Dynamics of oscillatory phenotypes in Saccharomyces cerevisiae reveal a network of genome-wide transcriptional oscillators.

Genetic and environmental factors are well-studied influences on phenotype; however, time is a variable that is rarely considered when studying changes in cellular phenotype. Time-resolved microarray data revealed genome-wide transcriptional oscillation in a yeast continuous culture system with ∼ 2 and ∼ 4 h periods. We mapped the global patterns of transcriptional oscillations into a 3D map to represent different cellular phenotypes of redox cycles. This map shows the dynamic nature of gene expression in that transcripts are ordered and coupled to each other through time and concentration space. Although cells differed in oscillation periods, transcripts involved in certain processes were conserved in a deterministic way. When oscillation period lengthened, the peak to trough ratio of transcripts increased and the fraction of cells in the unbudded (G0/G1) phase of the cell division cycle increased. Decreasing the glucose level in the culture medium was one way to increase the redox cycle, possibly from changes in metabolic flux. The period may be responding to lower glucose levels by increasing the fraction of cells in G1 and reducing S-phase gating so that cells can spend more time in catabolic processes. Our results support that gene transcripts are coordinated with metabolic functions and the cell division cycle.

Collective behavior in gene regulation: the cell is an oscillator, the cell cycle a developmental process.

The finding of a genome-wide oscillation in transcription that gates cells into S phase and coordinates mitochondrial and metabolic functions has altered our understanding of how the cell cycle is timed and how stable cellular phenotypes are maintained. Here we present the evidence and arguments in support of the idea that everything oscillates, and the rationale for viewing the cell as an attractor from which deterministic noise can be tuned by appropriate coupling among the many feedback loops, or regulons, that make up the transcriptional-respiratory attractor cycle. The existence of this attractor also explains many of the dynamic macroscopic properties of the cell cycle and appears to be the timekeeping oscillator in both cell cycles and circadian rhythms. The path taken by this primordial oscillator in the course of differentiation or drug response may involve period-doubling behavior. Evidence for a relatively high-frequency timekeeping oscillator in yeast and mammalian cells comes from expression array analysis, and GC/MS in the case of yeast, and primarily from macroscopic measures of phase response to perturbation in the case of mammalian cells. Low-amplitude, genome-wide oscillations, a ubiquitous but often unrecognized attribute of phenotype, may be a source of seemingly intractable biological noise in microarray and proteomic studies. These oscillations in transcript and protein levels and the repeated cycles of synthesis and degradation they require, represent a high energy cost to the cell which must, from an evolutionary point of view, be recovered as essential information. We suggest that the information contained in this genome-wide oscillation is the dynamic code that organizes a stable phenotype from an otherwise passive genome.

Signal processing and the design of microarray time-series experiments.

Recent findings of a genome-wide oscillation involving the transcriptome of the budding yeast Saccharomyces cerevisiae suggest that the most promising path to an understanding of the cell as a dynamic system will proceed from carefully designed time-series sampling followed by the development of signal-processing methods suited to molecular biological datasets. When everything oscillates, conventional biostatistical approaches fall short in identifying functional relationships among genes and their transcripts. Worse, based as they are on steady-state assumptions, such approaches may be misleading. In this chapter, we describe the continuous gated synchrony system and the experiments leading to the concept of genome-wide oscillations, and suggest methods of analysis better suited to dissection of oscillating systems. Using a yeast continuous-culture system, the most precise and stable biological system extant, we explore analytical tools such as wavelet multiresolution decomposition, Fourier analysis, and singular value decomposition to uncover the dynamic architecture of phenotype.

A rapid genome-scale response of the transcriptional oscillator to perturbation reveals a period-doubling path to phenotypic change.

Perturbation of the gated-synchrony system in yeast with phenelzine, an antidepressant drug used in the treatment of affective disorders in humans, leads to a rapid lengthening in the period of the genome-wide transcriptional oscillation. The effect is a concerted, genome-scale change in expression that is first seen in genes maximally expressed in the late-reductive phase of the cycle, doubling the length of the reductive phase within two cycles after treatment. Clustering of genes based on their temporal patterns of expression yielded just three super clusters whose trajectories through time could then be mapped into a simple 3D figure. In contrast to transcripts in the late-reductive phase, most transcripts do not show transients in expression relative to others in their temporal cluster but change their period in a concerted fashion. Mapping the trajectories of the transcripts into low-dimensional surfaces that can be represented by simple systems of differential equations provides a readily testable model of the dynamic architecture of phenotype. In this system, period doubling may be a preferred pathway for phenotypic change. As a practical matter, low-amplitude, genome-wide oscillations, a ubiquitous but often unrecognized attribute of phenotype, could be a source of seemingly intractable biological noise in microarray studies.

A genomewide oscillation in transcription gates DNA replication and cell cycle.

Microarray analysis from a yeast continuous synchrony culture system shows a genomewide oscillation in transcription. Maximums in transcript levels occur at three nearly equally spaced intervals in this approximately 40-min cycle of respiration and reduction. Two temporal clusters (4,679 of 5,329) are maximally expressed during the reductive phase of the cycle, whereas a third cluster (650) is maximally expressed during the respiratory phase. Transcription is organized functionally into redox-state superclusters with genes known to be important in respiration or reduction being synthesized in opposite phases of the cycle. The transcriptional cycle gates synchronous bursts in DNA replication in a constant fraction of the population at 40-min intervals. Restriction of DNA synthesis to the reductive phase of the cycle may be an evolutionarily important mechanism for reducing oxidative damage to DNA during replication.

Generation and maintenance of synchrony in Saccharomyces cerevisiae continuous culture.

Cultures of Saccharomyces cerevisiae grown continuously produce an autonomous oscillation in many metabolic outputs. The most conveniently measured variable, i.e., dissolved oxygen concentration, oscillates with a period of 40-55 min. Previously we have identified two compounds capable of resetting phase, acetaldehyde and hydrogen sulfide. The phase-response curves constructed for acetaldehyde show a strong (Type 0) response at 3.0 mM and a weak (Type 1) response at 1.0 mM. Ammonium sulfide phase-response curves (pulse injected at 1.0 microM and 3.0 microM) revealed that sulfide is only an effective perturbation agent when endogenous sulfide concentrations are at a maximum. Also only Type 1 phase responses were observed. When the phase-response curve for sulfite (at 3.0 M) was constructed, phase responses were at a maximum at 60 degrees, indicating the possible involvement of sulfite in cell synchronization. It is concluded that endogenously produced acetaldehyde and sulfite tune the oscillation of mitochondrial energization state whereas sulfide mediates population synchrony.