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.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Circadian clocks enable organisms to predict and align their behaviors and physiologies to constant daily day-night environmental cycle. Because the ubiquitin ligase Siah2 has been identified as a ...potential regulator of circadian clock function in cultured cells, we have used SIAH2-deficient mice to examine its function in vivo. Our experiments demonstrate a striking and unexpected sexually dimorphic effect of SIAH2-deficiency on the regulation of rhythmically expressed genes in the liver. The absence of SIAH2 in females, but not in males, altered the expression of core circadian clock genes and drastically remodeled the rhythmic transcriptome in the liver by increasing the number of day-time expressed genes, and flipping the rhythmic expression from nighttime expressed genes to the daytime. These effects are not readily explained by effects on known sexually dimorphic pathways in females. Moreover, loss of SIAH2 in females, not males, preferentially altered the expression of transcription factors and genes involved in regulating lipid and lipoprotein metabolism. Consequently, SIAH2-deficient females, but not males, displayed disrupted daily lipid and lipoprotein patterns, increased adiposity and impaired metabolic homeostasis. Overall, these data suggest that SIAH2 may be a key component of a female-specific circadian transcriptional output circuit that directs the circadian timing of gene expression to regulate physiological rhythms, at least in the liver. In turn, our findings imply that sex-specific transcriptional mechanisms may closely interact with the circadian clock to tailor overt rhythms for sex-specific needs.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Over the last decades, researchers have characterized a set of "clock genes" that drive daily rhythms in physiology and behavior. This arduous work has yielded results with far-reaching consequences ...in metabolic, psychiatric, and neoplastic disorders. Recent attempts to expand our understanding of circadian regulation have moved beyond the mutagenesis screens that identified the first clock components, employing higher throughput genomic and proteomic techniques. In order to further accelerate clock gene discovery, we utilized a computer-assisted approach to identify and prioritize candidate clock components. We used a simple form of probabilistic machine learning to integrate biologically relevant, genome-scale data and ranked genes on their similarity to known clock components. We then used a secondary experimental screen to characterize the top candidates. We found that several physically interact with known clock components in a mammalian two-hybrid screen and modulate in vitro cellular rhythms in an immortalized mouse fibroblast line (NIH 3T3). One candidate, Gene Model 129, interacts with BMAL1 and functionally represses the key driver of molecular rhythms, the BMAL1/CLOCK transcriptional complex. Given these results, we have renamed the gene CHRONO (computationally highlighted repressor of the network oscillator). Bi-molecular fluorescence complementation and co-immunoprecipitation demonstrate that CHRONO represses by abrogating the binding of BMAL1 to its transcriptional co-activator CBP. Most importantly, CHRONO knockout mice display a prolonged free-running circadian period similar to, or more drastic than, six other clock components. We conclude that CHRONO is a functional clock component providing a new layer of control on circadian molecular dynamics.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The protozoan parasite, Toxoplasma, like many intracellular pathogens, suppresses interferon gamma (IFN-γ)-induced signal transducer and activator of transcription 1 (STAT1) activity. We exploited ...this well-defined host-pathogen interaction as the basis for a high-throughput screen, identifying nine transcription factors that enhance STAT1 function in the nucleus, including the orphan nuclear hormone receptor TLX. Expression profiling revealed that upon IFN-γ treatment TLX enhances the output of a subset of IFN-γ target genes, which we found is dependent on TLX binding at those loci. Moreover, infection of TLX deficient mice with the intracellular parasite Toxoplasma results in impaired production of the STAT1-dependent cytokine interleukin-12 by dendritic cells and increased parasite burden in the brain during chronic infection. These results demonstrate a previously unrecognized role for this orphan nuclear hormone receptor in regulating STAT1 signaling and host defense and reveal that STAT1 activity can be modulated in a context-specific manner by such "modifiers."
