"Omics” analysis of mutations C. elegans and mice

Genetic mutations in humans, mice, and other organisms can lead to different outcomes. At times, distinct phenotype can be readily observed. Nevertheless, some of these phenotypes are difficult to study since mutations can result in lethality or prevent cells from proliferating.  On the other hand, in many cases no obvious phenotype can be observed. The simplistic conclusion is that such genes are not essential but this does not mean that such a gene has no important functions. Given that evolution conserves only genes that contribute to the fitness of organisms, the functions of ‘non-essential’ genes are likely compensated by homologous genes.
We aim to address this fundamental question focusing on cell cycle regulation and by combining mouse and C. elegans genetics and genomics. The generation time of these organisms is different and C. elegans, which has a live cycle of 3-4 days, can also be studied when phenotypes only become apparent after several generations. We will take advantage of well-characterized mouse knockouts that are viable. For example, Cdk2KO mice are viable but are sterile1. We have shown that the functions of Cdk2 are compensated by Cdk12 and Cdk43 but we cannot exclude other genes too. Using CRISPR/Cas9 technology we will make either deletions, kinase-dead mutations, and mutations changing key regulatory residues
(tyrosine 15) in C. elegans Cdks (and cyclin genes). Once these Cdk2 mutants are made, we will analyse single, double and triple mutant phenotypes. In case of immediate lethality of single or compound double mutants, we will analyse the exact nature of the cell cycle defects.  Double and triple mutants can easily be generated in the worm and allow for uncovering redundant pathways. In case of lethality conferred by Cdk kinase dead mutations, we will generate point mutations in the ATP binding sites that allow for rapid and specific inhibition by the addition of appropriate ATP analogues (Shockat approach4). Thus genes can be conditionally switched off and the consequences of this can be studied at various
developmental stages. Viable worm Cdk and cyclin mutations will be followed for up to 40 generation to assess the consequences of reduced robustness of the core cell cycle regulatory circuits. These measurements will include progressively reduced reproductive capacity and chromosome mis-segregation as well as genetic integrity. To measure genetic integrity, next generation sequencing of the entire genome will be conducted after the first, 20th and 40th
generation to see if the rate of mutagenesis, which is typically at about 1 nucleotide per worm generation, is increased. The Gartner lab in collaboration with the Sanger Institute already set up appropriate experimental procedures, which in principle can also be adapted to mouse tissue culture cells. Results obtained in the C. elegans system will guide our efforts to investigate analogous defects in the corresponding mouse mutations, many of which are already available in the Kaldis laboratory. Besides addressing the consequence of reducing the robustness of cell cycle regulation, we also envision that we might uncover novel unexpected connections between cell cycle regulation and other cellular processes.

1 Berthet, C., Aleem, E., Coppola, V., Tessarollo, L. & Kaldis, P. Cdk2 knockout mice are viable. Curr Biol 13, 1775-1785 (2003).
2 Aleem, E., Kiyokawa, H. & Kaldis, P. Cdc2-cyclin E complexes regulate the G1/S phase transition. Nat Cell Biol 7, 831-836 (2005).
3 Berthet, C. et al. Combined loss of Cdk2 and Cdk4 results in embryonic lethality and Rb hypophosphorylation. Dev Cell 10, 563-573 (2006).
4 Specht, K. M. & Shokat, K. M. The emerging power of chemical genetics. Curr Opin Cell Biol 14, 155-159 (2002).

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