BET Bromodomain Inhibition Releases the Mediator Complex from Select cis-Regulatory Elements

Report

BET Bromodomain Inhibition Releases the Mediator Complex from Select cis-Regulatory Elements Graphical Abstract

Authors Anand S. Bhagwat, Jae-Seok Roe, Beverly Y.L. Mok, Anja F. Hohmann, Junwei Shi, Christopher R. Vakoc

Correspondence [email protected]

In Brief In this study, Bhagwat et al. show that the Mediator complex and BRD4 are linked coactivators that support gene-specific transcriptional activation in leukemia cells. They provide evidence that smallmolecule inhibitors of BRD4 exert antileukemia effects by interfering with Mediator function to suppress transcription.

Highlights d

BET inhibitors release the Mediator complex from specific enhancers and promoters

d

Mediator eviction correlates with transcriptional changes caused by JQ1

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Genetic knockdown of specific Mediator subunits phenocopies BRD4 inhibition

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BRD4 and Mediator maintain expression of a common gene regulatory network

Bhagwat et al., 2016, Cell Reports 15, 519–530 April 19, 2016 ª2016 The Authors http://dx.doi.org/10.1016/j.celrep.2016.03.054

Accession Numbers GSE74651 GSE74536 GSE78221 GSE74650 GSE78224

Cell Reports

Report BET Bromodomain Inhibition Releases the Mediator Complex from Select cis-Regulatory Elements Anand S. Bhagwat,1,2 Jae-Seok Roe,1 Beverly Y.L. Mok,1 Anja F. Hohmann,1 Junwei Shi,1,3 and Christopher R. Vakoc1,* 1Cold

Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA of Molecular Genetics and Microbiology 3Molecular and Cellular Biology Program Stony Brook University, Stony Brook, NY 11794, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.celrep.2016.03.054 2Department

SUMMARY

The bromodomain and extraterminal (BET) protein BRD4 can physically interact with the Mediator complex, but the relevance of this association to the therapeutic effects of BET inhibitors in cancer is unclear. Here, we show that BET inhibition causes a rapid release of Mediator from a subset of cis-regulatory elements in the genome of acute myeloid leukemia (AML) cells. These sites of Mediator eviction were highly correlated with transcriptional suppression of neighboring genes, which are enriched for targets of the transcription factor MYB and for functions related to leukemogenesis. A shRNA screen of Mediator in AML cells identified the MED12, MED13, MED23, and MED24 subunits as performing a similar regulatory function to BRD4 in this context, including a shared role in sustaining a block in myeloid maturation. These findings suggest that the interaction between BRD4 and Mediator has functional importance for gene-specific transcriptional activation and for AML maintenance. INTRODUCTION The bromodomain and extraterminal (BET) protein BRD4 is a therapeutic target in several human malignancies, including acute myeloid leukemia (AML) (Shi and Vakoc, 2014). While the efficacy of BET inhibitors in mouse models has motivated ongoing clinical trials in hematologic malignancies (e.g., https:// clinicaltrials.gov: NCT01713582), the underlying molecular mechanism of BRD4 function in supporting cancer progression remains poorly understood. BRD4 uses tandem bromodomain modules to recognize acetyl-lysine side chains on histones and transcription factors (TFs), thereby localizing to hyper-acetylated promoter and enhancer regions of the genome (Dey et al., 2003; Roe et al., 2015). Moreover, chemical inhibitors of BET bromodomains (e.g., JQ1 and IBET) cause a global release of BRD4 from the genome (Filippakopoulos et al., 2010; Nicodeme et al., 2010). When bound to chromatin, BRD4 recruits various proteins, including P-TEFb, JMJD6, and NSD3, to activate its target genes

