The quantitative landscape of the mitotic checkpoint: from genes to function

Virginia Polytechnic Institute And State University, Blacksburg VA

Investigators

Abstract

Regulation of the cell cycle is critical for the growth and development of all organisms and the information needed to control this process is encoded in the DNA of the genome. The genome not only specifies which biomolecules are being produced, but also specifies their quantities, which is important since too little or too much of a biomolecule can lead to cellular malfunction. Despite this importance, it is still incompletely understood how the information regulating levels of biomolecules is specified in the genome sequence. This project will examine one particular regulatory pathway, the "mitotic checkpoint", which controls the inheritance of chromosomes during cellular division. This pathway only functions properly if the proteins that make up the pathway are present at the optimal concentrations. By combining laboratory experiments and computational analyses, this project will identify critical regions on the genome that control the quantity of proteins in the mitotic checkpoint and thereby control checkpoint function. This research will enhance our ability to interpret and purposefully modify genome sequences, with benefits for gene technology and synthetic biology. Furthermore, the project will provide training opportunities for graduate and undergraduate students that prepare them for careers in biotechnology or basic research and enhance their skills to work at the interface of traditional disciplines. Critical transitions in the eukaryotic cell cycle are guarded by checkpoints. The mitotic checkpoint prevents the precocious separation of duplicated chromosomes and thereby protects genome integrity. To function properly, the mitotic checkpoint needs precise concentrations of the proteins that act in this signaling pathway. Yet, hardly anything is known about how transcription and translation of the checkpoint genes is quantitatively controlled to yield these levels. This work will determine how checkpoint protein levels are kept within their permissive range and why checkpoint functionality is restricted to this range. Cas9-mediated genome engineering in fission yeast will be used to map the genetic regions that are critical to control checkpoint protein levels. This experimental analysis will be complemented by a bioinformatic analysis of these genes. Checkpoint functionality assays will be used to address how different checkpoint protein levels affect the activity and critical dynamics of the checkpoint. These results will be combined into a predictive computational model for checkpoint activity that will shed light on how this checkpoint operates. Overall, this project will provide new insights into the dynamics of checkpoint signaling and into the relation between genotype, quantitative gene expression control and phenotype.