Circadian hepatocyte clocks keep synchrony in the absence of a master pacemaker in the suprachiasmatic nucleus or other extrahepatic clocks.

It has been assumed that the suprachiasmatic nucleus (SCN) synchronizes peripheral circadian oscillators. However, this has never been convincingly shown, since biochemical time series experiments are not feasible in behaviorally arrhythmic animals. By using long-term bioluminescence recording in freely moving mice, we show that the SCN is indeed required for maintaining synchrony between organs. Surprisingly, however, circadian oscillations persist in the livers of mice devoid of an SCN or oscillators in cells other than hepatocytes. Hence, similar to SCN neurons, hepatocytes can maintain phase coherence in the absence of Zeitgeber signals produced by other organs or environmental cycles.

A molecular mechanism for circadian clock negative feedback.

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.

Identification of RACK1 and protein kinase Calpha as integral components of the mammalian circadian clock.

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-alpha (PKCalpha) were recruited in a circadian manner into a nuclear BMAL1 complex during the negative feedback phase of the cycle. Overexpression of RACK1 and PKCalpha suppressed CLOCK-BMAL1 transcriptional activity, and RACK1 stimulated phosphorylation of BMAL1 by PKCalpha in vitro. Depletion of endogenous RACK1 or PKCalpha 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.

The estrogen-related receptor alpha (ERRalpha) functions in PPARgamma coactivator 1alpha (PGC-1alpha)-induced mitochondrial biogenesis.

Estrogen-related receptor alpha (ERRalpha) is one of the first orphan nuclear receptors to be identified, yet its physiological functions are still unclear. We show here that ERRalpha is an effector of the transcriptional coactivator PGC-1alpha [peroxisome proliferator-activated receptor gamma (PPARgamma) coactivator 1alpha], and that it regulates the expression of genes involved in oxidative phosphorylation and mitochondrial biogenesis. Inhibition of ERRalpha compromises the ability of PGC-1alpha to induce the expression of genes encoding mitochondrial proteins and to increase mitochondrial DNA content. A constitutively active form of ERRalpha is sufficient to elicit both responses. ERRalpha binding sites are present in the transcriptional control regions of ERRalpha/PGC-1alpha-induced genes and contribute to the transcriptional response to PGC-1alpha. The ERRalpha-regulated genes described here have been reported to be expressed at reduced levels in humans that are insulin-resistant. Thus, changes in ERRalpha activity could be linked to pathological changes in metabolic disease, such as diabetes.

The transcriptional coactivator PGC-1 regulates the expression and activity of the orphan nuclear receptor estrogen-related receptor alpha (ERRalpha).

The estrogen-related receptor alpha (ERRalpha) is one of the first orphan nuclear receptors identified. Still, we know little about the mechanisms that regulate its expression and its activity. In this study, we show that the transcriptional coactivator PGC-1, which is implicated in the control of energy metabolism, regulates ERRalpha at two levels. First, PGC-1 induces the expression of ERRalpha. Consistent with this induction, levels of ERRalpha mRNA in vivo are highest in PGC-1 expressing tissues, such as heart, kidney, and muscle, and up-regulated in response to signals that induce PGC-1, such as exposure to cold. Second, PGC-1 interacts physically with ERRalpha and enables it to activate transcription. Strikingly, we find that PGC-1 converts ERRalpha from a factor with little or no transcriptional activity to a potent regulator of gene expression, suggesting that ERRalpha is not a constitutively active nuclear receptor but rather one that is regulated by protein ligands, such as PGC-1. Our findings suggest that the two proteins act in a common pathway to regulate processes relating to energy metabolism. In support of this hypothesis, adenovirus-mediated delivery of small interfering RNA for ERRalpha, or of PGC-1 mutants that interact selectively with different types of nuclear receptors, shows that PGC-1 can induce the fatty acid oxidation enzyme MCAD (medium-chain acyl-coenzyme A dehydrogenase) in an ERRalpha-dependent manner.

The PGC-1-related protein PERC is a selective coactivator of estrogen receptor alpha.

Peroxisome proliferator-activated receptor gamma coactivator-1 (PGC-1) is a tissue-specific coactivator that enhances the activity of many nuclear receptors and coordinates transcriptional programs important for energy metabolism. We describe here a novel PGC-1-related coactivator that is expressed in a similar tissue-specific manner as PGC-1, with the highest levels in heart and skeletal muscle. In contrast to PGC-1, the new coactivator shows high receptor specificity. It enhances potently the activity of estrogen receptor (ER) alpha, while having only small effects on other receptors. Because of its nuclear receptor selectivity, we have termed the new protein PERC (PGC-1 related Estrogen Receptor Coactivator). We show here that the coactivation function of PERC relies on a bipartite transcriptional activation domain and two LXXLL motifs that interact with the AF2 domain of ERalpha in an estrogen-dependent manner. PERC and PGC-1 are likely to have different functions in ER signaling. Whereas PERC acts selectively on ERalpha and not on the second estrogen receptor ERbeta, PGC-1 coactivates strongly both ERs. Moreover, PERC and PGC-1 show distinct preferences for enhancing ERalpha in different promoter contexts. Finally, PERC enhances the ERalpha-mediated response to the partial agonist tamoxifen, while PGC-1 modestly represses it. The two coactivators are likely to mediate distinct, tissue-specific responses to estrogens.