(Jang et al., 2005; Liu et al., 2013; Rahman et al., 2011; Shen et al., 2015; Yang et al., 2005). Proteomic analyses of BRD4 complexes have revealed numerous other associated factors (Dawson et al., 2011; Jang et al., 2005; Rahman et al., 2011); however, the relevance of such interactions to the cancer maintenance function of BRD4 is largely unstudied. A physical association between the Mediator complex and BRD4 has been shown in several prior studies (Donner et al., 2010; Jang et al., 2005; Jiang et al., 1998; Wu and Chiang, 2007). Mediator is a 30-subunit coactivator complex that interacts with TFs and participates in the recruitment and activation of RNA polymerase II (Pol II) (Allen and Taatjes, 2015; Malik and Roeder, 2010). Because the precise binding surface that links BRD4 and Mediator has yet to be defined, the functional importance of this physical interaction is currently unclear. In support of a functional link between BRD4 and Mediator, it has been observed that both factors colocalize at super-enhancers (clusters of highly active enhancers) and BET inhibition can perturb BRD4 and Mediator occupancy at such sites (Di Micco et al., 2014; Love´n et al., 2013). In addition, embryonic stem cells require both BRD4 and Mediator to maintain Oct4 expression and the pluripotent cell state (Di Micco et al., 2014; Kagey et al., 2010; Wu et al., 2015). However, a recent study has shown that the kinase subunits of Mediator (CDK8 and CDK19) function in opposition to BRD4 to repress super-enhancer-associated genes (Pelish et al., 2015). Taken together, these prior studies raise two key questions: (1) at what locations of the genome is Mediator released following BET inhibitor treatment? and (2) does perturbation of Mediator contribute to the transcriptional effects and therapeutic activity of BET inhibition in cancer and other diseases? Here, we show that JQ1 causes a dramatic loss of Mediator occupancy at a subset of cis elements in the genome of AML cells, which only partially overlaps with the location of superenhancers. Notably, Mediator eviction tracked closely with the sensitivity of gene expression to JQ1, which suggests that release of Mediator from the genome contributes to the transcriptional effects of BET inhibition. In support of this model, a Mediator-focused short hairpin RNA (shRNA) screen performed in AML cells revealed that BRD4 and Mediator coordinate a common gene-regulatory network that maintains a blocked state of differentiation. Because Mediator is preferentially evicted by JQ1 near genes that promote leukemogenesis, our findings

Cell Reports 15, 519–530, April 19, 2016 ª2016 The Authors 519 This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

implicate release of Mediator from the genome as a contributor to the therapeutic activity of BET inhibition in AML. RESULTS The Mediator Complex Is Released from the Leukemia Genome in a Variable Manner following JQ1 Exposure We tested the hypothesis that BET inhibition with JQ1 elicits antileukemia effects by interfering with the Mediator complex. To this end, we first performed chromatin immunoprecipitation sequencing (ChIP-seq) analysis comparing the chromatin occupancy profiles of BRD4 and MED1 (a Mediator subunit) in cells derived from a mouse model of MLL-AF9;NrasG12D AML (the RN2 cell line; Zuber et al., 2011a). This revealed that BRD4 and MED1 colocalized across the AML genome in a pattern that overlapped with H3K27 hyper-acetylation (Figure 1A). Moreover, the tag counts of MED1 and BRD4 at each individual peak were highly correlated (R2 = 0.91; Figure S1A). The close correlation between BRD4 and MED1 across the AML genome is consistent with a physical interaction between these regulators occurring in this cell type. Next, we performed ChIP-seq analysis of BRD4 and MED1 in RN2 cells following a 2-hr exposure to 500 nM JQ1 or to vehicle control (DMSO). Transcription of the Myc proto-oncogene in RN2 cells is highly sensitive to JQ1 and is regulated by BRD4 via a super-enhancer (also known as E1–E5) located 1.7 megabases downstream of the Myc promoter (Shi et al., 2013; Zuber et al., 2011c). As expected, we found that BRD4 was completely evicted from this location by JQ1 (Figure 1B). In addition, we observed an equally dramatic reduction of MED1 occupancy at this same region (Figure 1B). To ensure that the entirety of the Mediator complex was released from this site, we complemented our ChIP-seq studies with ChIP-qPCR analysis of BRD4, MED1, MED12, and MED23, which revealed parallel reductions of all four factors at the Myc super-enhancer following JQ1 treatment (Figures S1B–S1G). Because MED1, MED12, and MED23 reside in distinct modules of Mediator (middle, kinase, and tail, respectively), this result suggests that the entire Mediator complex was released following BET bromodomain inhibition. Because prior studies have shown that BET inhibition reduces MED1 occupancy at several super-enhancers (Di Micco et al., 2014; Love´n et al., 2013), we considered whether the release of Mediator from the genome in RN2 cells was restricted to this class of elements. For this purpose, we applied the rank ordering of super-enhancers (ROSE) algorithm to MED1 ChIPseq data to define super-enhancers in RN2 cells and inspected the sensitivity of MED1 at these locations to JQ1-mediated eviction (Figure 1C). Cdk6 has previously been implicated as a BRD4 target gene in AML and is regulated by an intronic superenhancer (Dawson et al., 2011; Roe et al., 2015). Similar to the Myc locus, we observed reductions of both BRD4 and MED1 at the Cdk6 super-enhancer following BET inhibition (Figure 1D). However, we unexpectedly observed that not all super-enhancers were susceptible to JQ1-mediated MED1 eviction. The Lrrfip1 gene harbors a promoter-proximal super-enhancer that displayed no apparent reduction of MED1 following exposure to JQ1 (Figures 1C and 1E). As another example, a large

