ID2 (inhibitor of DNA binding 2) is a rhythmically expressed transcriptional repressor required for circadian clock output in mouse liver

Abstract

Id2 is a helix-loop-helix transcription factor gene expressed in a circadian manner in multiple tissues with a phase-locked relationship with canonical clock genes. Our previous studies have identified circadian phenotypes in Id2 null mice, including enhanced photo-entrainment and disruption of activity rhythms, and have demonstrated a potent inhibitory effect of ID proteins upon CLOCK-BMAL1 transactivation of clock gene and clock-controlled gene activity. We have now begun to explore the potential role that ID2 may play in specifically regulating clock output. Here we show that ID2 protein is rhythmically expressed in mouse liver. Time-of-day-specific liver gene expression in Id2(+/+) and Id2(-/-) mice under circadian conditions was studied using DNA microarray analysis, identifying 651 differentially expressed genes, including a subset of 318 genes deemed rhythmically expressed in other studies. Examination of individual time courses reveals that these genes are dysregulated in a highly time-specific manner. A cohort of different functional groups were identified, including genes associated with glucose and lipid metabolism, e.g. serum protein Igfbp1 and lipoprotein lipase. We also reveal that the Id2(-/-) mice show a reduction in lipid storage in the liver and white adipose tissue, suggesting that disruption of normal circadian activity of components of lipid metabolism can result in overt physiological alterations. These data reveal a role for the transcriptional repressor ID2 as a circadian output regulator in the periphery.

FIGURE 1.

A, real time qRT-PCR of Id2 mouse liver (n ≥ 3) showing rhythmic profiles of Id2. The values are the means ± S.E. fold change of expression relative to the lowest expression value (Id2 and ARP using SYBR green, ABI 7700, normalized to ARP). One-way ANOVA was performed to determine the significance of the Id2 transcript rhythm with post-hoc Dunnett's t-tests (*, p < 0.05). B, representative blots for determining ID2 protein rhythm (n = 3–5) using 40 and 80 μg of total protein extracts. Visualization of β-actin was used to ensure equal protein loading. A positive control was Cos7 cells transfected with human Id2-FLAG. C, quantification of ID2 protein rhythm. Levels of ID2 were determined by normalizing ID2 blots to their respective β-actin signal. Internal normalization was conducted by making the nadir of the rhythm equal to 1.0. One-way ANOVA was performed to determine the significance of the ID2 protein rhythm with post-hoc Dunnett's t-tests (*, p < 0.05).

FIGURE 2.

qRT-PCR of Bmal1 (A), mPer1 (B), and mPer2 (C) in mouse liver (n ≥ 3). The values are the means ± S.E. fold change of expression relative to the lowest expression value (SYBR green, ABI 7700, normalized to ARP). Two-tailed Student's t test was performed at individual time points, and it was determined that there was no statistical difference between WT and Id2^−/− samples. The data from circadian time 0/24 is double plotted.

FIGURE 3.

A, genes involved in various cellular processes were identified by microarray experiment as differentially expressed in the absence of Id2 at CT8/12, CT20, CT8, and CT12. Microarray experiments (n = 3/genotype) at CT8, CT12, and CT20 identified genes involved in various cellular processes that are differentially expressed ≥1.3-fold change in the absence of Id2. B, 38 and 62% of genes identified as differentially expressed were up- and down-regulated, respectively, in the absence of Id2. C, distribution of Id2^−/− differentially expressed CCGs clustered according to phase of peak expression; 60% of these genes had a peak phase in subjective morning, whereas 40% of genes had a peak phase in the subjective night; CCGs were identified from published reports and microarray data bases (17, 20–23).

FIGURE 4.

qRT-PCR reveals time-specific disruption of clock output in the absence of Id2. Shown are Igfbp1, LpL, Acot1, Csad, and HexB in mouse liver (n ≥ 3 per time point and genotype) at CT0–20. The values are the means ± S.E. fold change of expression relative to the lowest expression value (SYBR green, ABI 7700, normalized to ARP). Two-tailed Student's t tests were performed at individual time points to determine the statistical significance between WT and Id2^−/− samples. *, p < 0.05. For Csad at CT12, a one-tailed Student's t test was performed because it was identified in the microarray experiment as up-regulated in Id2^−/− liver. #, p value < 0.05. For HexB at CT12, a two-tailed Student's t test was performed because it was not identified in the microarray experiment as differentially expressed at CT12, and it was determined to be not significant. ¶, p = 0.065. The data from circadian time 0/24 is double plotted.

FIGURE 5.

A, reduced white adipose tissue density in absence of Id2. Gonadal fat pads from WT, Id2^+/−, and Id2^−/− male and female mice were weighed and compared based on the percentage gonadal fat per body mass per age (weeks). One-factor ANOVA followed by post-hoc Dunnett's t-tests. *, p value < 0.05; **, p value < 0.01. B, Id2^−/− mice have significantly lower quantities of fat deposits in the liver. Oil red O staining of WT and Id2^−/− mouse liver (n = 3 mice/genotype). The image shows sections from representative sections from each of three WT (left) and three Id2^−/− (right) mice. Scale bar, 100 μm. C, quantification of fat deposits in WT and Id2^−/− mouse liver sections (n = 10) from three different mice/genotype. The values are the means ± S.E. total area of oil O red droplets occupied in square pixels. Two-tailed Student's t test was performed to determine statistical significance between WT and Id2^−/− samples. t₄₂ = 2.0. **, p < 0.01.

FIGURE 6.

A model for the action of ID2 in the circadian clock. Expression of Id2 is controlled by the clock, and ID2 in turn feeds back to regulate the expression of input and output genes yet is not required for circadian rhythmicity. Its action describes an autoregulatory feedback loop that closes outside the core oscillator but that affects aspects of circadian timing, including photic entrainment through the regulation of light responses (1) and regulation of lipid metabolism. Its actions may include interference with CLOCK-BMAL1 transactivation activity in both input and output regulation (1) and/or act through other bHLH transcription factor targets. C, CLOCK; B, BMAL1; pCREB, Ca²⁺/cAMP response element-binding protein phosphorylated on Ser¹³³.