Single-Cell Analysis of Transcriptional Dynamics During Cell Cycle Arrest

Abstract

In the past decade, a challenge to the canonical model of cell cycle transcriptional control has been posed by a series of high-throughput gene expression studies in budding yeast. Using genetic methods to inhibit or lock the activity of the cyclin-CDK/APC oscillator, these population studies demonstrated that a significant proportion of cell cycle transcription persists in the absence of cyclin-CDK/APC oscillations. To account for these findings, a network of serially activating transcription factors with sources of negative feedback from transcriptional repressors (referred to as a \say{TF network}) was proposed to drive cyclin-CDK/APC independent gene expression.However, population studies of cell cycle gene expression are limited due to loss of phase synchrony that limits the timescale of measurement of gene expression and due to expression averaging that limits assessment of heterogeneity of expression within the population. To circumvent these limitations I used a single-cell timelapse microscopy approach to assess transcriptional dynamics of cell cycle regulated genes during extended cell cycle arrests in both the Gl/S and early mitosis (metaphase) phases of the cell cycle. During G1/S arrest, transcriptional dynamics of four cell cycle regulated genes was assessed and activation of out-of-phase cell cycle transcription was observed in two of these genes. Though budding oscillations were observed in G1/S arrested cells, robust transcriptional oscillations were not seen for any of the four genes and budding dynamics were uncoupled from transcriptional dynamics after the first bud emergence. During cell cycle arrest in early mitosis, transcriptional dynamics of ten cell cycle regulated genes was assessed and activation of out-of-phase transcription was observed for four genes. All four genes activated once with canonical ordering but robust oscillations were not observed during mitotic arrest. Together these studies demonstrate activation, but not oscillation, of cell cycle transcription in the absence of cyclin-CDK/APC oscillations.