Maria S. Robles

A molecular mechanism for circadian clock negative feedback.

New cogs in the mammalian circadian clock are identified. Circadian rhythms in mammals are generated by a feedback loop in which the three PERIOD (PER) proteins, acting in a large complex, inhibit the transcriptional activity of the CLOCK-BMAL1 dimer, which represses their own expression. Although fundamental, the mechanism of negative feedback in the mammalian clock, or any eukaryotic clock, is unknown. We analyzed protein constituents of PER complexes purified from mouse tissues and identified PSF (polypyrimidine tract–binding protein–associated splicing factor). Our analysis indicates that PSF within the PER complex recruits SIN3A, a scaffold for assembly of transcriptional inhibitory complexes and that the PER complex thereby rhythmically delivers histone deacetylases to the Per1 promoter, which repress Per1 transcription. These findings provide a function for the PER complex and a molecular mechanism for circadian clock negative feedback.

In-Vivo Quantitative Proteomics Reveals a Key Contribution of Post-Transcriptional Mechanisms to the Circadian Regulation of Liver Metabolism

Circadian clocks are endogenous oscillators that drive the rhythmic expression of a broad array of genes, orchestrating metabolism and physiology. Recent evidence indicates that post-transcriptional and post-translational mechanisms play essential roles in modulating temporal gene expression for proper circadian function, particularly for the molecular mechanism of the clock. Due to technical limitations in large-scale, quantitative protein measurements, it remains unresolved to what extent the circadian clock regulates metabolism by driving rhythms of protein abundance. Therefore, we aimed to identify global circadian oscillations of the proteome in the mouse liver by applying in vivo SILAC mouse technology in combination with state of the art mass spectrometry. Among the 3000 proteins accurately quantified across two consecutive cycles, 6% showed circadian oscillations with a defined phase of expression. Interestingly, daily rhythms of one fifth of the liver proteins were not accompanied by changes at the transcript level. The oscillations of almost half of the cycling proteome were delayed by more than six hours with respect to the corresponding, rhythmic mRNA. Strikingly we observed that the length of the time lag between mRNA and protein cycles varies across the day. Our analysis revealed a high temporal coordination in the abundance of proteins involved in the same metabolic process, such as xenobiotic detoxification. Apart from liver specific metabolic pathways, we identified many other essential cellular processes in which protein levels are under circadian control, for instance vesicle trafficking and protein folding. Our large-scale proteomic analysis reveals thus that circadian post-transcriptional and post-translational mechanisms play a key role in the temporal orchestration of liver metabolism and physiology.

Identification of RACK1 and Protein Kinase Cα as Integral Components of the Mammalian Circadian Clock

Late-Running Clock Components Many mammalian cells contain a well-characterized biological clock with a 24-hour cycle. In the latter part of the day, transcription mediated by one of the clock components, the transcription factor made up of the CLOCK and BMAL1 proteins, is inhibited, but the mechanism of inhibition has been unclear. Robles et al. (p. 463) used mass spectrometry to identify proteins that RACK1 (receptor for activated C kinase–1), a scaffold protein that brings protein kinase C–α (PKCα ) into contact with its substrates, caused to be associated with BMAL1 at the time of day when its transcription-activating function was inhibited. Further studies implicated PKCα and RACK1 as integral components of the clock, without which the clocks free-running period was shortened. Rhythmic activation of signaling occurs by core components of the biological clock mechanism. At the core of the mammalian circadian clock is a negative feedback loop in which the dimeric transcription factor CLOCK-BMAL1 drives processes that in turn suppress its transcriptional activity. To gain insight into the mechanisms of circadian feedback, we analyzed mouse protein complexes containing BMAL1. Receptor for activated C kinase–1 (RACK1) and protein kinase C–α (PKCα) were recruited in a circadian manner into a nuclear BMAL1 complex during the negative feedback phase of the cycle. Overexpression of RACK1 and PKCα suppressed CLOCK-BMAL1 transcriptional activity, and RACK1 stimulated phosphorylation of BMAL1 by PKCα in vitro. Depletion of endogenous RACK1 or PKCα from fibroblasts shortened the circadian period, demonstrating that both molecules function in the clock oscillatory mechanism. Thus, the classical PKC signaling pathway is not limited to relaying external stimuli but is rhythmically activated by internal processes, forming an integral part of the circadian feedback loop.

