Decades of research in human cells and model organisms have characterized a set of core components of the cell cycle regulatory network, which includes cyclins, CDKs, and the APC/C. However, extending our understanding the cell cycle regulatory network in higher eukaryotes faces several major challenges. One challenge is the diversity of cell cycle regulation in different tissue and developmental contexts. A second challenge is using experimental approaches that reflect in vivo biology as closely as possible. A third challenge is developing unbiased, large-scale assays to comprehensively characterize cell cycle regulatory networks at a system-wide level.
We have tried to meet these challenges by using large-scale MS-based proteomics and RNA-Seq to characterize gene expression in a minimally perturbed cell cycle in a myeloid leukemia cell line. Populations of cells enriched for different cell cycle stages were obtained using centrifugal elutriation. This avoids complications of indirect effects on gene expression arising from either genetic depletion of key cell cycle factors, inhibition of metabolism or DNA synthesis, or activation of cell cycle checkpoints. This data set, one of the deepest surveys to date of gene expression in human cells, is presented in an online, searchable database, the Encyclopedia of Proteome Dynamics (http://www.peptracker.com/epd/).
We identify myeloid-specific gene expression and variations in protein abundance, isoform expression and phosphorylation at different cell cycle stages. We dissect the relationship between protein and mRNA levels for both bulk gene expression and for over ~6,000 genes individually across the cell cycle, revealing complex, gene-specific patterns. We highlight ARHGAP11A, a novel cell cycle regulated protein whose abundance is low in G1 and peaks in G2 and M, and show that the levels of ARHGAP11A protein are regulated by targeted degradation by the APC/C.
We are continuing to analyse variations in phosphorylation levels and other post translational modifications across the minimally perturbed cell cycle. We are also interested in comparing the minimal perturbation centrifugal elutriation with more commonly used synchronization approaches, including checkpoint activation (serum starvation, hydroxyurea, small molecule CDK1 inhibitors), and arrest-and-release (thymidine block).