520 Cell Reports 15, 519–530, April 19, 2016

domain of MED1 occupancy at the HoxA locus qualifies as a super-enhancer (Figure 1C) and yet is entirely unaffected by JQ1 (Figure 1F). Conversely, we found that MED1 was released from several regions that fell below the threshold of being called as super-enhancers, such as intronic locations of Mgat5 (Figures 1C and 1G). Across all genomic sites, we observed a weak correlation between the magnitude of MED1 and BRD4 release following JQ1 exposure (R2 = 0.13; Figure S1H). However, we also identified a small subset of sites that exhibit severe decreases of both MED1 and BRD4 following JQ1 treatment (Figure S1H). Collectively, these findings show that JQ1 causes variable Mediator release at cis-regulatory elements in AML cells. JQ1-Induced Mediator Eviction Correlates with Transcriptional Suppression Next, we sought to determine whether MED1 eviction was correlated with the gene expression changes caused by JQ1. 10,604 reproducible MED1 peaks were defined using model-based analysis of ChIP-seq (MACS) software and rank ordered based on the average fold change of MED1 tag counts following exposure to JQ1 in two biological replicates (Zhang et al., 2008). This revealed that the constituent MED1 peaks within the Myc superenhancer were outliers in the RN2 genome with regard to the severity of MED1 loss (Figure 2A). This result was striking, because Myc is also among the most-downregulated mRNAs in AML cells after JQ1 treatment (Dawson et al., 2011; Zuber et al., 2011c; Figure 2B). Furthermore, the magnitude of MED1 loss at peaks located near Cdk6, Lrrfip1, Hoxa9, and Mgat5 correlated well with the relative effect of JQ1 on expression of these genes (Figures 2A and 2B). This analysis raised the possibility that release of Mediator from the genome may contribute to the effect of BET inhibition on transcription. To test this hypothesis in a systematic manner, we defined 200 MED1 peaks in RN2 cells that exhibited the greatest sensitivity to JQ1 (Figures 2A and S2A; Table S1). We next applied the genomic regions enrichment of annotations tool (GREAT) to link each of these peaks to the nearest expressed gene as its presumed target (McLean et al., 2010; Table S2). We performed an analogous GREAT analysis to link each superenhancer in RN2 cells to the nearest expressed gene (Figure 1C; Table S2). These two gene sets were then independently evaluated in RNA-seq data derived from RN2 cells treated with 500 nM JQ1 for 6 hr (Roe et al., 2015). Consistent with prior observations, we found that super-enhancer genes were more suppressed, on average, by JQ1 than randomly chosen expressed genes (Figure 2C; Love´n et al., 2013). However, we found that genes located near JQ1-sensitive MED1 peaks were suppressed to a significantly greater extent than genes located in proximity to super-enhancers (Figures 2C). This result prompted us to consider whether super-enhancers encompass two distinct classes of cis elements that differ with regard to their sensitivity to JQ1-mediated perturbation. Indeed, we found that 75 of the 178 super-enhancers in RN2 cells overlapped with at least one JQ1-sensitive MED1 peak (Figure 2D), and only this subset of super-enhancers was associated with transcriptional suppression by JQ1 (Figure 2E). Importantly, this result suggests that a large fraction of super-enhancers (103 out of 178) are not

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Figure 1. The Mediator Complex Is Released from the AML Genome in a Variable Manner following JQ1 Exposure (A) Density plot of BRD4, MED1, and H3K27Ac ChIP-seq datasets in murine MLL-AF9/NrasG12D AML. Data are centered on 5,135 high-confidence BRD4occupied elements (Roe et al., 2015). Each row represents a 10-kilobase interval surrounding a single peak. (B) ChIP-seq occupancy profiles of BRD4 and MED1 at the Myc locus following a 2-hr treatment with DMSO (vehicle) or 500 nM JQ1. (C) 178 super-enhancers defined by MED1 occupancy using the ROSE algorithm. (D) ChIP-seq occupancy profiles of BRD4 and MED1 at the Cdk6 locus following a 2-hr treatment with DMSO or 500 nM JQ1. (E) ChIP-seq occupancy profiles of BRD4 and MED1 at the Mgat5 locus following a 2-hr treatment with DMSO or 500 nM JQ1. (F) ChIP-seq occupancy profiles of BRD4 and MED1 at the Lrrfip1 locus following a 2-hr treatment with DMSO or 500 nM JQ1. (G) ChIP-seq occupancy profiles of BRD4 and MED1 at the HoxA cluster following a 2-hr treatment with DMSO or 500 nM JQ1. See also Figure S1 and Table S4.

