Distinguishing between stochasticity and determinism: Examples from cell cycle duration variability.

We describe a recent approach for distinguishing between stochastic and deterministic sources of variability, focusing on the mammalian cell cycle. Variability between cells is often attributed to stochastic noise, although it may be generated by deterministic components. Interestingly, lineage information can be used to distinguish between variability and determinism. Analysis of correlations within a lineage of the mammalian cell cycle duration revealed its deterministic nature. Here, we discuss the sources of such variability and the possibility that the underlying deterministic process is due to the circadian clock. Finally, we discuss the "kicked cell cycle" model and its implication on the study of the cell cycle in healthy and cancerous tissues.

Single-cell analysis of circadian dynamics in tissue explants.

Tracking molecular dynamics in single cells in vivo is instrumental to understanding how cells act and interact in tissues. Current tissue imaging approaches focus on short-term observation and typically nonendogenous or implanted samples. Here we develop an experimental and computational setup that allows for single-cell tracking of a transcriptional reporter over a period of >1 wk in the context of an intact tissue. We focus on the peripheral circadian clock as a model system and measure the circadian signaling of hundreds of cells from two tissues. The circadian clock is an autonomous oscillator whose behavior is well described in isolated cells, but in situ analysis of circadian signaling in single cells of peripheral tissues is as-yet uncharacterized. Our approach allowed us to investigate the oscillatory properties of individual clocks, determine how these properties are maintained among different cells, and assess how they compare to the population rhythm. These experiments, using a wide-field microscope, a previously generated reporter mouse, and custom software to track cells over days, suggest how many signaling pathways might be quantitatively characterized in explant models.

A positive feedback loop links circadian clock factor CLOCK-BMAL1 to the basic transcriptional machinery.

Circadian clocks in mammals are built on a negative feedback loop in which the heterodimeric transcription factor circadian locomotor output cycles kaput (CLOCK)-brain, muscle Arnt-like 1 (BMAL1) drives the expression of its own inhibitors, the PERIOD and CRYPTOCHROME proteins. Reactivation of CLOCK-BMAL1 occurs at a specific time several hours after PERIOD and CRYPTOCHROME protein turnover, but the mechanism underlying this process is unknown. We found that mouse BMAL1 complexes include TRAP150 (thyroid hormone receptor-associated protein-150; also known as THRAP3). TRAP150 is a selective coactivator for CLOCK-BMAL1, which oscillates under CLOCK-BMAL1 transcriptional control. TRAP150 promotes CLOCK-BMAL1 binding to target genes and links CLOCK-BMAL1 to the transcriptional machinery at target-gene promoters. Depletion of TRAP150 caused low-amplitude, long-period rhythms, identifying it as a positive clock element. The activity of TRAP150 defines a positive feedback loop within the clock and provides a potential mechanism for timing the reactivation of circadian transcription.

The critical discussion group: fostering personal and scientific growth.

As scientists, we greatly benefit from discussing our work with our peers. Informal, unstructured interactions often yield highly creative feedback. With this in mind, we created a group that fosters discussion of its members' work. The group engages us in new research fields and ways of thinking, and provides us with an opportunity for co-mentoring. Three key components were essential for making this group work: the emphasis on non-hierarchical debate; the diversity of the group members; and the mutual respect existing among the participants.

Shifts in replication timing actively affect histone acetylation during nucleosome reassembly.

The entire genome is replicated in a programmed manner, with specific regions undergoing DNA synthesis at different times in S phase. Active genes generally replicate in early S phase, while repressed genes replicate late, and for some loci this process is developmentally regulated. Using a nuclear microinjection system, we demonstrate that DNA sequences originally packaged into nucleosomes containing deacetylated histones during late S become reassembled with acetylated histones after undergoing replication in early S. Conversely, a change from early to late replication timing is accompanied by repackaging into nucleosomes containing deacetylated histones. This is carried out by differential cell-cycle-controlled acetylation and deacetylation of histones H3 and H4. These studies provide strong evidence that switches in replication timing may play a role in the regulation of nucleosome structure during development.

Role of DNA methylation in stable gene repression.

A large fraction of the animal genome is maintained in a transcriptionally repressed state throughout development. By generating viable Dnmt1(-)(/)(-) mouse cells we have been able to study the effect of DNA methylation on both gene expression and chromatin structure. Our results confirm that the underlying methylation pattern has a profound effect on histone acetylation and is the major effector of me-H3(K4) in the animal genome. We demonstrate that many methylated genes are subject to additional repression mechanisms that also impact on histone acetylation, and the data suggest that late replication timing may play an important role in this process.

Silence of the genes--mechanisms of long-term repression.

A large fraction of genes in the mammalian genome is repressed in every cell throughout development. Here, we propose that this long-term silencing is carried out by distinct molecular mechanisms that operate in a global manner and, once established, can be maintained autonomously through DNA replication. Both individually and in combination these mechanisms bring about repression, mainly by lowering gene accessibility through closed chromatin structures.