Biophysics and Systems Pharmacology [BSP], Cancer Biology [CAB]
MD, Stanford University
PhD, Stanford University
My laboratory studies control of cell division and regulation of gene expression in eukaryotic cells, with a focus on the roles of protein phosphorylation by cyclin-dependent kinases (CDKs). CDKs were discovered because of their essential functions at the major transition points of the cell cycle: the commitment to duplicate genetic information by replication of DNA in S phase, and the decision to segregate the duplicated chromosomes to the two daughter cells in mitosis. Subsequently, CDK family members were found to regulate the transcription cycle of RNA polymerase (Pol) II (which transcribes protein-coding genes into mRNA), by phosphorylating the carboxyl-terminal domain (CTD) of Pol II to promote elongation, recruit regulatory proteins to the transcription complex, and coordinate RNA synthesis with co-transcriptional mRNA-processing events such as capping, splicing and polyadenylation.
We take a chemical-genetic approach to dissect the networks of CDKs and their targets involved in cell division and gene expression, both in yeast and mammalian cells. We created human cell lines and yeast strains that express only a mutant version of a particular CDK, which has been engineered to be analog-sensitive (AS)--susceptible to inhibition by bulky purine analogs that do not interfere with the functions of other, wild-type kinases. By modifying a single kinase in a cell, we can precisely determine its functions and targets. By combining mutations in more than one enzyme, we can determine the order of pathways that contain multiple CDKs, and uncover genetic interactions between separate CDK-containing pathways. In human colon cancer cells, we have targeted Cdk7, which has dual functions in cell cycle control and in transcription, to uncover its essential requirements at the entry to both S phase and mitosis, and to reveal its role in Pol II dynamics on actively transcribed genes in vivo. In the fission yeast Schizosaccharomyces pombe, we made AS versions of three different CDKs that phosphorylate the Pol II CTD with distinct but overlapping specificities; by inhibiting each CDK individually or in combinations, we defined an ordered, two-CDK pathway of phosphorylation that couples elongation with capping of the 5' end of a nascent transcript--a potential quality control mechanism reminiscent of the checkpoints that ensure the integrity and fidelity of genome transmission during mitotic cell division.
In our current work, we continue to expand our map of the CDK network, and to use chemical genetics--switching CDK activity ON and OFF in vivo with small molecules--to test predictions of network function generated by computational modeling. In this way, we hope to reveal new functions and targets of the network, and potentially promising targets for anticancer chemotherapy.
Visit the Fisher Laboratory
Schachter MM, Merrick KA, Larochelle S, Hirschi A, Zhang C, Shokat KM, Rubin SM, Fisher RP. A Cdk7-Cdk4 T-loop phosphorylation cascade promotes G1 progression. Molecular cell 2013 Apr; 50(2).
Larochelle S, Amat R, Glover-Cutter K, Sansó M, Zhang C, Allen JJ, Shokat KM, Bentley DL, Fisher RP. Cyclin-dependent kinase control of the initiation-to-elongation switch of RNA polymerase II. Nature structural & molecular biology 2012 Nov; 19(11).
Wohlbold L, Merrick KA, De S, Amat R, Kim JH, Larochelle S, Allen JJ, Zhang C, Shokat KM, Petrini JH, Fisher RP. Chemical genetics reveals a specific requirement for Cdk2 activity in the DNA damage response and identifies Nbs1 as a Cdk2 substrate in human cells. PLoS genetics 2012 Aug; 8(8).
St Amour CV, Sansó M, Bösken CA, Lee KM, Larochelle S, Zhang C, Shokat KM, Geyer M, Fisher RP. Separate domains of fission yeast Cdk9 (P-TEFb) are required for capping enzyme recruitment and primed (Ser7-phosphorylated) Rpb1 carboxyl-terminal domain substrate recognition. Molecular and cellular biology 2012 Jul; 32(13).
Sansó M, Lee KM, Viladevall L, Jacques PE, Pagé V, Nagy S, Racine A, St Amour CV, Zhang C, Shokat KM, Schwer B, Robert F, Fisher RP, Tanny JC. A positive feedback loop links opposing functions of P-TEFb/Cdk9 and histone H2B ubiquitylation to regulate transcript elongation in fission yeast. PLoS genetics 2012; 8(8).