Cell Reports 15, 519–530, April 19, 2016 521

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522 Cell Reports 15, 519–530, April 19, 2016

perturbed by JQ1 and appear to be unrelated to the transcriptional effects of BET inhibition. Next, we evaluated whether JQ1-sensitive MED1 peaks harbor unique sequences features. A set of hematopoietic TFs (C/EBPa, C/EBPb, ERG, FLI1, MYB, and PU.1) has been shown to facilitate BRD4 recruitment in AML cells (Roe et al., 2015). When compared to all MED1 peaks, JQ1 sensitivity was associated with a higher motif density and genomic occupancy of MYB and, to a lesser extent, C/EBPa and C/EBPb (Figures 2F, S2C, and S2D). In contrast, sequence motifs and overall occupancy for PU.1, ERG, and FLI1 were not dramatically elevated at JQ1-sensitive versus JQ1-insenstive MED1 peaks. We also evaluated whether the genes located near JQ1-sensitive MED1 peaks were enriched for any particular biological or molecular pathways. For this purpose, we used GREAT to compare the ontology of genes located near 200 JQ1-sensitive MED1 peaks with genes located near 200 random MED1 peaks. This revealed that Mediator was released disproportionately near genes involved in leukemia pathogenesis (Figure 2G). In this same analysis, MYB was the most-enriched TF network among the JQ1sensitive MED1 peaks, in agreement with the higher relative level of MYB occupancy at this class of cis elements (Figures 2F and 2G). These results suggest that Mediator is preferentially evicted by JQ1 near MYB targets genes and near genes involved in the pathogenesis of leukemia. Knockdown of Select Mediator Tail and Kinase Module Subunits Triggers Differentiation of Leukemic Blasts The genomic correlations described above support a model in which BRD4 and Mediator function as linked coactivators for a common set of target genes in AML cells. One prediction of this model is that targeting of specific subunits of Mediator may lead to similar cellular phenotypes and transcriptional changes as observed when targeting BRD4. Inhibition of BRD4 with either JQ1 or shRNA impairs the proliferation of RN2 cells and triggers differentiation of leukemic blasts into macrophage-like cells, which express higher levels of Mac1 and lower levels of cKit on their cell surface (Zuber et al., 2011c). To evaluate the phenotypic consequences of Mediator perturbation in

this cell type, we constructed a custom library of 190 shRNAs targeting all of the known Mediator subunits (approximately six shRNAs/gene; Table S3) and carried out a negative-selection screen to identify essential subunits for cell proliferation and/or viability. The relative growth arrest observed over 10 days in culture was quantified using GFP reporters in a competition-based assay (Figure 3A). This screen revealed that RN2 cell proliferation was hypersensitive to targeting of select subunits of the Mediator complex. This includes components of the head (MED8, MED28, and MED30), middle (MED9 and MED26), tail (MED16, MED23, MED24, and MED25), and kinase (MED12 and MED13) modules (Figures 3A and 3B). On-target knockdown of the intended target gene of each scoring shRNA was validated using qRT-PCR (Figure S3A). These findings confirm that several Mediator subunits are essential for AML proliferation. To evaluate the specificity of these growth-arrest phenotypes, we transduced immortalized mouse embryonic fibroblast cells (iMEFs) with shRNAs targeting each of the essential Mediator subunits identified in the screen above and performed competition-based proliferation assays. We found that the knockdown of MED12, MED13, MED23, and MED24 led to no apparent growth defect in iMEFs, whereas MED8 and MED30 were each found to be essential for iMEF growth (Figures S3B and S3C). This heterogeneity of growth-arrest phenotypes following perturbation of individual Mediator subunits was unrelated to the level of expression of these subunits (data not shown). Collectively, our findings suggest that AML cell proliferation is uniquely hypersensitive to targeting of Mediator subunits MED12, MED13, MED23, and MED24. Interestingly, MED12 and MED23 have each been implicated previously in the physical interaction with BRD4 (Jang et al., 2005; Wang et al., 2013). Next, we evaluated whether knockdown of specific Mediator subunits in RN2 cells caused myeloid maturation in an analogous manner to BRD4 inhibition (Zuber et al., 2011c). When expressed conditionally via a doxycycline (dox)-regulated promoter, we found that MED12, MED13, MED23, and MED24 shRNAs, but not MED8 and MED30 shRNAs, altered the cell surface expression of cKit and Mac1 in a manner that resembled the phenotype provoked by BRD4 knockdown (Figures 3C and 3D). To confirm

