Robert Fisher, MD, PhD

Cell Cycle Arrest in CAK-deficient Fission Yeast Cells Cells lacking CAK, shown here stained with anti-tubulin antibodies, are unable to divide but continue to grow to cell lengths many times that of wild type cells.

How do cells decide to divide? How is cell division coordinated with cell growth, and in multicellular organisms, with development, differentiation and death? How is that coordination disrupted or disturbed in cancer cells? By understanding the molecular mechanisms that control the cell division cycle, we hope to gain insights into these fundamental questions.

At the heart of the cellular machinery governing cell division are the cyclin-dependent kinases (CDKs), key regulatory enzymes that trigger all the major transitions of the eukaryotic cell cycle, including the onset of DNA replication, and both entry into and exit from mitosis. A major focus of our laboratory is the molecular mechanisms that regulate the periodic activation and inactivation of CDKs to ensure the orderly and coordinated progression through the phases of the cell cycle.

	[Enlarge Image] A network of partially redundant CAKs in fission yeast The Mcs6 complex is homologous to metazoan Cdk7, whereas Csk1 is the ortholog of budding yeast Cak1. Inactivation of both is required to block activation of Cdc2 and prevent G1/S or G2/M progression.

Precise control of cell division depends on the sequential activation of distinct CDKs at different points in the cell cycle. All CDKs, however, share a common mechanism of activation, which requires binding of a positive regulatory subunit — the cyclin — and phosphorylation of a conserved threonine (or serine) residue by a CDK-activating kinase (CAK). Surprisingly, the conserved phosphorylation step in CDK activation is carried out by divergent enzymes in different species. In metazoans, including humans and Drosophila (fruit flies), CAK is itself a CDK, consisting of the Cdk7 catalytic subunit in complexes with cyclin H and an assembly factor, Mat1. In contrast, the CAK of the budding yeast Saccharomyces cerevisiae is a distinct, single-subunit (and thus, cyclin-independent) kinase.

We have shown that the fission yeast, Schizosaccharomyces pombe, has evolved a strategy for activating CDKs based on two partially redundant enzymes, one of which resembles metazoan CAK, whereas the other is more like budding yeast CAK. Because CDK activation in fission yeast appears to have more in common with the same pathway in higher eukaryotes, we are pursuing both genetic and biochemical strategies to elucidate the regulation of the two CAKs of S. pombe.

[Enlarge Image] The CAK network: linking cell division with transcription In fission yeast, the CAK-CDK network controls the G1/S and G2/M transitions, but also coordinates transcriptional programs important at a later stage of cell division.

A long-term goal of our laboratory is to understand how decisions concerning cell division are made within the context of cell growth and differentiation. The CDK activation pathway may hold crucial clues as to how this is achieved at the molecular level. Cdk7/Mcs6, in addition to its direct role in activating CDKs, plays a critical role in the transcription of mRNAs as the component of TFIIH required to phosphorylate the carboxyl-terminal domain (CTD) of Pol II. Why does the cell use the same enzyme to perform two seemingly unrelated regulatory functions? A possibility we are actively exploring is that Cdk7/Mcs6 plays a role in coordinating cell growth — which depends on mRNA synthesis — with cell division, which depends on CDK activation.

	[Enlarge Image] Mechanisms of transcriptional repression by chromatin Packaging into nucleosomes can: 1) occlude key DNA-binding sites; 2) sterically block elongation by Pol II; or 3) generate negative (and positive) signals (a histone code) written by histone-modiying enzymes and read by transcriptional regulators.

In vivo, Pol II must contend with the generally repressive packaging of DNA into chromatin in order to transcribe protein-coding genes. The wrapping of the DNA around nucleosomes in chromatin poses barriers to Pol II transcription by: 1) occlusion of binding sites on the DNA for activators and components of the core Pol II machinery; 2) steric blockade of Pol II elongation through nuclosomes; and 3) by covalent modifications of the core histones (building blocks of the nucleosomes), which can either repress or promote transcription. Although Pol II-mediated transcription responsive to activator proteins has been reconstituted and extensively characterized on naked templates in vitro, the requirements for regulated transcription of chromatin templates is still incompletely understood. We have identified the histone chaperone and suspected oncogene product TAF-I/SET/INHAT as a factor required to remodel chromatin templates in order to facilitate their activated transcription by Pol II in vitro.