Robert P Fisher, MD, PhD

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