Figure 2. JQ1-Induced Mediator Eviction Correlates with JQ1-Induced Transcriptional Suppression (A) Fold change in occupancy of MED1 at 10,604 individual MED1 peaks in AML following 2 hr treatment with 500 nM JQ1. The peaks are ranked in order of increasing fold change (JQ1/DMSO). The blue box highlights the 200 most-JQ1-sensitive MED1 elements. Fold changes presented are the average of two independent biological replicates. (B) Fold change in FPKM (fragments per kilobase of transcript per million) for 8,118 expressed genes in AML (defined by FPKM > 5 in DMSO sample) following 6 hr of 500 nM JQ1 treatment. The genes are ranked in order of increasing fold change. The numbers in parentheses are the fold change expression rank of the indicated genes. (C) Average fold change in FPKM after JQ1 treatment for all expressed genes (left), for genes associated with super-enhancers (center), and for genes associated with JQ1-sensitive MED1 peaks (right). The numbers in parentheses represent the number of genes matched to the class of peaks indicated. *** represents a p value < 0.0001, the result of a Mann-Whitney test. (D) Stratification of 178 MED1 super-enhancers based on whether or not they overlap with at least one of the 200 JQ1-sensitive MED1 peaks (minimum overlap 1 bp). (E) Average fold change in FPKM for genes associated with JQ1-sensitive MED1 super-enhancers and for genes associated with JQ1-insensitive MED1 superenhancers (as delineated in D). The numbers in parentheses represent the number of genes matched to the subclass of super-enhancer indicated. *** represents a p value < 0.0001, the result of a Mann-Whitney test. (F) ChIP-seq meta-profiles for hematopoietic TF occupancy at 200 JQ1-sensitive MED1 peaks or the remaining 10,404 MED1 peaks. (G) GREAT ontology analysis binomial p values for 200 JQ1-sensitive MED1 peaks versus 200 random MED1 peaks. Top-ranking ontology terms for the JQ1sensitive peaks are displayed alongside values for the same ontology terms in the random peaks. Terms in parentheses represent the ontology identifiers in the GREAT database. See also Figure S2 and Tables S2 and S3.

Cell Reports 15, 519–530, April 19, 2016 523

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Figure 3. Genetic Knockdown of Select Mediator Subunits Triggers Differentiation of AML Blasts (A) Summary of negative-selection shRNA screen targeting the indicated Mediator subunits. Bars represent the average of all hairpins for each gene. Black bars highlight subunits having at least 3-fold loss of GFP-positive cells with at least two independent hairpins. shRNAs are expressed using the LMN vector. Data are represented as the mean of all hairpins for the corresponding gene ± SEM. (B) Negative-selection experiments using the indicated shRNAs chosen from the screen in (A). GFP+/shRNA+ percentages were normalized to values taken on day 2 and tracked for 8 days. Data are presented as mean ± SEM and n = 3. (C) Flow cytometric analysis of cell-surface cKit following 96 hr of doxycycline-induced expression of the indicated shRNAs. shRNAs were expressed using the TRMPV-Neo vector. Gating was performed on GFP+/shRNA+ cell populations. (D) Flow cytometric analysis of cell-surface Mac1 following 96 hr of doxycycline-induced expression of the indicated shRNAs. shRNAs were expressed using the TRMPV-Neo vector. Gating was performed on GFP+/shRNA+ cell populations. €nwald-Giemsa-stained RN2 cells after 96 hr of doxycycline-induced expression of the indicated shRNAs. The images (E) Light microscopy analysis of May-Gru were taken with 403 objective. See also Figure S3 and Table S3.

524 Cell Reports 15, 519–530, April 19, 2016

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shMed23 vs shRen

Figure 4. MED12 and MED23 Are Required to Sustain Expression of BRD4, MYC, and MYB Target Gene Signatures in AML Cells (A) Fold change in FPKM for 8,393 expressed genes in AML (defined by FPKM > 5 in shRen sample) following 48 hr of doxycycline treatment to induce two independent shRNAs targeting Med12 (401 and 5,755) versus shRen.713. Fold change values for each gene are the average value of the independent hairpins targeting the indicated Mediator subunit. The genes are ranked in order of increasing fold change. The numbers in parentheses represent the fold change expression rank of the indicated genes. (B) Fold change in FPKM for 8,393 expressed genes in AML (defined by FPKM > 5 in shRen sample) following 48 hr of doxycycline treatment to induce two independent shRNAs targeting Med23 (678 and 4,061) versus shRen.713. Fold change values for each gene are the average value of the independent hairpins targeting the indicated Mediator subunit. The genes are ranked in order of increasing fold change. The numbers in parentheses represent the fold change expression rank of the indicated genes. (C) Gene set enrichment analysis (GSEA) of shMed12 versus shRen RNA-seq using 10,379 gene sets, including all gene sets in the Molecular Signatures Database. Signatures are ranked by their normalized enrichment scores and false discovery rate (FDR) q values according to GSEA.

