Regulated large-scale nucleosome density patterns and precise nucleosome positioning correlate with V(D)J recombination.

We show that the physical distribution of nucleosomes at antigen receptor loci is subject to regulated cell type-specific and lineage-specific positioning and correlates with the accessibility of these gene segments to recombination. At the Ig heavy chain locus (IgH), a nucleosome in pro-B cells is generally positioned over each IgH variable (VH) coding segment, directly adjacent to the recombination signal sequence (RSS), placing the RSS in a position accessible to the recombination activating gene (RAG) recombinase. These changes result in establishment of a specific chromatin organization at the RSS that facilitates accessibility of the genomic DNA for the RAG recombinase. In contrast, in mouse embryonic fibroblasts the coding segment is depleted of nucleosomes, which instead cover the RSS, thereby rendering it inaccessible. Pro-T cells exhibit a pattern intermediate between pro-B cells and mouse embryonic fibroblasts. We also find large-scale variations of nucleosome density over hundreds of kilobases, delineating chromosomal domains within IgH, in a cell type-dependent manner. These findings suggest that developmentally regulated changes in nucleosome location and occupancy, in addition to the known chromatin modifications, play a fundamental role in regulating V(D)J recombination. Nucleosome positioning-which has previously been observed to vary locally at individual enhancers and promoters-may be a more general mechanism by which cells can regulate the accessibility of the genome during development, at scales ranging from several hundred base pairs to many kilobases.

Time of feeding and the intrinsic circadian clock drive rhythms in hepatic gene expression.

In mammals, the circadian oscillator generates approximately 24-h rhythms in feeding behavior, even under constant environmental conditions. Livers of mice held under constant darkness exhibit circadian rhythm in abundance in up to 15% of expressed transcripts. Therefore, oscillations in hepatic transcripts could be driven by rhythmic food intake or sustained by the hepatic circadian oscillator, or a combination of both. To address this question, we used distinct feeding and fasting paradigms on wild-type (WT) and circadian clock-deficient mice. We monitored temporal patterns of feeding and hepatic transcription. Both food availability and the temporal pattern of feeding determined the repertoire, phase, and amplitude of the circadian transcriptome in WT liver. In the absence of feeding, only a small subset of transcripts continued to express circadian patterns. Conversely, temporally restricted feeding restored rhythmic transcription of hundreds of genes in oscillator-deficient mouse liver. Our findings show that both temporal pattern of food intake and the circadian clock drive rhythmic transcription, thereby highlighting temporal regulation of hepatic transcription as an emergent property of the circadian system.

Reciprocity between phase shifts and amplitude changes in the mammalian circadian clock.

Circadian rhythms help organisms adapt to predictable daily changes in their environment. Light resets the phase of the underlying oscillator to maintain the organism in sync with its surroundings. Light also affects the amplitude of overt rhythms. At a critical phase during the night, when phase shifts are maximal, light can reduce rhythm amplitude to nearly zero, whereas in the subjective day, when phase shifts are minimal, it can boost amplitude substantially. To explore the cellular basis for this reciprocal relationship between phase shift and amplitude change, we generated a photoentrainable, cell-based system in mammalian fibroblasts that shares several key features of suprachiasmatic nucleus light entrainment. Upon light stimulation, these cells exhibit calcium/cyclic AMP responsive element-binding (CREB) protein phosphorylation, leading to temporally gated acute induction of the Per2 gene, followed by phase-dependent changes in phase and/or amplitude of the PER2 circadian rhythm. At phases near the PER2 peak, photic stimulation causes little phase shift but enhanced rhythm amplitude. At phases near the PER2 nadir, on the other hand, the same stimuli cause large phase shifts but dampen rhythm amplitude. Real-time monitoring of PER2 oscillations in single cells reveals that changes in both synchrony and amplitude of individual oscillators underlie these phenomena.