In this work, we have identified seven consensus clusters of genes, whose transcripts show periodic time-series during both, the 0.7 h 
and the 5 h 
period respiratory oscillations. Specifically, clusters A, AB, B, C and D define a common temporal gene expression program ( & ). Their relation to respiratory activity and their functional enrichment profiles (, S3
) support a distinction of two superclusters. The cell growth supercluster (A
B) is expressed during the oxidative phase, and the energy-mobilizing supercluster (C
D) is expressed in the reductive phase. Each supercluster develops from predominantly TATA-less and TFIID-controlled genes that encode for ribosome biogenesis (A/AB: cytoplasmic or C: mitochondrial), to gene groups that are enriched in TATA Boxes and SAGA-control and encode for metabolic functions (B: amino acid synthesis or D: catabolism and stress-response) ().
Summary of results & proposed feedback model.
Clusters C and D are co-expressed in the 0.7 h but anti-phase in the 5 h system, accompanied by differential regulation of the amphibolic core carbon backbone of metabolism and DNA replication machineries in clusters B.C and B.D. These differences may be due to differential extent of S-phase synchrony (
50%) in the two systems. This difference is reflected in differential association of average cluster C transcript levels in the transcription factor mutant dataset of 
). Genes encoding for mitochondrial functions, i
.e., cluster C, were switched from wide (cluster A-like) to narrow NDR configurations, concurrent with the evolution of the respiro-fermentative lifestyle after a whole genome duplication event 
, and could also be distinguished in a detailed analysis of stress-response cohorts 
. In line with the direct feedback interactions discussed below, mitochondrial activity, reactive oxygen species or, more specifically, NAD+
-mediated regulation of both chromatin 
and the flux direction along the core carbon backbone 
may well play a role for the differential regulation. Further data on systems with different extent of S-phase synchrony or an experimental system to reproducibly vary the oscillation periods will be required to go beyond this only descriptive discussion of the differences between the two analyzed systems.
Common to both systems, however, is the antiphase relation of the two superclusters. This and their anti-correlation in our transcriptome meta-analysis () and the correlation with the “environmental stress response” 
() point to a common regulator with opposing effects on the expression of the two superclusters. A detailed analysis of the functional annotations of co-regulated gene groups lead to an interpretation of the stress response as a general reaction to energy-limitation, where the costly translation program is downregulated and concurrently energy-mobilizing processes are upregulated 
. We have previously shown that various measures of the energetic flux of the cell strongly vary over the cycle, e
.g., the cytochrome oxidation state and mitochondrial morphology 
. Anabolism is, however, energetically driven by a concentration gradient between ATP and ADP. We report here an oscillation of the ATP:ADP ratio () that is compatible with this energetic interpretation of the stress response. When ATP:ADP is high (
5–6), the growth supercluster is expressed. A subsequent activity of this growth program, concurrent with low respiratory activity, would explain the decrease of the ATP:ADP ratio in the reductive phase (down to
1–2). This phase is paralleled by increase in expression of catabolic and respiratory genes whose activity subsequently would replenish ATP in the next cycle. These consequences of the metabolic activity of the two superclusters are depicted as positive or negative influence on ATP in . Could, in turn, the energetic state or specifically the ATP:ADP ratio directly and differentially feed back on the expression of the anabolic and catabolic superclusters?
