Mediator is a common target of DNA-binding transcription factors (TFs) and also interacts with various Pre-Initiation Complex (PIC) factors. The PIC consists of TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, Mediator, and RNA polymerase II (pol II). As a central integrator of both general and TF-specific regulatory signals, Mediator plays a prominent role in controlling transcription on a genome-wide scale. Because Mediator interacts with both DNA-binding TFs and the general transcription machinery (including pol II), Mediator acts as a molecular bridge to link these factors and facilitate TF-dependent regulation. A fundamentally important consequence of TF-Mediator interactions may be the establishment of enhancer-promoter loops. That is, Mediator may enable the genome to be "stitched together" in cell-type or stimulus-specific ways; this, in turn, may promote expression of specific sets of genes. These biological roles may be enhanced through liquid-liquid phase separation (LLPS) in human cells; in fact, Mediator, pol II, and TFs themselves appear to have evolved to form molecular condensates. This allows an ability to "compartmentalize" with other proteins and biochemicals, which likely contributes to their biological function.
Furthermore, Mediator tailors its response in TF-specific ways. Analysis of Mediator using electron microscopy indicated that Mediator undergoes structural changes when it binds a TF activation domain. These structural changes appear to govern Mediator function, but their precise mechanistic roles remain incompletely understood, in part because of limited high resolution structural data for TF-induced structural changes. An equally important observation was that when different TFs (which regulate distinct sets of genes) bind Mediator, they induce different conformational states in the complex. These TF-specific structural states may direct gene-specific regulatory events; in other words, Mediator—a general transcription factor—may acquire gene-specific functions that are triggered by TF binding. One focus of the lab is to study how TFs regulate Mediator structure and function. To do this, we are employing a variety of biochemical, biophysical, and chemical biology techniques.
Sequence-specific, DNA-binding TFs are major regulators of cell identity and physiology. As examples, expression of select TFs can revert differentiated cells back to a pluripotent state and mutations in a single TF can drive tumor formation. Notably, sequence-specific DNA-binding TFs represent a major class of targets for the Mediator kinases CDK8 and CDK19. Our lab is using a variety of approaches to better understand how TF activity is controlled by CDK8 and CDK19 (e.g. Top Figure). Given their central roles in development and disease, DNA-binding TFs represent quintessential targets for molecular therapeutics. However, TFs are generally considered “undruggable” due to few successes with small molecule compounds, despite large-scale efforts. A common means by which DNA-binding TFs activate transcription is through the Mediator complex; thus, the TF-Mediator interface represents a key control point for transcription (Bottom Figure). Through collaborative efforts, we are working to develop chemical probes to target key TF-Mediator interfaces. Although our primary goal is to utilize these probes for detailed mechanistic studies in vitro and in cells, these efforts may help establish Mediator as a viable therapeutic target.
The Figure above highlights how different TFs bind different subunits/surfaces on Mediator. The TF binding sites shown are for illustrative purposes but reflect general locations of Mediator subunits. Mediator structure shown is based upon cryo-EM data from the Yanhui Xu lab (Science 2021). The CDK8 module structure is from yeast, determined by the Tsai lab (Sci Adv 2021). Note that paralogs for CDK8, MED12, and MED13 are shown in parenthesis, and are specific to vertebrates.
Finally, the lab focuses on the p53 TF in particular, which is best known as a tumor suppressor but also plays fundamental roles in aging, metabolism, and stem cell biology. A naturally occurring p53 isoform, called Δ40p53 (or ΔNp53), causes accelerated aging in mice when expressed together with WTp53. We generated genome-edited human cell lines that express Δ40p53:WTp53 from the native TP53 locus, ensuring a 2:2 tetramer stoichiometry. This strategy, which is based off the structure of the native WTp53 tetramer (Orlova et al. EMBO 2006), eliminates confounding issues associated with Δ40p53 and WTp53 co-expression and allows clear delineation of cellular changes triggered by Δ40p53:WTp53 vs. WTp53.
Recent publications related to this topic:
Nayak, S; Taatjes DJ. SnapShot: Mediator Complex Structure. Cell 2022, 185: 3458.
Richter, WF; Nayak, S; Iwasa, J; Taatjes, DJ. The Mediator complex as a master regulator of transcription by RNA polymerase II. Nat Rev Mol Cell Biol 2022, 23: 732 - 749.
Allen, BL; Quach, K; Jones, T; Levandowski, CB; Ebmeier, CC; Rubin, JD; Read, T; Dowell, RD; Schepartz, A*; Taatjes, DJ*. Suppression of p53 response by targeting p53–Mediator binding with a stapled peptide. Cell Rep 2022, 39: 110630.