Feedback Regulation of Transcriptional Termination by the Mammalian Circadian Clock PERIOD Complex

The Inner Workings of a Clock Eukaryotic circadian clocks are built at least in part on transcriptional feedback loops, but the mechanisms underlying circadian feedback are poorly understood. Padmanabhan et al. (p. 599, published online 5 July) explored the transcriptional feedback mechanism at the heart of the mammalian circadian clock. The proteins PERIOD (PER) and CRYPTOCHROME suppress transcription of their own genes. PER complexes do so in part by recruiting a histone deacetylase to promoters of clock genes. But PER is also present on DNA in a complex with Senataxin, a helicase that functions in transcriptional termination. Senataxin appears to be inhibited in the PER complex, thus inhibiting termination and further reducing the rate of transcription. A circadian rhythm regulator acts by altering the elongation stage of gene expression. Eukaryotic circadian clocks are built on transcriptional feedback loops. In mammals, the PERIOD (PER) and CRYPTOCHROME (CRY) proteins accumulate, form a large nuclear complex (PER complex), and repress their own transcription. We found that mouse PER complexes included RNA helicases DDX5 and DHX9, active RNA polymerase II large subunit, Per and Cry pre-mRNAs, and SETX, a helicase that promotes transcriptional termination. During circadian negative feedback, RNA polymerase II accumulated near termination sites on Per and Cry genes but not on control genes. Recruitment of PER complexes to the elongating polymerase at Per and Cry termination sites inhibited SETX action, impeding RNA polymerase II release and thereby repressing transcriptional reinitiation. Circadian clock negative feedback thus includes direct control of transcriptional termination.

Circadian control of oscillations in mitochondrial rate-limiting enzymes and nutrient utilization by PERIOD proteins

Significance Mitochondria are major cellular energy suppliers and have to cope with changes in nutrient supply and energy demand that naturally occur throughout the day. We obtained the first, to our knowledge, comprehensive mitochondrial proteome around the clock and identified extensive oscillations in mitochondrial protein abundance that predominantly peak during the early light phase. Remarkably, several rate-limiting mitochondrial enzymes that process different nutrients accumulate in a diurnal manner and are dependent on the clock proteins PER1/2. Concurrently, we uncovered daily oscillations in mitochondrial respiration that are substrate-specific and peak during different times of the day. We propose that the circadian clock PERIOD proteins regulate the diurnal utilization of different nutrients by the mitochondria and thus, optimize mitochondrial function to daily changes in energy supply/demand. Mitochondria are major suppliers of cellular energy through nutrients oxidation. Little is known about the mechanisms that enable mitochondria to cope with changes in nutrient supply and energy demand that naturally occur throughout the day. To address this question, we applied MS-based quantitative proteomics on isolated mitochondria from mice killed throughout the day and identified extensive oscillations in the mitochondrial proteome. Remarkably, the majority of cycling mitochondrial proteins peaked during the early light phase. We found that rate-limiting mitochondrial enzymes that process lipids and carbohydrates accumulate in a diurnal manner and are dependent on the clock proteins PER1/2. In this conjuncture, we uncovered daily oscillations in mitochondrial respiration that peak during different times of the day in response to different nutrients. Notably, the diurnal regulation of mitochondrial respiration was blunted in mice lacking PER1/2 or on a high-fat diet. We propose that PERIOD proteins optimize mitochondrial metabolism to daily changes in energy supply/demand and thereby, serve as a rheostat for mitochondrial nutrient utilization.

Degradation of cellular mRNA is a general early apoptosis-induced event.