Merrick KA, Wohlbold L, Zhang C, Allen JJ, Horiuchi D, Huskey NE, Goga A, Shokat KM, Fisher RP. Switching Cdk2 on or off with small molecules to reveal requirements in human cell proliferation. Molecular cell 2011 Jun; 42(5).
Glover-Cutter K, Larochelle S, Erickson B, Zhang C, Shokat K, Fisher RP, Bentley DL. TFIIH-associated Cdk7 kinase functions in phosphorylation of C-terminal domain Ser7 residues, promoter-proximal pausing, and termination by RNA polymerase II. Molecular and cellular biology 2009 Oct; 29(20).
Viladevall L, St Amour CV, Rosebrock A, Schneider S, Zhang C, Allen JJ, Shokat KM, Schwer B, Leatherwood JK, Fisher RP. TFIIH and P-TEFb coordinate transcription with capping enzyme recruitment at specific genes in fission yeast. Molecular cell 2009 Mar; 33(6).
Merrick KA, Larochelle S, Zhang C, Allen JJ, Shokat KM, Fisher RP. Distinct activation pathways confer cyclin-binding specificity on Cdk1 and Cdk2 in human cells. Molecular cell 2008 Dec; 32(5).
Gamble MJ, Fisher RP. SET and PARP1 remove DEK from chromatin to permit access by the transcription machinery. Nature structural & molecular biology 2007 Jun; 14(6).
Larochelle S, Merrick KA, Terret ME, Wohlbold L, Barboza NM, Zhang C, Shokat KM, Jallepalli PV, Fisher RP. Requirements for Cdk7 in the assembly of Cdk1/cyclin B and activation of Cdk2 revealed by chemical genetics in human cells. Molecular cell 2007 Mar; 25(6).
Pei Y, Du H, Singer J, Stamour C, Granitto S, Shuman S, Fisher RP. Cyclin-dependent kinase 9 (Cdk9) of fission yeast is activated by the CDK-activating kinase Csk1, overlaps functionally with the TFIIH-associated kinase Mcs6, and associates with the mRNA cap methyltransferase Pcm1 in vivo. Molecular and cellular biology 2006 Feb; 26(3).
Larochelle S, Batliner J, Gamble MJ, Barboza NM, Kraybill BC, Blethrow JD, Shokat KM, Fisher RP. Dichotomous but stringent substrate selection by the dual-function Cdk7 complex revealed by chemical genetics. Nature structural & molecular biology 2006 Jan; 13(1).
Lee KM, Miklos I, Du H, Watt S, Szilagyi Z, Saiz JE, Madabhushi R, Penkett CJ, Sipiczki M, Bähler J, Fisher RP. Impairment of the TFIIH-associated CDK-activating kinase selectively affects cell cycle-regulated gene expression in fission yeast. Molecular biology of the cell 2005 Jun; 16(6).
Saiz JE, Fisher RP. A CDK-activating kinase network is required in cell cycle control and transcription in fission yeast. Current biology : CB 2002 Jul; 12(13).
Larochelle S, Chen J, Knights R, Pandur J, Morcillo P, Erdjument-Bromage H, Tempst P, Suter B, Fisher RP. T-loop phosphorylation stabilizes the CDK7-cyclin H-MAT1 complex in vivo and regulates its CTD kinase activity. The EMBO journal 2001 Jul; 20(14).
Garrett S, Barton WA, Knights R, Jin P, Morgan DO, Fisher RP. Reciprocal activation by cyclin-dependent kinases 2 and 7 is directed by substrate specificity determinants outside the T loop. Molecular and cellular biology 2001 Jan; 21(1).
Lee KM, Saiz JE, Barton WA, Fisher RP. Cdc2 activation in fission yeast depends on Mcs6 and Csk1, two partially redundant Cdk-activating kinases (CAKs). Current biology : CB 1999 Apr; 9(8).
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