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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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High temporal resolution profiling of NIH3T3 and U2OS cells reveals severely dampened circadian output.NIH3T3 and U2OS cells were grown to confluence and shocked with either forskolin (NIH3T3) or dexamethasone (U2OS) to synchronize their circadian clocks. mRNA samples were collected every h for 48 h and profiled on Affymetrix expression arrays. Rhythmic genes were identified using both COSOPT and Fisher's G-test at a false-discovery rate of <0.05. The period length of every rhythmic transcript was plotted as a histogram (A–B). To demonstrate that core clock genes cycle well in these data sets, panels C–F show the microarray intensity from two representative genes was plotted against CT time for both NIH3T3 and U2OS cells. NR1D2 (C–D) and Per3 (E–F) expression profiles show examples of cycling 24 h genes.
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pgen-1000442-g002: High temporal resolution profiling of NIH3T3 and U2OS cells reveals severely dampened circadian output.NIH3T3 and U2OS cells were grown to confluence and shocked with either forskolin (NIH3T3) or dexamethasone (U2OS) to synchronize their circadian clocks. mRNA samples were collected every h for 48 h and profiled on Affymetrix expression arrays. Rhythmic genes were identified using both COSOPT and Fisher's G-test at a false-discovery rate of <0.05. The period length of every rhythmic transcript was plotted as a histogram (A–B). To demonstrate that core clock genes cycle well in these data sets, panels C–F show the microarray intensity from two representative genes was plotted against CT time for both NIH3T3 and U2OS cells. NR1D2 (C–D) and Per3 (E–F) expression profiles show examples of cycling 24 h genes.

Mentions: These tests were corrected for multiple comparisons post hoc using the method described by Storey and colleagues[27],[28]. Briefly, by examining the distribution of p-values from a given data set, an estimate of the proportion that are truly non-rhythmic can be derived. Using this approach to model the rate of false-discoveries, the p-value for each transcript, which estimates the frequency that a truly observation will be labeled as significant, can be converted to a more stringent q-value which instead estimates the frequency that significant observations are truly non-rhythmic. At a false discovery rate [27] of <0.05, over 3000 transcripts were found to oscillate by both statistical tests in liver, while fewer than a dozen were found in NIH3T3 and U2OS cells. As expected, the majority of cycling transcripts from liver (and all from NIH3T3 and U2OS cells) had period lengths of approximately 24 h (Figure 1A, Figure 2A and B, Table 1).


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)

High temporal resolution profiling of NIH3T3 and U2OS cells reveals severely dampened circadian output.NIH3T3 and U2OS cells were grown to confluence and shocked with either forskolin (NIH3T3) or dexamethasone (U2OS) to synchronize their circadian clocks. mRNA samples were collected every h for 48 h and profiled on Affymetrix expression arrays. Rhythmic genes were identified using both COSOPT and Fisher's G-test at a false-discovery rate of <0.05. The period length of every rhythmic transcript was plotted as a histogram (A–B). To demonstrate that core clock genes cycle well in these data sets, panels C–F show the microarray intensity from two representative genes was plotted against CT time for both NIH3T3 and U2OS cells. NR1D2 (C–D) and Per3 (E–F) expression profiles show examples of cycling 24 h genes.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2654964&req=5

pgen-1000442-g002: High temporal resolution profiling of NIH3T3 and U2OS cells reveals severely dampened circadian output.NIH3T3 and U2OS cells were grown to confluence and shocked with either forskolin (NIH3T3) or dexamethasone (U2OS) to synchronize their circadian clocks. mRNA samples were collected every h for 48 h and profiled on Affymetrix expression arrays. Rhythmic genes were identified using both COSOPT and Fisher's G-test at a false-discovery rate of <0.05. The period length of every rhythmic transcript was plotted as a histogram (A–B). To demonstrate that core clock genes cycle well in these data sets, panels C–F show the microarray intensity from two representative genes was plotted against CT time for both NIH3T3 and U2OS cells. NR1D2 (C–D) and Per3 (E–F) expression profiles show examples of cycling 24 h genes.
Mentions: These tests were corrected for multiple comparisons post hoc using the method described by Storey and colleagues[27],[28]. Briefly, by examining the distribution of p-values from a given data set, an estimate of the proportion that are truly non-rhythmic can be derived. Using this approach to model the rate of false-discoveries, the p-value for each transcript, which estimates the frequency that a truly observation will be labeled as significant, can be converted to a more stringent q-value which instead estimates the frequency that significant observations are truly non-rhythmic. At a false discovery rate [27] of <0.05, over 3000 transcripts were found to oscillate by both statistical tests in liver, while fewer than a dozen were found in NIH3T3 and U2OS cells. As expected, the majority of cycling transcripts from liver (and all from NIH3T3 and U2OS cells) had period lengths of approximately 24 h (Figure 1A, Figure 2A and B, Table 1).

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