The fate of cellular mRNAs was analyzed in several cell lines of lymphoid origin, after induction of apoptosis by different mechanisms. Cytoplasmic mRNAs are specifically degraded as part of the early apoptotic response. This degradation is not species restricted and is independent of the cell line, the apoptotic stimulus, the intrinsic half‐life of the mRNAs, and the transcriptional status of the gene (constitutive or inducible). mRNA degradation precedes DNA fragmentation and correlates with the appearance of phosphatidylserine in the outer cell membrane. In addition, apoptosis‐induced mRNA degradation is an active process that can be dissected from other apoptotic hallmarks (degradation of annexin V, DNA, and poly(ADP‐ribose) polymerase [PARP]), which suggests that apoptosis‐induced mRNA degradation is controlled by a distinct signaling pathway. Furthermore, mRNA degradation also occurs in vivo, specifically during thymocyte apoptosis. Taken together, these data support the notion that degradation of mRNA is a general early apoptotic event that may become a new apoptotic hallmark.

Histone monoubiquitination by Clock–Bmal1 complex marks Per1 and Per2 genes for circadian feedback

Circadian rhythms in mammals are driven by a feedback loop in which the transcription factor Clock–Bmal1 activates expression of Per and Cry proteins, which together form a large nuclear complex (Per complex) that represses Clock–Bmal1 activity. We found that mouse Clock–Bmal1 recruits the Ddb1–Cullin-4 ubiquitin ligase to Per (Per1 and Per2), Cry (Cry1 and Cry2) and other circadian target genes. Histone H2B monoubiquitination at Per genes was rhythmic and depended on Bmal1, Ddb1 and Cullin-4a. Depletion of Ddb1–Cullin-4a or an independent decrease in H2B monoubiquitination caused defective circadian feedback and decreased the association of the Per complex with DNA-bound Clock–Bmal1. Clock–Bmal1 thus covalently marks Per genes for subsequent recruitment of the Per complex. Our results reveal a chromatin-mediated signal from the positive to the negative limb of the clock that provides a licensing mechanism for circadian feedback.

Cell death during lymphocyte development and activation

The development and homeostasis of the immune system requires an exquisite balance between cell proliferation and cell death. In this review, we discuss several in vivo and in vitro models that have been developed to help understand the importance of apoptosis during B and T cell development and activation.

Guidelines for Genome-Scale Analysis of Biological Rhythms

Genome biology approaches have made enormous contributions to our understanding of biological rhythms, particularly in identifying outputs of the clock, including RNAs, proteins, and metabolites, whose abundance oscillates throughout the day. These methods hold significant promise for future discovery, particularly when combined with computational modeling. However, genome-scale experiments are costly and laborious, yielding “big data” that are conceptually and statistically difficult to analyze. There is no obvious consensus regarding design or analysis. Here we discuss the relevant technical considerations to generate reproducible, statistically sound, and broadly useful genome-scale data. Rather than suggest a set of rigid rules, we aim to codify principles by which investigators, reviewers, and readers of the primary literature can evaluate the suitability of different experimental designs for measuring different aspects of biological rhythms. We introduce CircaInSilico, a web-based application for generating synthetic genome biology data to benchmark statistical methods for studying biological rhythms. Finally, we discuss several unmet analytical needs, including applications to clinical medicine, and suggest productive avenues to address them.

Proteomic approaches in circadian biology

Circadian clocks are endogenous oscillators that drive the rhythmic expression of a broad array of genes that orchestrate metabolism and physiology. Recent evidence indicates that posttranscriptional and posttranslational mechanisms play essential roles in modulating circadian gene expression, particularly for the molecular mechanism of the clock. In contrast to genetic technologies that have long been used to study circadian biology, proteomic approaches have so far been limited and, if applied at all, have used two-dimensional gel electrophoresis (2-DE). Here, we review the proteomics approaches applied to date in the circadian field, and we also discuss the exciting potential of using cutting-edge proteomics technology in circadian biology. Large-scale, quantitative protein abundance measurements will help to understand to what extent the circadian clock drives system wide rhythms of protein abundance downstream of transcription regulation.