Expression profiling by high throughput sequencing
Summary
The molecular circadian clock, which controls rhythmic 24-hour oscillation of genes, proteins, and metabolites, is disrupted across many human cancers. Deregulated expression of MYC oncoprotein has been shown to alter expression of molecular clock genes, leading to a disruption of molecular clock oscillation across cancer types. However, it remained unclear how this loss of molecular clock oscillation impacted global gene expression and metabolism in cancer, and what benefit cancer cells might gain from suppressing clock oscillation. We hypothesized that MYC suppresses oscillation of gene expression and metabolism to instead upregulate pathways involved in biosynthesis in a static, non-oscillatory fashion. To test this, we utilized cells from distinct cancer types with inducible MYC or the closely related N-MYC to determine, using detailed time-series RNA-sequencing and metabolomics, the extent to which MYC activation disrupts global oscillation of genes, gene expression, programs, and metabolites. We focused our analyses on genes, pathways, and metabolites that changed in common across multiple cancer cell line models. We report here that MYC disrupted over 85% of oscillating genes, while instead promoting enhanced ribosomal and mitochondrial biogenesis and suppressed cell attachment pathways. Notably, when MYC is activated, biosynthetic programs that were formerly circadian flipped to being upregulated in an oscillation-free manner. Further, activation of MYC ablates the oscillation of nutrient transporter glycosylation while greatly upregulating transporter expression, cell surface localization, and intracellular amino acid pools. Finally, we report that MYC disrupts metabolite oscillations and the temporal segregation of amino acid metabolism from nucleotide metabolism. Our results demonstrate that MYC disruption of the molecular circadian clock releases metabolic and biosynthetic processes from circadian control, which may provide a distinct advantage to cancer cells.
Overall design
We activated N-MYC-ER in cells for 24 hours with 4-hydroxytamoxifen (4OHT, MYC-ON), or used vehicle control (MYC-OFF). We then entrained cells circadian rhythms with dexamethasone. 24 hours after dexamethasone treatment, cells were then collected every 4 hours for up to 52 hours, and RNA was extracted. CT refers to circadian time, ie, hours after dexamethasone entrainment. RNA at approximately 37 ng/µL was submitted to Novogene Co., was analyzed by BioAnalyzer and determined to have an average RNA Integrity Number (RIN) of 9.8. RNA-sequencing was performed on polyadenylated mRNAs using an Illumina NovaSeq 6000 as 150 base paired end sequencing, yielding an average of 54.7 million reads per sample (when adding together both paired ends). Reads were mapped with Salmon, and collapsed to gene level with Tximport. Two biological replicate experiments were performed in SHEP, and a single replicate was performed in SKNAS.
Less...
More...