Super-enhancers (SEs), which are composed of large clusters of enhancers densely loaded with the Mediator complex, transcription factors (TFs), and chromatin regulators, drive high expression of genes implicated in cell identity and disease, such as lineage-controlling TFs and oncogenes 1, 2. BRD4 and CDK7 are positive regulators of SE-mediated transcription3,4,5. In contrast, negative regulators of SE-associated genes have not been well described. Here we report that Mediator-associated kinases cyclin-dependent kinase 8 (CDK8) and CDK19 restrain increased activation of key SE-associated genes in acute myeloid leukaemia (AML) cells. We determined that the natural product cortistatin A (CA) selectively inhibited Mediator kinases, had antileukaemic activity in vitro and in vivo, and disproportionately induced upregulation of SE-associated genes in CA-sensitive AML cell lines but not in CA-insensitive cell lines. In AML cells, CA upregulated SE-associated genes with tumour suppressor and lineage-controlling functions, including the TFs CEBPA, IRF8, IRF1 and ETV6 6, 7, 8. The BRD4 inhibitor I-BET151 downregulated these SE-associated genes, yet also has antileukaemic activity. Individually increasing or decreasing expression of these TFs suppressed AML cell growth, providing evidence that leukaemia cells are sensitive to dosage of SE-associated genes. Our results demonstrate that Mediator kinases can negatively regulate SE-associated gene expression in specific cell types and can be pharmacologically targeted as a therapeutic approach to AML.

We report that azides capable of copper-chelation undergo much faster “Click chemistry” (copper-accelerated azide-alkyne cycloaddition, or CuAAC) than nonchelating azides under a variety of biocompatible conditions. This kinetic enhancement allowed us to perform site-specific protein labeling on the surface of living cells with only 10–40 µM CuI/II and much higher signal than could be obtained using the best previously-reported live-cell compatible CuAAC labeling conditions. Detection sensitivity was also increased for CuAAC detection of alkyne-modified proteins and RNA labeled by metabolic feeding.

The inverse-electron-demand Diels-Alder cycloaddition between trans-cyclooctenes and tetrazines is biocompatible and exceptionally fast. We utilized this chemistry for site-specific fluorescence labeling of proteins on the cell surface and inside living mammalian cells by a two-step protocol. E. coli lipoic acid ligase site-specifically ligates a trans-cyclooctene derivative onto a protein of interest in the first step, followed by chemoselective derivatization with a tetrazinefluorophore conjugate in the second step. On the cell surface, this labeling was fluorogenic and highly sensitive. Inside the cell, we achieved specific labeling of cytoskeletal proteins with green and red fluorophores. By incorporating the Diels-Alder cycloaddition, we have broadened the panel of fluorophores that can be targeted by lipoic acid ligase.

SUMMARY Cortistatin A (CA) is a highly selective inhibitor of the Mediator kinases CDK8 and CDK19. Using CA, we now report a large-scale identification of Mediator kinase substrates in human cells (HCT116). We identified over 16,000 quantified phosphosites including 78 high-confidence Mediator kinase targets within 64 proteins, including DNA-binding transcription factors and proteins associated with chromatin, DNA repair, and RNA polymerase II. Although RNA-Seq data correlated with Mediator kinase targets, the effects of CA on gene expression were limited and distinct from CDK8 or CDK19 knockdown. Quantitative proteome analyses, tracking around 7,000 proteins across six time points (0 – 24h), revealed that CA selectively affected pathways implicated in inflammation, growth, and metabolic regulation. Contrary to expectations, increased turnover of Mediator kinase targets was not generally observed. Collectively, these data support Mediator kinases as regulators of chromatin and RNA polymerase II activity and suggest their roles extend beyond transcription to metabolism and DNA repair.

The copper-catalyzed azide-alkyne cycloaddition, or CuAAC, has been used extensively for the conjugation, immobilization, and purification of biomolecules. [1] Despite excellent reaction kinetics, high specificity, and bioorthogonality, CuAAC has been used to a far lesser extent in the cellular context because of toxicity caused by the Cu Imediated generation of reactive oxygen species (ROS) from O 2 . [2] One way to address this problem is to remove the Cu I requirement, by using alkynes activated by ring strain. [3][4][5] However, even the fastest of the strained cyclooctynes [6] react with azides more than tenfold slower than terminal alkynes in the presence of Cu I (k obs % 1m À1 s À1 for (aza)dibenzocyclo octyne [6] compared to k obs % 10-100 m À1 s À1 per 10-100 mm Cu I / Cu II for CuAAC [7] ). A second approach to improve cell compatibility is to use water-soluble ligands such as tris-(hydroxypropyltriazolylmethyl)amine (THPTA), [8] bis[(tertbutyltriazoyl)methyl]-[(2-carboxymethyltriazoyl)methyl]amine (BTTAA), [9] or bis(l-histidine) [10] for Cu I . These ligands both accelerate the cycloaddition reaction and act as sacrificial reductants, helping to protect cells and biomolecules from ROS. [8] Here we explore a third approach to improve the cell compatibility and performance of CuAAC. In general, decreasing the copper concentration lowers the toxicity of CuAAC to cells, but this is accompanied by a large decrease in reaction kinetics. [9] We reasoned that it might be possible to compensate for this decrease by using an azide reaction partner that contains an internal copper-chelating moiety (Figure 1 A), which would raise the effective copper concentration at the reaction site. This concept has been explored for azide-alkyne reactions in organic solvents, with Cu II rather than Cu I species, and at very high copper (10 mm) and reactant (200-400 mm) concentrations, [11,12] but never before under conditions relevant to biomolecular labeling. The goal of our study was to examine the effect of substrate chelation assistance on CuAAC kinetics and biocompatibility.The rate-determining step of CuAAC is postulated to be the formation of the metallacycle from the Cu I acetylide and the organic azide. [15] We decided to test whether an organic azide containing an internal Cu I ligand could accelerate formation of the metallacycle and hence the overall rate of the CuAAC reaction. We prepared two azides with proximal pyridine nitrogen atoms to chelate the Cu I ion (picolyl azides 2 and 4), as well as their nonchelating carbocyclic analogues, 1 and 3 ( Figure 2).CuAAC reaction timecourses were measured using 7ethynylcoumarin, a fluorogenic alkyne whose quantum yield (QY) increases from 1 % to 25 % upon reaction with azides [4] (Figure 2 A). Assays were first performed with 10 mm CuSO 4 in the absence of Cu I ligands. Reaction timecourses are shown in Figure S1 (see Supporting Information) and values for percent conversion into product after 10 and 30 minutes are given in Figure 2 B. Whereas the conventional azides 1 a...