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that differentiation was induced following Mediator subunit €nwald/Giemsa-stained RN2 knockdown, we imaged May-Gru cells using light microscopy. Consistent with the flow cytometry analysis, knockdown of MED12, MED13, MED23, or MED24, but not of MED8 or MED30, led to similar morphologic changes toward mature macrophages as observed with BRD4 knockdown (Figure 3E). These findings suggest that targeting select Mediator subunits can trigger differentiation of AML blasts. shRNA-based knockdown of the homologous kinase subunits CDK8 and CDK19 of Mediator failed to elicit proliferation arrest of RN2 cells (Figures 3A, S3D, and S3E). However, we found that dual shRNA-based targeting of these two kinases led to an arrest in the proliferation of RN2 cells (Figure S3E). This result suggests a redundant function for these two kinase subunits, which is consistent with a recent study showing that dual CDK8/CDK19 inhibition with cortistatin A suppressed AML cell growth (Pelish et al., 2015). Mediator Subunits Are Required to Sustain Expression of BRD4 Target Genes in AML Cells Next, we considered whether a common gene regulatory network depends on BRD4 and Mediator for expression in RN2 cells using RNA-seq analysis. In an analogous manner to JQ1 treatment, knockdown of MED12 or MED23 resulted in reduced mRNA levels of Myc, Mgat5, and Cdk6, but not of Lrrfip1 or Hoxa9 (Figures 4A and 4B). To provide an unbiased evaluation of gene signatures altered by knockdown, we performed gene set enrichment analysis (GSEA) to interrogate a database of 10,379 gene sets (Subramanian et al., 2005). Remarkably, several of the most-downregulated gene signatures in MED12or MED23-deficient RN2 cells corresponded to the target genes of MYB, MYC, and BRD4 (Figures 4C–4F). Among the positively enriched gene sets were those related to myeloid cell maturation, consistent with the phenotypic changes observed following MED12 and MED23 knockdown (Figures 4C–4F). Knockdown of MED8 also led to suppression of BRD4 target genes such as Myc; however, the global pattern of gene expression was different from what was observed following MED12 or MED23 knockdown, consistent with the differing phenotypes upon knockdown of these subunits (Figures S4A–S4C). GSEA revealed that, while BRD4 and MYC signatures were affected by MED8 knockdown, a number of transcriptional pathways not suppressed by JQ1 treatment or by MED12 or MED23 shRNA

were suppressed in MED8-deficient cells (Figures S4B and S4D). Whereas it appears that individual Mediator subunits perform distinct transcriptional functions, our findings support a role for the Mediator complex in supporting BRD4-dependent gene activation. Next, we explored the mechanism by which BRD4 and Mediator regulate Pol II activity at their co-regulated target genes. Mediator and BRD4 have each been shown to interact with the kinase P-TEFb, comprised of CDK9 and cyclin T, which can stimulate the release of paused Pol II near the transcriptional start site (TSS) (Donner et al., 2010; Jang et al., 2005; Takahashi et al., 2011; Yang et al., 2005). Using ChIP-qPCR, we found that BET inhibition and MED12 knockdown each led to a loss of CDK9 and Pol II from the Myc super-enhancer and gene body (Figures 5A–5H; Table S4). Notably, the loss of Pol II near the Myc TSS was less severe (3.7-fold for JQ1 and 1.3-fold for shMED12) relative to the gene body (15.8-fold for JQ1 and 5.4-fold for shMED12; Figures 5D and 5H). This result is consistent with BRD4 and Mediator promoting the transition of Pol II from initiation to elongation by facilitating P-TEFb recruitment. A recent study showed that Mediator can facilitate BRD4 chromatin localization in the setting of acquired resistance to BET inhibitors in breast cancer (Shu et al., 2016). We therefore considered whether Mediator plays a reciprocal role in stabilizing BRD4 occupancy in RN2 cells. Because MED12 and MED23 knockdown each mimicked the phenotypic and transcriptional effects of BRD4 inhibition, we performed BRD4 ChIP-seq following knockdown of these subunits. This revealed that MED12 and MED23 knockdown resulted in a modest reduction in BRD4 chromatin occupancy at the same cis elements at which MED1 is perturbed following BET inhibition (Figures S5A–S5E). This result suggests that BRD4 and Mediator can mutually stabilize one another’s chromatin occupancy. DISCUSSION The findings presented in this study support a model in which BRD4 regulates its downstream target genes, at least in part, via a physical interaction with the Mediator complex. We have previously shown that BRD4 directly interacts with the short isoform of NSD3, which acts as a bridge to recruit the CHD8 chromatin remodeler (Shen et al., 2015). In addition, BRD4 is known to recruit P-TEFb to promote transcription elongation of

