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Harmonics of circadian gene transcription in mammals.

Hughes ME, DiTacchio L, Hayes KR, Vollmers C, Pulivarthy S, Baggs JE, Panda S, Hogenesch JB - PLoS Genet. (2009)

Bottom Line: The circadian clock is a molecular and cellular oscillator found in most mammalian tissues that regulates rhythmic physiology and behavior.Strikingly, we found two clusters of genes that cycle at the second and third harmonic of circadian rhythmicity in liver, but not cultured cells.These harmonics are lost ex vivo, as well as under restricted feeding conditions.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Institute for Translational Medicine and Therapeutics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, United States of America.

ABSTRACT
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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12 h rhythmic transcription is dampened in ex vivo hepatocytes.Primary hepatocytes were prepared from Per2-luciferase mice and shocked with dexamethasone to synchronize their circadian clocks. Real-time luciferase measurements revealed a circadian oscillation which dampens over the course of three days in vitro for two replicates shown in red and blue (A). Starting four h after dexamethasone shock, mRNA samples from these cells were collected every two h for an entire day and quantitative PCR was used to assess the levels of endogenous mRNAs. Core clock genes, including NR1D1, Dbp, Per2 and Bmal1, were rhythmic over the analyzed time points (B–E); however, 12 h genes were either severely dampened (F) or were completely arrhythmic (G–M). Error bars are +/−S.E.M.; thick purple traces represent the average of three replicates, thin traces show the result of each individual replicate.
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pgen-1000442-g006: 12 h rhythmic transcription is dampened in ex vivo hepatocytes.Primary hepatocytes were prepared from Per2-luciferase mice and shocked with dexamethasone to synchronize their circadian clocks. Real-time luciferase measurements revealed a circadian oscillation which dampens over the course of three days in vitro for two replicates shown in red and blue (A). Starting four h after dexamethasone shock, mRNA samples from these cells were collected every two h for an entire day and quantitative PCR was used to assess the levels of endogenous mRNAs. Core clock genes, including NR1D1, Dbp, Per2 and Bmal1, were rhythmic over the analyzed time points (B–E); however, 12 h genes were either severely dampened (F) or were completely arrhythmic (G–M). Error bars are +/−S.E.M.; thick purple traces represent the average of three replicates, thin traces show the result of each individual replicate.

Mentions: An obvious caveat to this observation is the possibility that tissue-specific cues may drive sub-circadian oscillations in hepatocytes, but not in fibroblasts or osteosarcoma cells. To examine this, disassociated cultures of primary hepatocytes were prepared from Per2:Luc [9] mice and synchronized with dexamethasone. Real-time imaging of luciferase (Figure 6A) as well as qPCR of core clock genes (Figure 6B–E) demonstrated their oscillations with a period of approximately 24 h. The gene expression pattern of Per2 may reflect its role as an immediate early gene; however, the expression patterns of Bmal1, Dbp and Nr1d1 all suggest the presence of an oscillating 24 h clock. Similar to NIH3T3 and U2OS cells, the amplitude of the core clock genes in this system is dampened relative to samples taken from intact liver in vivo. However, 12 h oscillations were either severely dampened (Figure 6F) or entirely absent (Figure 6G–M, statistical analysis: Table S7). Combined with the results from NIH3T3 and U2OS cells, these data show dampening of both circadian rhythms and their harmonics in three different isolated cellular models. Given the sensitivity of gene expression assays, it is impossible to distinguish between loss of harmonic oscillations and extremely low amplitude cycling, but for practical purposes, these cellular models are not useful for the study of ultradian rhythms.


Harmonics of circadian gene transcription in mammals.

Hughes ME, DiTacchio L, Hayes KR, Vollmers C, Pulivarthy S, Baggs JE, Panda S, Hogenesch JB - PLoS Genet. (2009)

12 h rhythmic transcription is dampened in ex vivo hepatocytes.Primary hepatocytes were prepared from Per2-luciferase mice and shocked with dexamethasone to synchronize their circadian clocks. Real-time luciferase measurements revealed a circadian oscillation which dampens over the course of three days in vitro for two replicates shown in red and blue (A). Starting four h after dexamethasone shock, mRNA samples from these cells were collected every two h for an entire day and quantitative PCR was used to assess the levels of endogenous mRNAs. Core clock genes, including NR1D1, Dbp, Per2 and Bmal1, were rhythmic over the analyzed time points (B–E); however, 12 h genes were either severely dampened (F) or were completely arrhythmic (G–M). Error bars are +/−S.E.M.; thick purple traces represent the average of three replicates, thin traces show the result of each individual replicate.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2654964&req=5

pgen-1000442-g006: 12 h rhythmic transcription is dampened in ex vivo hepatocytes.Primary hepatocytes were prepared from Per2-luciferase mice and shocked with dexamethasone to synchronize their circadian clocks. Real-time luciferase measurements revealed a circadian oscillation which dampens over the course of three days in vitro for two replicates shown in red and blue (A). Starting four h after dexamethasone shock, mRNA samples from these cells were collected every two h for an entire day and quantitative PCR was used to assess the levels of endogenous mRNAs. Core clock genes, including NR1D1, Dbp, Per2 and Bmal1, were rhythmic over the analyzed time points (B–E); however, 12 h genes were either severely dampened (F) or were completely arrhythmic (G–M). Error bars are +/−S.E.M.; thick purple traces represent the average of three replicates, thin traces show the result of each individual replicate.
Mentions: An obvious caveat to this observation is the possibility that tissue-specific cues may drive sub-circadian oscillations in hepatocytes, but not in fibroblasts or osteosarcoma cells. To examine this, disassociated cultures of primary hepatocytes were prepared from Per2:Luc [9] mice and synchronized with dexamethasone. Real-time imaging of luciferase (Figure 6A) as well as qPCR of core clock genes (Figure 6B–E) demonstrated their oscillations with a period of approximately 24 h. The gene expression pattern of Per2 may reflect its role as an immediate early gene; however, the expression patterns of Bmal1, Dbp and Nr1d1 all suggest the presence of an oscillating 24 h clock. Similar to NIH3T3 and U2OS cells, the amplitude of the core clock genes in this system is dampened relative to samples taken from intact liver in vivo. However, 12 h oscillations were either severely dampened (Figure 6F) or entirely absent (Figure 6G–M, statistical analysis: Table S7). Combined with the results from NIH3T3 and U2OS cells, these data show dampening of both circadian rhythms and their harmonics in three different isolated cellular models. Given the sensitivity of gene expression assays, it is impossible to distinguish between loss of harmonic oscillations and extremely low amplitude cycling, but for practical purposes, these cellular models are not useful for the study of ultradian rhythms.

Bottom Line: The circadian clock is a molecular and cellular oscillator found in most mammalian tissues that regulates rhythmic physiology and behavior.Strikingly, we found two clusters of genes that cycle at the second and third harmonic of circadian rhythmicity in liver, but not cultured cells.These harmonics are lost ex vivo, as well as under restricted feeding conditions.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Institute for Translational Medicine and Therapeutics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, United States of America.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus