Derivation of the human cell cycle transcriptional signature

Abstract

Duplication of the genome and successful mitotic cell division requires the coordinated activity of hundreds of proteins. Many are known, but a complete list of the components of the cell cycle machinery is still lacking. This thesis describes a series of data driven analyses to assemble a comprehensive list of genes induced during the human cell cycle. To start with, a meta-analysis of previous transcriptomics studies revealed a larger number of cell cycle genes consistently expressed across multiple human cell types than previously reported. Following this observation, the cell cycle transcriptome was further investigated with the generation of a new time-course microarray dataset on normal human dermal fibroblasts (NHDF) undergoing synchronised cell division. Network cluster analysis of these data identified transcripts whose expression was associated with different stages of cell cycle progression. Co-expression of these transcripts was then analysed using a complementary dataset that included genome-wide promoter expression of a wide range of human primary cells. This resulted in the identification of a core set of 545 cell cycle genes, mainly associated with G1/S to M phases, which showed a high degree of co-expression across all cell types. Expression of 75% of these genes was also found conserved in mouse, as revealed by the analysis of a new microarray experiment generated from mouse fibroblasts. Gene Ontology and motif enrichment analysis validated the list with significant enrichments for terms and transcription factor biding sites linked with cell cycle biology. Toward a better interpretation of these 545 genes, a meticulous manual annotation exercise was carried out. Unsurprisingly, the majority of these genes were known to be involved in S and M phases-associated processes, however 50 genes were functionally uncharacterised. A subset of 36 of these were then taken forward for subcellular localisation assays. These studies were performed by transfection of human embryonic kidney cells (HEK293T) with GFP-tagged cDNA clones leading to the finding of four uncharacterised proteins co-localising with the centrosome, a crucial organelle for normal cell cycle progression. This thesis represents an attempt in documenting the genes actively transcribed and therefore likely involved in the processes associated with cell cycle, hence providing a comprehensive catalogue of its key components. In so doing, I have also identified a significant number of new genes likely to contribute to this central process vital in health and disease.