Circadian variation of the response of T cells to antigen.

Circadian clocks regulate many important aspects of physiology, and their disturbance leads to various medical conditions. Circadian variations have been found in immune system variables, including daily rhythms in circulating WBC numbers and serum concentration of cytokines. However, control of immune functional responses by the circadian clock has remained relatively unexplored. In this study, we show that mouse lymph nodes exhibit rhythmic clock gene expression. T cells from lymph nodes collected over 24 h show a circadian variation in proliferation after stimulation via the TCR, which is blunted in Clock gene mutant mice. The tyrosine kinase ZAP70, which is just downstream of the TCR in the T cell activation pathway and crucial for T cell function, exhibits rhythmic protein expression. Lastly, mice immunized with OVA peptide-loaded dendritic cells in the day show a stronger specific T cell response than mice immunized at night. These data reveal circadian control of the Ag-specific immune response and a novel regulatory mode of T cell proliferation, and may provide clues for more efficient vaccination strategies.

Clock-dependent and independent transcriptional control of the two isoforms from the mouse Rorgamma gene.

Accumulating evidence indicate that molecular mechanisms generating circadian rhythms display some degree of tissue-specificity. More specifically, distinct patterns of expression for nuclear receptors of the ROR family indicate that the transcriptional control of the clock gene Bmal1 differs among tissues. This study aims to investigate the expression of Rorgammaisoforms (Rorgamma and Rorgammat) and characterize the molecular mechanisms underlying their tissue-specific expression. The expression of Rorgamma isoforms was assessed in mouse liver, muscle, thymus and testis throughout 24 h using quantitative RT-PCR. Although the expression of Rorgamma was rhythmic in the liver and thymus, it was constitutively expressed in muscle and testis. In contrast, the expression of Rorgammat was constitutive in all four tissues. Furthermore, rhythmic expression of Rorgamma was impaired in Clock mutant mice whereas the mutation had no effect on Rorgammat expression. In line with these findings, luciferase assays revealed that transcription of the Rorgamma promoter is clock-controlled whereas that of Rorgammat promoter is essentially clock-independent. Our results provide insights into the molecular mechanisms that lead to differential expression of Rorgamma and Rorgammat and are suggestive of a framework that might account for tissue-specific circadian regulation.

Cryptochromes impair phosphorylation of transcriptional activators in the clock: a general mechanism for circadian repression.

CLOCK and BMAL1 [brain and muscle ARNT (arylhydrocarbon receptor nuclear translocator)-like protein 1] are central components of the molecular clock in mammals and belong to the bHLH (basic helix-loop-helix)/PAS [PER (Period)/ARNT/SIM (single-minded)] family. Features of their dimerization have never been investigated. Here, we demonstrate that PAS domain function requires regions extending over the short PAS core repeats. Strikingly, while deleting PAS core repeats does not overtly affect dimerization, it abolishes the transcriptional activity of the heterodimer. Interestingly, these deletions also abolish co-dependent phosphorylation of CLOCK and BMAL1, suggesting a link between the phosphorylation status of the heterodimer and its transactivation potential. We demonstrate that NPAS2 (neuronal PAS domain protein 2) and BMAL2 also undergo similar posttranslational modifications, thereby establishing the mechanism proposed for CLOCK-BMAL1 as a common feature of transcriptional activators in the circadian clock. The discovery of two novel splice variants of BMAL2 confirms the crucial role of the PAS domain and further strengthens the view that co-dependent phosphorylation is of functional significance. In agreement with this, we demonstrate that CRY1-2 (cryptochromes 1-2) affect transactivation and phosphorylation of transcriptional activators of the clock. Furthermore, CRY proteins stabilize the unphosphorylated forms of BMAL1(BMAL2) thereby shifting the phosphorylated/unphosphorylated ratio towards a predominantly unphosphorylated (transcriptionally inactive) form. In contrast, PER proteins, which are weak repressors, are without effect. From these results, we propose a general mechanism for the inhibition of CLOCK(NPAS2)-BMAL1(BMAL2) circadian transcriptional activation by CRY1-2.