Cell-cycle dependence of transcriptome gene modules: comparison of regression lines.

The transcriptome consists of various gene modules that can be mutually dependent, and ignoring these dependencies may lead to misinterpretation. The most important problem is module dependence on cell-cycle activity. Using meta-analysis of over 30 000 single-cell transcriptomes, we show gene module dependencies on cell-cycle signature, which can be consistently observed in various normal and cancer cells. Transcript levels of receptors, plasma membrane, and differentiation-related genes are negatively regressed on cell-cycle signature. Pluripotency, stress response, DNA repair, chromatin remodeling, proteasomal protein degradation, protein network connectivity, and unicellular evolutionary origin are regressed positively. These effects cannot be explained by partial overlap of corresponding gene sets because they remain if the overlapped genes were removed. We propose a visual analysis of gene module-specific regression lines as complement to an uncurated enrichment analysis. The different lines for a same gene module indicate different cell conditions. The approach is tested on several problems (polyploidy, pluripotency, cancer, phylostratigraphy). Intriguingly, we found variation in cell-cycle activity, which is independent of cell progression through the cycle. The upregulation of G2/M checkpoint genes with downregulation of G2/M transition and cytokinesis is revealed in polyploid cells. A temporal increase in cell-cycle activity at transition from pluripotent to more differentiated state is found in human embryonic stem cells. The upregulation of unicellular interactome cluster in human cancers is shown in single cells with control for cell-cycle activity. The greater scatter around regression line in cancer cells suggests greater heterogeneity caused by deviation from a line of normal cells.