Abstract
The pulsatile nature of gene activity has recently emerged as a general property of the transcriptional process. It has been shown that the frequency and amplitude of transcriptional bursts can be subjected to extrinsic regulation. Here we have investigated if these parameters were constant throughout the cell cycle using the single molecule RNA FISH technique. We found evidence of transcriptional spikes upon mitotic exit in three different human cell lines. Recording of cell growth prior to hybridization and immuno-RNA FISH analysis revealed that these spikes were short-lived and subsided before completion of cytokinesis. The transient post-mitotic increase in transcriptional output was found to be the result of cells displaying a higher number of active alleles and/or an increased number of nascent transcripts per active allele, indicating that both the burst fraction and the amplitude of individual bursts can be increased upon mitotic exit. Our results further suggest that distinct regulatory mechanisms are at work shortly after mitotic exit and during the rest of interphase. We speculate that transcriptional spikes are associated with chromatin decondensation, a hallmark of post-mitotic cells that might alter the dynamics of transcriptional regulators and effectors.
Highlights
Single cell studies revealed that transcription of most genes is a discontinuous process, with periods of activity interspersed with periods of inactivity[1]
To image the dynamics of transcriptional pulsing throughout the cell cycle, we performed smRNA FISH on established human cell lines (HepG2, HT-1080 and U-2-OS) using fluorescent oligonucleotide probes against the transferrin receptor (TFRC) and the large subunit of RNA polymerase II (POLR2A)
A cell was considered to be in telophase if it and one of its neighbors showed obvious signs of chromatin decondensation and mitotic exit, i.e. the shape of the DAPI signal was irregular, its intensity lied between that of metaphase and interphase cells, and cells had not yet flattened
Summary
Single cell studies revealed that transcription of most genes is a discontinuous process, with periods of activity interspersed with periods of inactivity[1]. This property, referred to as transcriptional bursting (or pulsing), helps to explain the cell-to-cell variability in the distribution of mRNA counts that is often observed in isogenic cell populations[2]. Numerous studies have investigated gene expression during the cell cycle and subsets of genes that are periodically expressed at one point or another of the cell cycle have been readily identified[18,19,20] Most of these studies relied on measuring steady-state expression levels of cytoplasmic mRNAs in large cell populations, making it impossible to reach conclusions about nascent transcription at the single cell level. Similar observations of dosage compensation after DNA replication were made in yeast cells[24]
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