Transcription Factors and PIC function
Mediator is a common target of DNA-binding transcription factors (TFs) and also interacts with various听Pre-Initiation Complex (PIC) factors. The PIC听consists of TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, Mediator, and RNA polymerase II (pol II). 听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. 听Because Mediator interacts with both DNA-binding TFs and the general transcription machinery (including pol听II), Mediator acts as a molecular bridge to link these factors and facilitate TF-dependent regulation. 听A fundamentally important consequence of TF-Mediator interactions may be the establishment of enhancer-promoter loops. 听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.听 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.听听This allows an ability to "compartmentalize" with other proteins and biochemicals, which听likely contributes to their biological function.
Furthermore, Mediator tailors its response in TF-specific ways. 听Analysis of Mediator using electron microscopy indicated that Mediator undergoes听structural changes when it binds a TF activation domain. 听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. 听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. 听These TF-specific structural states may direct gene-specific regulatory events; in other words, Mediator鈥攁 general transcription factor鈥攎ay acquire gene-specific functions that are triggered by TF binding. 听One focus of the lab is to study how TFs听regulate Mediator structure and function. 听To do this, we are employing a variety of biochemical, biophysical, and chemical biology techniques. 听
Sequence-specific, DNA-binding TFs are major regulators of cell identity and physiology. 听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. 听Notably, sequence-specific DNA-binding TFs represent a major class of targets for the Mediator kinases CDK8 and CDK19. 听Our lab is using a variety of approaches to better understand how TF activity is controlled by CDK8 and CDK19 (e.g. Top听Figure). 听Given their central roles in development and disease, DNA-binding TFs represent quintessential targets for molecular therapeutics. 听However, TFs are generally considered 鈥渦ndruggable鈥 due to few successes with small molecule compounds, despite large-scale efforts. 听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听for transcription (Bottom听Figure). 听Through collaborative efforts, we are working to develop chemical probes to target key TF-Mediator interfaces. 听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.听
The Figure above highlights how different TFs bind different subunits/surfaces on Mediator. 听The TF binding sites shown are for illustrative purposes but reflect general locations of Mediator subunits. 听Mediator structure shown is based upon cryo-EM data from the Yanhui Xu lab (Science 2021). 听The CDK8 module structure is from yeast, determined by the Tsai lab (Sci Adv 2021). 听Note that paralogs for CDK8, MED12, and MED13 are shown in parenthesis, and are specific to vertebrates.听
Finally,听the lab听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.听 A naturally occurring p53 isoform, called 螖40p53 (or 螖Np53), causes accelerated aging in mice when expressed together with WTp53. 听We generated genome-edited human cell lines that express听螖40p53:WTp53 from the native TP53 locus, ensuring a 2:2 tetramer stoichiometry. 听This strategy, which is based off the structure of the native WTp53 tetramer (Orlova et al. EMBO 2006), eliminates听confounding issues associated with 螖40p53 and WTp53 co-expression and allows clear delineation of cellular changes triggered by 螖40p53:WTp53 vs. WTp53.
Recent publications related to this topic:
Nayak, S; Taatjes DJ. SnapShot: Mediator Complex Structure.听Cell听2022, 185: 3458.
Richter, WF; Nayak, S; Iwasa, J; Taatjes, DJ. The Mediator complex as a master regulator of transcription by RNA polymerase II.听Nat Rev Mol Cell Biol听2022, 23: 732 - 749.听
Allen, BL; Quach, K; Jones, T; Levandowski, CB; Ebmeier, CC; Rubin, JD; Read, T; Dowell, RD; Schepartz, A*; Taatjes, DJ*.听听Suppression of p53 response by targeting p53鈥揗ediator binding with a stapled peptide.听Cell Rep听2022, 39: 110630.
Levandowski, CB; Jones, T; Gruca, M; Ramamoorthy, S; Dowell, RD;* Taatjes, DJ.*听听The听鈭40p53 isoform inhibits p53-dependent eRNA transcription and enables regulation by signal-specific transcription factors during p53 activation.听PLoS Biol听2021, 19: e3001364.
Schier, A; Taatjes, DJ.听Everything at once: Cryo-EM yields remarkable insights about human RNA polymerase II transcription.听Nat Struct Mol Biol听2021, 28: 540 - 543.听
Rubin, JD; Stanley, JT; Sigauke, RS; Levandowski, CB; Maas, ZL; Westfall, J; Taatjes, DJ; Dowell, RD.听Transcription factor enrichment analysis (TFEA) quantifies the activity of multiple transcription factors from a single experiment.听Commun Biol听2021, 4: 661.
Tomko, EJ; Luyties, O; Rimel, JK; Tsai, C; Fuss, JO; Fishburn, J; Hahn, S; Tsutakawa, SE; Taatjes, DJ; Galburt, EA.听The role of XPB/Ssl2 dsDNA translocation processivity in transcription start-site scanning.听J Mol Biol听2021;听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.*听听Selective inhibition of CDK7 reveals high-confidence targets and novel mechanisms for TFIIH function in transcription.听Genes Dev听2020; 34: 1452 鈥撎1473.听
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.听TFIID enables RNA polymerase II promoter-proximal pausing.听Mol Cell听2020, 78: 785 鈥 793.
Schier, AC; Taatjes, DJ.听Structure and mechanism of the RNA polymerase II transcription machinery.听Genes Dev.听2020, 34: 465 鈥 488.
Zamudio, AV; Dall鈥橝gnese, 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.听Mol Cell听2019, 76: 753 鈥 766.
Guo, YE; Manteiga, JC; Henninger, J; Sabari, BR; Dall'Agnese, A; Hannett, NM; Spille, J-H; Afeyan, LK; Zamudio, AV;听听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.听Nature听2019, 572: 543 鈥 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听require Mediator kinase-dependent pause release and mechanistically distinct CDK8 and CDK19 functions.听Mol Cell听2019, 76: 485 鈥 499.听听
Fant, CB; Taatjes, DJ. Regulatory functions of the Mediator kinases CDK8 and CDK19.听Transcription听2019, 10: 76 鈥 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;听Young, RA.听Transcription factors activate genes through the phase separation capacity of their activation domains. Cell 2018, 175: 1842-1855.
Taatjes, DJ. Transcription factor鈥揗ediator interfaces: multiple and multi-valent. J Mol Biol.听2017, 429: 2996 - 2998.