(D) Gene set enrichment analysis (GSEA) of shMed23 versus shRen RNA-seq using 10,379 gene sets, including all gene sets in the Molecular Signatures Database. Signatures are ranked by their normalized enrichment scores and false discovery rate (FDR) q values according to GSEA. (E) GSEA plots from the shMed12 RNA-seq showing enrichment of a BRD4 target gene set. Normalized enrichment scores (NESs) and nominal p values (Nom p value), as calculated by GSEA, are provided. (F) GSEA plots from the shMed12 RNA-seq showing enrichment of a MYC target gene set. Normalized enrichment scores (NESs) and nominal p values (Nom p value), as calculated by GSEA, are provided. (G) GSEA plots from the shMed12 RNA-seq showing enrichment of a myeloid differentiation gene set. Normalized enrichment scores (NESs) and nominal p values (Nom p value), as calculated by GSEA, are provided. (H) GSEA plots from the shMed23 RNA-seq showing enrichment of a BRD4 target gene set. Normalized enrichment scores (NESs) and nominal p values (Nom p value), as calculated by GSEA, are provided. (I) GSEA plots from the shMed23 RNA-seq showing enrichment of a MYC target gene set. Normalized enrichment scores (NESs) and nominal p values (Nom p value), as calculated by GSEA, are provided. (J) GSEA plots from the shMed23 RNA-seq showing enrichment of a myeloid differentiation gene set. Normalized enrichment scores (NESs) and nominal p values (Nom p value), as calculated by GSEA, are provided. See also Figure S4 and Table S4.

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A

B

C

D

E

F

G

H

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its target genes (Jang et al., 2005; Yang et al., 2005). Notably, Mediator has also been shown to recruit P-TEFb to promote transcription elongation (Donner et al., 2010; Takahashi et al., 2011). Taken together, these observations reinforce a model in which BRD4 acts as a scaffold that recruits multiple regulatory machineries to cis elements bound by acetylated TFs and nucleosomes to promote transcription of genes that sustain the aberrant growth and self-renewal properties of AML cells. Our dual shRNA-targeting experiments are in agreement with a recent study, which found that AML cells are sensitive to chemical inhibition of CDK8/CDK19 with cortistatin A (Pelish et al., 2015). In this study, it was shown that these kinases act to restrain super-enhancer activity, thereby antagonizing the activation function of BRD4 (Pelish et al., 2015). Importantly, our shRNA-screening results suggest that this repressive function is likely to be confined to the CDK8/CDK19 subunits of Mediator, whereas other subunits, including the kinase-associated subunit MED12, are involved in supporting BRD4-dependent transcriptional activation. While it is surprising to observe opposing effects of targeting individual subunits within the same module of the Mediator complex, other studies have also observed contrasting phenotypic and transcriptional consequences after knockdown of MED12 versus CDK8 subunits (Gobert et al., 2010; Kuuluvainen et al., 2014). Nevertheless, our study and Pelish et al. (2015) reinforce how distinct perturbations of Mediator can destabilize the AML cell state. Our study provides insights into how JQ1 exerts disproportionate effects on cancer-relevant genes while sparing housekeeping gene expression, a property that likely underlies the therapeutic efficacy of this agent in AML and other malignancies. It has previously been proposed that the location of superenhancers harboring exceptional levels of BRD4 and Mediator provides the molecular basis for the hypersensitivity of specific genes to JQ1-mediated transcriptional suppression (Love´n et al., 2013). We have shown here that the hypersensitivity of Mediator to JQ1-mediated displacement can be uncoupled from the pre-existing levels of Mediator occupancy, which is, in turn, reflected in the heterogeneous sensitivity of superenhancer-linked genes to JQ1-mediated suppression. Our study shows that less than half of all super-enhancers in AML cells exhibit JQ1-mediated displacement of the Mediator complex (Love´n et al., 2013). Importantly, our findings suggest that the degree of Mediator eviction can serve as a useful biomarker for revealing the critical cis elements that are functionally suppressed by BET inhibition. Measurements of MED1 eviction

