize = 75, and p-value threshold of 10–5. To perform the average gene profile, BED and WIG files from MACS output were used in CEAS.54 ChIP-seq analysis was conducted based on the mouse genome built on July 2007 (NCBI37/mm9).

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(sc-292), NELF-E (sc-32912), SPT5 (sc-28678), CDK9 (sc8338), and GST (sc-459) antibodies were purchased from Santa Cruz Biotechnology. MED23 antibody was purchased from BD PharMingen. β-actin and FLAG antibodies were purchased from Sigma-Aldrich. Ser5P (ab5131) and Ser2P (ab5095) antibodies were purchased from Abcam. CDK8 antibody was purchased from Neomarkers. Brd4 antibodies were kindly provided by Dr. Cheng-Ming Chiang. Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed. Acknowledgments

We thank our colleagues for discussion and critically reading the manuscript. This work was supported in part by Grants from CAS (XDA01010401), China MOST (2009CB941100 and 2011CB510104), and CNSF (81030047 and 30770452) to GW. GW is a scholar of the “Hundred Talent Program.”

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by centrifugation, resuspended in PBS, lysed by sonication, and mixed with TritonX-100 (1% final concentration). After the cell debris was removed by centrifugation, GST fusion proteins were adsorbed to glutathione-agarose beads (Sigma). The beads were collected and rinsed in PBS. The fusion proteins were eluted with 10 mM glutathione in 50 mM Tris (pH 8.0) and dialyzed against PBS. GST and GST-CDK9 fusion proteins were immobilized on glutathione agarose beads, blocked with PBS containing 0.1% NP-40 (v/v), 5 mM EDTA, and protease inhibitor plus 5% nonfat milk, and incubated with the same amount H-F-MED23 for 2 h at 4°C. After washing with PBS containing 0.1% NP-40 (v/v) five times, the bound proteins were dissolved in SDS loading buffer, separated by SDS-PAGE, and subjected to western blot analysis. Antibodies. Elk1(sc-355), phospho-Elk1(sc-8406), MED1 (sc-5334), MED17 (sc-12453), RNAP II (sc-898), TBP (sc273), TFIIA (sc-5316), TFIIB (sc-225), TFIIE (sc-237), TFIIH

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