Julie E. Baggs

Harmonics of Circadian Gene Transcription in Mammals

The circadian clock is a molecular and cellular oscillator found in most mammalian tissues that regulates rhythmic physiology and behavior. Numerous investigations have addressed the contribution of circadian rhythmicity to cellular, organ, and organismal physiology. We recently developed a method to look at transcriptional oscillations with unprecedented precision and accuracy using high-density time sampling. Here, we report a comparison of oscillating transcription from mouse liver, NIH3T3, and U2OS cells. Several surprising observations resulted from this study, including a 100-fold difference in the number of cycling transcripts in autonomous cellular models of the oscillator versus tissues harvested from intact mice. Strikingly, we found two clusters of genes that cycle at the second and third harmonic of circadian rhythmicity in liver, but not cultured cells. Validation experiments show that 12-hour oscillatory transcripts occur in several other peripheral tissues as well including heart, kidney, and lungs. These harmonics are lost ex vivo, as well as under restricted feeding conditions. Taken in sum, these studies illustrate the importance of time sampling with respect to multiple testing, suggest caution in use of autonomous cellular models to study clock output, and demonstrate the existence of harmonics of circadian gene expression in the mouse.

Feedback repression is required for mammalian circadian clock function

Direct evidence for the requirement of transcriptional feedback repression in circadian clock function has been elusive. Here, we developed a molecular genetic screen in mammalian cells to identify mutants of the circadian transcriptional activators CLOCK and BMAL1, which were uncoupled from CRYPTOCHROME (CRY)-mediated transcriptional repression. Notably, mutations in the PER-ARNT-SIM domain of CLOCK and the C terminus of BMAL1 resulted in synergistic insensitivity through reduced physical interactions with CRY. Coexpression of these mutant proteins in cultured fibroblasts caused arrhythmic phenotypes in population and single-cell assays. These data demonstrate that CRY-mediated repression of the CLOCK/BMAL1 complex activity is required for maintenance of circadian rhythmicity and provide formal proof that transcriptional feedback is required for mammalian clock function.

Network Features of the Mammalian Circadian Clock

The mammalian circadian clock is a cell-autonomous system that drives oscillations in behavior and physiology in anticipation of daily environmental change. To assess the robustness of a human molecular clock, we systematically depleted known clock components and observed that circadian oscillations are maintained over a wide range of disruptions. We developed a novel strategy termed Gene Dosage Network Analysis (GDNA) in which small interfering RNA (siRNA)-induced dose-dependent changes in gene expression were used to build gene association networks consistent with known biochemical constraints. The use of multiple doses powered the analysis to uncover several novel network features of the circadian clock, including proportional responses and signal propagation through interacting genetic modules. We also observed several examples where a gene is up-regulated following knockdown of its paralog, suggesting the clock network utilizes active compensatory mechanisms rather than simple redundancy to confer robustness and maintain function. We propose that these network features act in concert as a genetic buffering system to maintain clock function in the face of genetic and environmental perturbation.

Asparagine Plays a Critical Role in Regulating Cellular Adaptation to Glutamine Depletion

Many cancer cells consume large quantities of glutamine to maintain TCA cycle anaplerosis and support cell survival. It was therefore surprising when RNAi screening revealed that suppression of citrate synthase (CS), the first TCA cycle enzyme, prevented glutamine-withdrawal-induced apoptosis. CS suppression reduced TCA cycle activity and diverted oxaloacetate, the substrate of CS, into production of the nonessential amino acids aspartate and asparagine. We found that asparagine was necessary and sufficient to suppress glutamine-withdrawal-induced apoptosis without restoring the levels of other nonessential amino acids or TCA cycle intermediates. In complete medium, tumor cells exhibiting high rates of glutamine consumption underwent rapid apoptosis when glutamine-dependent asparagine synthesis was suppressed, and expression of asparagine synthetase was statistically correlated with poor prognosis in human tumors. Coupled with the success of L-asparaginase as a therapy for childhood leukemia, the data suggest that intracellular asparagine is a critical suppressor of apoptosis in many human tumors.

The Role of Clock Genes in Pharmacology

The physiology of a wide variety of organisms is organized according to periodic environmental changes imposed by the earths rotation. This way, a large number of physiological processes present diurnal rhythms regulated by an internal timing system called the circadian clock. As part of the rhythmicity in physiology, drug efficacy and toxicity can vary with time. Studies over the past four decades present diurnal oscillations in drug absorption, distribution, metabolism, and excretion. On the other hand, diurnal variations in the availability and sensitivity of drug targets have been correlated with time-dependent changes in drug effectiveness. In this review, we provide evidence supporting the regulation of drug kinetics and dynamics by the circadian clock. We also use the examples of hypertension and cancer to show current achievements and challenges in chronopharmacology.