following JQ1 exposure could be applied more broadly to reveal the critical downstream genes that underlie the therapeutic effects of BET inhibition in various cellular contexts. Whereas BRD4 occupancy correlates genome-wide with the occupancy of several hematopoietic TFs (Roe et al., 2015), the sensitivity of Mediator to JQ1-mediated eviction correlates with cis elements specifically harboring higher levels of the TF MYB. In addition, our ontology analysis shows that genes associated with JQ1-sensitive MED1 also enriched for the MYB target gene network. Notably, a somatic mutation has been described in T cell acute lymphoblastic leukemia that produces a novel MYB-binding motif near the TAL1 gene, which leads to the formation of a MED1-occupied super-enhancer (Mansour et al., 2014). Interestingly, AML cells are known to be more sensitive to MYB knockdown than normal myeloblasts (Zuber et al., 2011b). Since MYB physically associates with BRD4 (Roe et al., 2015), our results suggest that MYB function might place unique demands on BRD4-Mediator complexes to sustain expression of genes with leukemogenic functions, which may contribute to the hypersensitivity of AML cell growth to BET bromodomain inhibition. EXPERIMENTAL PROCEDURES ChIP-Seq One hundred twenty million RN2 cells were crosslinked with 1% formaldehyde for 20 min and quenched for 10 min with 0.125 M glycine. Nuclear lysates were sonicated in 20 million cell batches using a BioRuptor water bath sonicator. Sonicated chromatin was pre-cleared using rabbit immunoglobulin G (IgG) and agarose beads or was incubated with the BRD4 antibody for 2 hr followed by addition of magnetic beads (BRD4 ChIP-seq in MED12/23 knockdown conditions). Immunoprecipitation (IP) with the relevant antibodies was performed overnight at 4 C with rotation. After extensive washing as previously described (Steger et al., 2008), crosslinks were reversed overnight at 65 C. DNA was treated with RNase A and Proteinase K and purified using the QIAGEN PCR purification kit. Libraries were constructed with the TruSeq ChIP Sample Prep kit from lllumina. Libraries underwent a final amplification step of 15 PCR cycles and were analyzed using a Bioanalyzer with a high-sensitivity chip (Agilent). Libraries were single-end sequenced on a HiSeq2000 with reads of 50 bp. A full description can be found in the Supplemental Experimental Procedures section. ACCESSION NUMBERS The accession number for the raw and processed sequencing data reported in this paper is GEO: GSE74651, with the subseries accession numbers GEO: GSE74536 and GEO: GSE78221 (ChIP-seq) and GEO: GSE74650 and GEO: GSE78224 (RNA-seq).

Figure 5. BET Inhibition and MED12 Knockdown Cause Loss of CDK9 Occupancy and Reduced Pol II Elongation at the Myc Locus (A) ChIP-qPCR analysis of CDK9 at the Myc super-enhancer following a 2-hr treatment with DMSO or with 500 nM JQ1. (B) ChIP-qPCR analysis of CDK9 across the Myc gene body following a 2-hr treatment with DMSO or 500 nM JQ1. (C) ChIP-qPCR analysis of Pol II at the Myc super-enhancer following a 2-hr treatment with DMSO or with 500 nM JQ1. (D) ChIP-qPCR analysis of Pol II at the Myc gene body following a 2-hr treatment with DMSO or with 500 nM JQ1. Numbers above +14-bp and +3,965-bp regions indicate fold change between DMSO and JQ1 ChIP-qPCR enrichment. (E) ChIP-qPCR analysis of CDK9 at the Myc super-enhancer locus following 0- or 48-hr induction of shMed12 with doxycycline (dox). TRMPV-Neo vector was used. (F) ChIP-qPCR analysis of CDK9 across the Myc gene body following 0- or 48-hr induction of shMed12 with dox. TRMPV-Neo vector was used. (G) ChIP-qPCR analysis of Pol II at the Myc enhancer locus following 0- or 48-hr induction of shMed12 with dox. TRMPV-Neo vector was used. (H) ChIP-qPCR analysis of Pol II across the Myc gene body following 0- or 48-hr induction of shMed12 with dox. Numbers above +14 bp and +3,965 bp data indicate fold change between control and shMed12 ChIPs at these regions. TRMPV-Neo vector was used. All experiments were performed in RN2 cells. All data are presented as mean ± SEM and n = 3.

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SUPPLEMENTAL INFORMATION Supplemental Information includes Supplemental Experimental Procedures, five figures, and four tables and can be found with this article online at http://dx.doi.org/10.1016/j.celrep.2016.03.054. AUTHOR CONTRIBUTIONS A.S.B., J.-S.R., J.S., and C.R.V. designed experiments and analyzed results. A.S.B., J.-S.R., B.Y.L.M., and A.F.H. carried out experiments. C.R.V. supervised the research. A.S.B. and C.R.V. wrote the manuscript. ACKNOWLEDGMENTS We thank James Bradner and Jun Qi for providing JQ1. We thank R. Wysocki for assistance with microscopy. This work was supported by Cold Spring Harbor Laboratory NCI Cancer Center Support grant CA455087 for developmental funds and shared resource support. Additional funding was provided by the Alex’s Lemonade Stand Foundation and the V Foundation. J.-S.R. is supported by the Martin Sass Foundation and the Lauri Strauss Leukemia Foundation. A.S.B. is supported by NIH T32 GM008444 and NCI F30 CA186632. C.R.V. is supported by a Burroughs-Wellcome Fund Career Award, NIH grant NCI RO1 CA174793, and a Leukemia & Lymphoma Society Scholar Award. Received: October 28, 2015 Revised: January 22, 2016 Accepted: March 14, 2016 Published: April 7, 2016 REFERENCES

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