Mitotic transcription and waves of gene reactivation during mitotic exit

Abstract

Although the genome is generally thought to be transcriptionally silent during mitosis, technical limitations have prevented sensitive mapping of transcription during mitosis and mitotic exit. Thus, the means by which the interphase expression pattern is transduced to daughter cells have been unclear. We used 5-ethynyluridine to pulse-label transcripts during mitosis and mitotic exit and find that many genes exhibit transcription during mitosis, as confirmed by FITC-UTP labeling, RNA FISH, and RT-qPCR. The first round of transcription immediately following mitosis primarily activates genes involved in the growth and rebuilding of daughter cells, rather than cell type-specific functions. We propose that the cell’s transcription pattern is largely retained at a low level through mitosis, whereas the amplitude of transcription observed in interphase is re-established during mitotic exit.

Low-Level, Global Transcription during Mitosis and Dynamic Gene Reactivation during Mitotic Exit

“…If the entirety of the mitotic signal was due to an interphase population, then we would expect the relative rank of genes within this population to be identical to that of the asynchronous population. However, a pairwise analysis of the Spearman rank correlation coefficient of all mitotic and asynchronous replicates indicated that the two populations were distinct (Palozola et al 2017). From these studies and other controls (Palozola et al 2017) we conclude that mitosis is not a period of transcriptional silence, as has long been considered the case.…”

“…Of these, 8074 were reproducibly expressed among all three mitotic replicates, accounting for 28% of the genes expressed in an asynchronous population. However, the transcriptome was expressed much lower in mitotic than in asynchronous cells, with a fivefold mean decrement in transcript levels (Palozola et al 2017). It is important to note that this low-level transcription is not purely the result of the ∼3% contaminating interphase cells in the mitotic population.…”

“…After global normalization between the asynchronous and mitotic samples, based on the relative spike-in sequence amplification in each replicate, there were approximately 28,000 transcripts expressed in the asynchronous population (Palozola et al 2017). Of these, 8074 were reproducibly expressed among all three mitotic replicates, accounting for 28% of the genes expressed in an asynchronous population.…”

“…To assess the profile of global mitotic transcription, we developed EU-RNA-seq as a way to capture and sequence nascent RNAs in vivo (Yokoyama et al 2016;Palozola et al 2017). Mitotic and asynchronous cells were pulselabeled with the cell permeable uridine analog, 5′-ethynyluridine (EU) for 40 min.…”

“…The total RNA from each sample was harvested and a click reaction was performed to allow the conjugation of biotin azide to the ethynyl group of EU (Jao and Salic 2008) on any EU-RNAs present in the total RNA harvested. Once the click reaction was complete, we added custom, biotin-RNA spike-in controls to both mitotic and asynchronous samples (Palozola et al 2017), and then streptavidin-coated magnetic beads were used to isolate biotinylated RNAs from unlabeled RNAs in each sample. We were then able to generate cDNA libraries directly off the magnetic beads for sequencing.…”