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Regulated degradation of proteins by the proteasome is often critical to their function in dynamic cellular pathways. The molecular clock underlying mammalian circadian rhythms relies on the rhythmic ...expression and degradation of its core components. However, because the tools available for identifying the mechanisms underlying the degradation of a specific protein are limited, the mechanisms regulating clock protein degradation are only beginning to be elucidated. Here we describe a cell-based functional screening approach designed to quickly identify the ubiquitin E3 ligases that induce the degradation of potentially any protein of interest. We screened the nuclear hormone receptor RevErbα (Nr1d1), a key constituent of the mammalian circadian clock, for E3 ligases that regulate its stability and found Seven in absentia2 (Siah2) to be a key regulator of RevErbα stability. Previously implicated in hypoxia signaling, Siah2 overexpression destabilizes RevErbα/β, and siRNA depletion of Siah2 stabilizes endogenous RevErbα. Moreover, Siah2 depletion delays circadian degradation of RevErbα and lengthens period length. These results demonstrate the utility of functional screening approaches for identifying regulators of protein stability and reveal Siah2 as a previously unidentified circadian clockwork regulator that mediates circadian RevErbα turnover.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
Abstract
Polycystin-1 (PC1), encoded by the PKD1 gene that is mutated in the autosomal dominant polycystic kidney disease, regulates a number of processes including bone development. Activity of the ...transcription factor RunX2, which controls osteoblast differentiation, is reduced in Pkd1 mutant mice but the mechanism governing PC1 activation of RunX2 is unclear. PC1 undergoes regulated cleavage that releases its C-terminal tail (CTT), which translocates to the nucleus to modulate transcriptional pathways involved in proliferation and apoptosis. We find that the cleaved CTT of PC1 (PC1-CTT) stimulates the transcriptional coactivator TAZ (Wwtr1), an essential coactivator of RunX2. PC1-CTT physically interacts with TAZ, stimulating RunX2 transcriptional activity in pre-osteoblast cells in a TAZ-dependent manner. The PC1-CTT increases the interaction between TAZ and RunX2 and enhances the recruitment of the p300 transcriptional co-regulatory protein to the TAZ/RunX2/PC1-CTT complex. Zebrafish injected with morpholinos directed against pkd1 manifest severe bone calcification defects and a curly tail phenotype. Injection of messenger RNA (mRNA) encoding the PC1-CTT into pkd1-morphant fish restores bone mineralization and reduces the severity of the curly tail phenotype. These effects are abolished by co-injection of morpholinos directed against TAZ. Injection of mRNA encoding a dominant-active TAZ construct is sufficient to rescue both the curly tail phenotype and the skeletal defects observed in pkd1-morpholino treated fish. Thus, TAZ constitutes a key mechanistic link through which PC1 mediates its physiological functions.
Loss of Pax3, a developmentally regulated transcription factor expressed in premigratory neural crest, results in severe developmental defects and embryonic lethality. Although Pax3 mutations produce ...profound phenotypes, the intrinsic transcriptional activation exhibited by Pax3 is surprisingly modest. We postulated the existence of transcriptional coactivators that function with Pax3 to mediate developmental functions. A high-throughput screen identified the Hippo effector proteins Taz and Yap65 as Pax3 coactivators. Synergistic coactivation of target genes by Pax3-Taz/Yap65 requires DNA binding by Pax3, is Tead independent, and is regulated by Hippo kinases Mst1 and Lats2. In vivo, Pax3 and Yap65 colocalize in the nucleus of neural crest progenitors in the dorsal neural tube. Neural crest deletion of Taz and Yap65 results in embryo-lethal neural crest defects and decreased expression of the Pax3 target gene, Mitf. These results suggest that Pax3 activity is regulated by the Hippo pathway and that Pax factors are Hippo effectors.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Soluble syntaxin 3 functions as a transcriptional regulator Giovannone, Adrian J.; Winterstein, Christine; Bhattaram, Pallavi ...
Journal of biological chemistry/The Journal of biological chemistry,
04/2018, Volume:
293, Issue:
15
Journal Article
Peer reviewed
Open access
Syntaxins are a conserved family of SNARE proteins and contain C-terminal transmembrane anchors required for their membrane fusion activity. Here we show that Stx3 (syntaxin 3) unexpectedly also ...functions as a nuclear regulator of gene expression. We found that alternative splicing creates a soluble isoform that we termed Stx3S, lacking the transmembrane anchor. Soluble Stx3S binds to the nuclear import factor RanBP5 (RAN-binding protein 5), targets to the nucleus, and interacts physically and functionally with several transcription factors, including ETV4 (ETS variant 4) and ATF2 (activating transcription factor 2). Stx3S is differentially expressed in normal human tissues, during epithelial cell polarization, and in breast cancer versus normal breast tissue. Inhibition of endogenous Stx3S expression alters the expression of cancer-associated genes and promotes cell proliferation. Similar nuclear-targeted, soluble forms of other syntaxins were identified, suggesting that nuclear signaling is a conserved, novel function common among these membrane-trafficking proteins.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The role of clock genes in pharmacology Paschos, Georgios K; Baggs, Julie E; Hogenesch, John B ...
Annual review of pharmacology and toxicology,
02/2010, Volume:
50
Journal Article
Peer reviewed
The physiology of a wide variety of organisms is organized according to periodic environmental changes imposed by the earth's 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.
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