The γ-Secretase Cleavage Product of Polycystin-1 Regulates TCF and CHOP-Mediated Transcriptional Activation through a p300-Dependent Mechanism

Mutations in Pkd1, encoding polycystin-1 (PC1), cause autosomal-dominant polycystic kidney disease (ADPKD). We show that the carboxy-terminal tail (CTT) of PC1 is released by γ-secretase-mediated cleavage and regulates the Wnt and CHOP pathways by binding the transcription factors TCF and CHOP, disrupting their interaction with the common transcriptional coactivator p300. Loss of PC1 causes increased proliferation and apoptosis, while reintroducing PC1-CTT into cultured Pkd1 null cells reestablishes normal growth rate, suppresses apoptosis, and prevents cyst formation. Inhibition of γ-secretase activity impairs the ability of PC1 to suppress growth and apoptosis and leads to cyst formation in cultured renal epithelial cells. Expression of the PC1-CTT is sufficient to rescue the dorsal body curvature phenotype in zebrafish embryos resulting from either γ-secretase inhibition or suppression of Pkd1 expression. Thus, γ-secretase-dependent release of the PC1-CTT creates a protein fragment whose expression is sufficient to suppress ADPKD-related phenotypes in vitro and in vivo.

Wnt ligands signal in a cooperative manner to promote foregut organogenesis

Endoderm-mesenchyme cross-talk is a central process in the development of foregut-derived organs. How signaling pathways integrate the activity of multiple ligands to guide organ development is poorly understood. We show that two Wnt ligands, Wnt2 and Wnt7b, cooperatively induce Wnt signaling without affecting the stabilization of the Wnt canonical effector β-catenin despite it being necessary for Wnt2–Wnt7b cooperativity. Wnt2–Wnt7b cooperation is specific for mesenchymal cell lineages and the combined loss of Wnt2 and Wnt7b leads to more severe developmental defects in the lung than loss of Wnt2 or Wnt7b alone. High-throughput small-molecule screens and biochemical assays reveal that the Pdgf pathway is required for cooperative Wnt2-Wnt7b signaling. Inhibition of Pdgf signaling in cell culture reduces Wnt2–Wnt7b cooperative signaling. Moreover, inhibition of Pdgf signaling in lung explant cultures results in decreased Wnt signaling and lung smooth-muscle development. These data suggest a model in which Pdgf signaling potentiates Wnt2–Wnt7b signaling to promote high levels of Wnt activity in mesenchymal progenitors that is required for proper development of endoderm-derived organs, such as the lung.

Genomics and systems approaches in the mammalian circadian clock.

The circadian clock is an endogenous oscillator that regulates daily rhythms in behavior and physiology. In recent years, systems biology and genomics approaches re-shaped our view of the clock. Our understanding of outputs that regulate behavior and physiology has been enhanced through gene expression profiling and proteomic analyses. Systems approaches uncovered underlying principles of transcriptional regulation and robustness of the oscillator through perturbation analysis and synthetic methods. Finally, new clock components and modifiers were identified through cell-based screening efforts and proteomics.

Generation of a Novel Allelic Series of Cryptochrome Mutants via Mutagenesis Reveals Residues Involved in Protein-Protein Interaction and CRY2-Specific Repression

ABSTRACT CRYPTOCHOME proteins are necessary for mammalian circadian rhythms and have many well-established biochemical roles within the molecular clock. While studies examining the effect of null Cry alleles have been informative, they have failed to dissect out the relative importance of, and the molecular mechanisms behind, the many roles of the CRY1 and CRY2 proteins. To address this, we created an allelic series of Cry mutants through random mutagenesis, followed by a cell-based screen to isolate mutants with aberrant repression of CLOCK-BMAL1. We identified 22 mutants with mutations resulting in single amino acid substitutions which cause a variety of deficiencies in different CRY functions. To illustrate the breadth and value of these new tools, we present an in-depth analysis of two of these mutants, CRY2G354D and CRY2G351D; the former shows deficiency in clock protein binding and is required for repression by both CRYs, while in contrast, the latter displays normal binding function but exhibits a CRY2-specific repression phenotype. Further, while overexpression of CRY2 in NIH 3T3 cells caused a dose-dependent decrease in rhythm amplitude, overexpression of CRY2G351D abolished rhythmicity. In summary, characterization of these unique alleles provides new opportunities for more-sophisticated insight into the multifaceted functions of the CRY proteins in circadian rhythms.

Circadian clocks are seeing the systems biology light

Circadian rhythms are those biological rhythms that have a periodicity of around 24 hours. Recently, the generation of a circadian transcriptional network - compiled from RNA-expression and promoter-element analysis and phase information - has led to a better understanding of the gene-expression patterns that regulate the precise 24-hour clock.