Such a direct feedback between energetic state and gene expression is known from bacteria, where the ATP:ADP ratio correlates with the extent of negative supercoiling that is introduced by ATP-dependent gyrase 
which in turn differentially affects transcription of the gene encoding for the gyrase 
and for anabolic and catabolic enzymes 
. While in Escherichia coli
the resulting feedback was interpreted in terms of a homeostatic regulation system, rhythmic changes in DNA structure were observed over the circadian cycle of the cyanobacterium Synechococcus elongatus
. Negative supercoiling is increased during the photosynthetic phase and is required for transcription from GC-rich genes 
. In our system, all clusters are significantly enriched in one of four distinct promoter nucleosome configurations ( & ) 
. Nucleosome occupancy partially depends on sequence properties, e
.g., the GC-content 
. Cluster A transcripts are purine-rich and cluster D genes are GC-rich (). Thus, the clusters may differ in sequence-dependent “default” nucleosome configurations or overall occupancy, which is also reflected in the differential in vitro
occupancy () 
and could lay the grounds for differential regulation. A candidate mechanism is ATP-dependent nucleosome remodeling, where ATP hydrolysis provides the mechanical force to generate negative superhelical torque 
and break DNA-histone contacts 
. The addition of ATP to naked DNA, histones and cell extract allowed the in vitro
reconstitution of in vivo
promoter nucleosome configurations, suggesting a major role of ATP-dependent remodeling in the establishment and maintenance of different types of promoter nucleosome configuration 
. The differential consequences of promoter nucleosome remodeling by the RSC- and Isw2-types of remodeling machineries, and their differential association with cluster genes (, , S5
) elegantly complement the proposed feedback model between anabolic and catabolic pathways (). At high ATP:ADP ratio, RSC would keep promoters of anabolic genes open and competent for transcription, while Isw2 would actively repress catabolic gene promoters. When the ATP:ADP ratio drops both remodelers may become less active, and gene expression would switch from growth to catabolic genes. ADP promotes the dissociation of Isw2 from DNA 
, further supporting a direct influence of the ATP:ADP ratio. In this scenario, ATP-dependent nucleosome remodeling literally gates gene expression by opening or closing promoter regions apt to the current energetic state of the cell. However, the diverse targets of RSC remain elusive and are difficult to establish experimentally 
. Interestingly, the step-length of RSC-mediated remodeling, i
.e., the distance over which a given nucleosome is moved along the DNA in one remodeling cycle, has recently been observed to depend on the ATP concentration in vitro
, which in vivo
could lead to differential rotational positioning, and thus exposure or covering, of regulatory motifs 
such as the TATA-Box in the metabolic cohorts B and D of the two superclusters. Oscillating levels of acetyl-CoA- and SAGA-dependent histone acetylation have been found to enable rapid transcription of growth genes (clusters AB, B) in the oxidative phase, while the SAGA complex binds to stress-regulated genes (D) during the reductive phase of a
5 h oscillation 
. Thus, RSC and SAGA, or ATP-dependent nucleosome remodeling and acetyl-CoA-dependent histone acetylation, may cooperate 
at both anabolic and catabolic gene clusters, and relate the metabolic state of the cell to an appropriate transcriptional output.
Nucleotide content & in vitro nucleosome occupancy.
The combined dataset provided by Badis et al.
clearly shows that indeed differential promoter occupancy of the cluster genes is associated with differential transcript levels ( & S13
), where the observed effects reach well beyond local binding sites of the tested mutants of DNA-binding proteins. The diverse detail observations in this dataset point to further processes involved. Upstream non-coding and antisense transcription around the stress-activated clusters D and B.D indicate a role of noncoding RNA transcription 
, potentially in transcriptional silencing 
. And finally, the global bias in nucleosome occupancy (
in all clusters) or positioning (periodic
Nucl.Occ. downstream of TSS) in some of the mutants may point towards genome-wide chromatin re-arrangements. We interpret this as further strong evidence of genome-wide chromatin remodeling cycles and complex transcriptional landscapes during the respiratory oscillation.
In summary, our systematic statistical comparison of large data compendia provide an integrated perspective on the possible interactions between metabolism, chromatin structure and transcription. Such direct links between central metabolism and chromatin dynamics have recently been implicated also in mammalian regulatory systems such as the circadian clock 
and cancer 
. Here, we proposed an analogy in prokaryote systems, i
.e., the circadian supercoiling dynamics in cyanobacteria 
. For the case of respiratory oscillation in yeast continuous culture, we defined a gene expression program () that is coherent in both, function and time, and proposed a first mechanistic interpretation of not only the oscillatory gene expression common to the 0.7 h and 5 h systems, but also for the often observed, yet still enigmatic stress response of transcription (). We expect that ATP-dependent nucleosome remodeling plays an important role, most likely in interaction with the co-factor dependences of post-translational histone modifications 
. It has recently been proposed that even in the absence of culture synchrony, individual cells may always undergo an oscillatory growth program, and that a given sample merely reflects a mixture of cells that are in either the reductive or the oxidative phase. An observed stress response would then just reflect a decreased overall growth rate where individual cells remain longer in the reductive phase 
. This would require a re-interpretation of all previous experiments on steady-state and batch cultures, including all chromatin-structural data analyzed herein. Our analysis and interpretations are fully compatible with this hypothesis. Time series data on chromatin structure over the respiratory cycle will be required to understand the dynamics of local and global chromatin and transcription landscapes. We predict that oscillatory continuous culture will become an invaluable experimental system for an integrative mechanistic understanding of both chromatin biology and growth regulation, since the synchronized culture naturally cycles between transcription from genes with both, complementary functions in cellular growth, and differential chromatin structure and dynamics.