Levandowski, CB; Jones, T; Gruca, M; Ramamoorthy, S; Dowell, RD;* Taatjes, DJ.* The ∆40p53 isoform inhibits p53-dependent eRNA transcription and enables regulation by signal-specific transcription factors during p53 activation. PLoS Biol 2021, 19: e3001364.
Schier, A; Taatjes, DJ. Everything at once: Cryo-EM yields remarkable insights about human RNA polymerase II transcription. Nat Struct Mol Biol 2021, 28: 540 - 543.
Rubin, JD; Stanley, JT; Sigauke, RS; Levandowski, CB; Maas, ZL; Westfall, J; Taatjes, DJ; Dowell, RD. Transcription factor enrichment analysis (TFEA) quantifies the activity of multiple transcription factors from a single experiment. Commun Biol 2021, 4: 661.
Tomko, EJ; Luyties, O; Rimel, JK; Tsai, C; Fuss, JO; Fishburn, J; Hahn, S; Tsutakawa, SE; Taatjes, DJ; Galburt, EA. The role of XPB/Ssl2 dsDNA translocation processivity in transcription start-site scanning. J Mol Biol 2021; 433: 166813.
Rimel, JK; Poss, ZC; Erickson, B; Maas, ZL; Ebmeier, CC; Johnson, JL; Decker, T-M; Yaron, TM; Bradley, MJ; Hamman, KB; Hu, S; Malojcic, G; Marineau, JJ; White, PW; Brault, M.; Tao, L.; DeRoy, P; Clavette, C; Nayak, S; Damon, LJ; Kaltheuner, IH; Bunch, H; Cantley, LC; Geyer, M; Iwasa, J; Dowell, RD; Bentley, DL; Old WM;* Taatjes, DJ.* Selective inhibition of CDK7 reveals high-confidence targets and novel mechanisms for TFIIH function in transcription. Genes Dev 2020; 34: 1452 – 1473.
Fant, CB; Levandowski, CB; Gupta, K; Maas, ZL; Moir, JT; Rubin, JD; Sawyer, A; Esbin, M; Rimel, JK; Luyties, O; Marr, MT; Berger, I; Dowell, RD; Taatjes, DJ. TFIID enables RNA polymerase II promoter-proximal pausing. Mol Cell 2020, 78: 785 – 793.
Schier, AC; Taatjes, DJ. Structure and mechanism of the RNA polymerase II transcription machinery. Genes Dev. 2020, 34: 465 – 488.
Zamudio, AV; Dall’Agnese, A; Henninger, JE; Manteiga, JC; Afeyan, LK; Hannett, NM; Coffey, EL; Li, CH; Oksuz, O; Boija, A; Klein, IA; Sabari, BR; Hawken, SW; Spille, JH; Decker, TM; Cisse, II; Abraham, BJ; Lee, TI; Taatjes, DJ; Schuijers, J; Young, RA. Mediator condensates localize signaling factors to key cell identity genes. Mol Cell 2019, 76: 753 – 766.
Guo, YE; Manteiga, JC; Henninger, J; Sabari, BR; Dall'Agnese, A; Hannett, NM; Spille, J-H; Afeyan, LK; Zamudio, AV; Shrinivas, K; Abraham, BJ; Boija, A; Decker, TM; Rimel, JK; Fant, CB; Lee, TI; Cisse, II; Sharp, PA; Taatjes, DJ; Young, RA. RNA polymerase II phosphorylation regulates a switch between transcriptional and splicing condensates. Nature 2019, 572: 543 – 548.
Steinparzer, I; Sedlyarov, V; Rubin, JD; Eislmayr, K; Galbraith MD; Levandowski, CB; Vcelkova, T; Sneezum, L; Wascher, F; Amman, F; Kleinova, R; Bender, H; Andrysik, Z; Espinosa, JM; Superti-Furga, G; Dowell, RD; Taatjes, DJ;* Kovarik, P.* Transcriptional responses to IFNg require Mediator kinase-dependent pause release and mechanistically distinct CDK8 and CDK19 functions. Mol Cell 2019, 76: 485 – 499.
Fant, CB; Taatjes, DJ. Regulatory functions of the Mediator kinases CDK8 and CDK19. Transcription 2019, 10: 76 – 90.
Boija, A; Klein, IA; Sabari, BR; Dall'Agnese, A; Coffey, EL; Zamudio, AV; Li, CH; Shrinivas, K; Manteiga, J; Hannett, NM; Abraham, BJ; Schuijers, J; Afeyan, L; Guo, YE; Rimel, JK; Fant, CB; Lee, TI; Taatjes, DJ; Young, RA. Transcription factors activate genes through the phase separation capacity of their activation domains. Cell 2018, 175: 1842-1855.
Taatjes, DJ. Transcription factor–Mediator interfaces: multiple and multi-valent. J Mol Biol. 2017, 429: 2996 - 2998.