Transcription dynamics during metabolic changes

Thesis

Temporal control of processes in cells such as metabolism, gene expression, and cell growth and division are ubiquitous in nature but how they are coordinated in single cells is poorly understood. The yeast Saccharomyces cerevisiae offers a tractable system in which to address these relationships, as growth rates, the number of cells in the cell division cycle (CDC), and their metabolism can be varied and linked to patterns of gene expression. Precise numbers of nascent and mature transcripts can be monitored in single cells using single-molecule RNA fluorescence in-situ hybridisation (smRNA-FISH). The Yeast Metabolic Cycle (YMC) is a model system of temporal compartmentalisation of cellular and metabolic processes in slow-growing synchronous cultures and can be compared with data from asynchronous cultures at different growth rates (exponential mid-log versus slow growth) and metabolic states.

These studies reveal that single cells from mid-log and YMC cultures possess an intrinsic metabolic and gene expression clock. Remarkably, individual cells are found to possess a high degree of variability in levels of expression of individual transcripts yet the oscillations in mean transcript levels over time are similar to those described at the population level using RNA-seq. Furthermore, although most yeast genes are proposed to show a constitutive mode of transcription initiation, the transcript distribution of oscillating transcripts along the YMC do not fit those predicted by a one-state mathematical model of constitutive transcription. Instead, the predicted “bursty” mode of expression could explain the variability in transcript levels in single cells. Cell-to-cell transcript heterogeneity was also observed for pre-rRNA transcripts during the YMC, despite population levels oscillating with periods of cell growth and ribosome biogenesis. The link to cell growth is supported by the observation that ribosomal RNA production is found to be temporally coordinated to TORC1-controlled Rps6 phosphorylation.

The examination of DNA content and cell diameters together with transcript numbers in single cells of YMC and mid-log populations indicates distinct sub-populations with different relationships between gene expression, proliferation and growth. In synchronous YMC cultures, metabolism and patterns of gene expression are proposed to alternate synchronously between phases of growth and quiescence, linked to metabolism by high or lower levels of oxygen consumption respectively. This study suggests that the organization of the culture is far more nuanced. It supports at least three distinct co-existing states in the population over each metabolic cycle, including small quiescent-like cells, slow-growing cells, and larger cells whose metabolism is remodelled, with increased oxygen consumption, to facilitate their entry and progression through the cell division cycle.

Authors: Wouters, M

• DOI: 10.5287/ora-oryzjkpvo
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: sub-populations; yeast metabolic cycle; saccharomyces cerevisiae; RNA fluorescence in-situ hybridization; transcription dynamics
• Subjects: Molecular biology