Materials and Methods

- System-Driven and Oscillator-Dependent Circadian Transcription in Mice with a Conditionally Active Liver Clock

TRE-Rev-erbα mice were generated by pronuclear injection as described in [46]. A cDNA containing the full-length REV-ERBα coding sequence was obtained from F. Damiola. This cDNA contains the first 134 bp of the mouse cDNA (up to the BamHI site) preceded by two HA tags and followed by the remaining of the rat REV-ERBα sequence (F. Damiola and U. Schibler, unpublished data). The cDNA sequence was then PCR amplified using the primers “Bcl1-HA-Revα” and “Bcl1-downstream-Revα” (see Table S1). The PCR product was cut with BclI and cloned into the BamHI site of the pTRE-2 plasmid (ClonTech, Mountain View, California, United States). This new plasmid was then cleaved with XhoI and AseI, and the resulting 3,645-bp fragment encompassing the seven TREs, the minimal CMV promoter, and the HA-tagged REV-ERBα ORF followed by the rabbit β-globin 3′UTR, was used for microinjection into the pronuclei of mouse zygotes. The transgenic mouse line used in this study was selected among 21 lines obtained from 21 different founder mice for its high and strictly Dox-dependent expression of the TRE-Rev-erbα transgene (as assessed by TaqMan real-time RT-PCR).

 

Dox-containing food pellets were produced as follows: powdered mouse chow (Provimi Kliba, Kaiseraugst, Switzerland) was mixed with an equal weight of water containing 3-g/l Dox (Ufamed, Sursee, Switzerland). The suspension was allowed to stand for a few hours in order to saturate the powder with the Dox solution. Small pellets were then formed, and the water was removed by vacuum lyophilization. Mice were fed with these food pellets for at least 1 wk before they were sacrificed for the analysis of RNA and protein.

 

We determined the chromosomal insertion site for the TRE-Rev-erbα and LAP-tTA transgenes in order to facilitate the genotyping analysis of transgenic mice by PCR experiments. To this end, transgenic genomic DNA was digested with a frequently cutting restriction enzyme that cleaves the transgene at defined sites (NlaIII for TRE-Rev-erbα and Sau3AI for LAP-tTA). After heat inactivation of the restriction enzyme, the DNA was diluted to a concentration of 2 ng/μl and ligated with T4 DNA ligase in order to circularize the DNA restriction fragments. These DNA fragments were then precipitated and re-linearized with an infrequently cutting restriction enzyme (SacI in both cases) that cuts the transgene between the restriction sites previously used for the production of circularized DNA fragments (composed of transgene and flanking genomic sequences). The DNA was then used for PCR amplification with primers “TRE-fwd” and “TRE-rev” for TRE-Rev-erbα and “pLAP-fwd” and “pLAP-rev” for LAP-tTA (see Table S1). The resulting PCR products were sequenced and the insertion sites determined.

Lap-tTA genotyping was performed by PCR using the primers “LAPtTAtg-fwd” for the transgenic allele, “LAP-tTAwt-fwd2” for the wild-type (WT) allele, and “LAPtTA-rev2” as common reverse primer (see Table S1). The resulting PCR products encompass 302 bp for the WT allele and 360 bp for the transgenic allele. Genotyping of TRE-Rev-erbα was performed by PCR using the primers “twdTRE” for the transgenic allele, ‘TREvalphaWT2” for the WT allele, and ‘TREvalpha-rev2” as common reverse primer (see Table S1). The resulting PCR products span 351 bp for the WT allele and 560 for the transgenic allele. All experiments shown in the main text of the paper were conducted using double homozygous mice for both Lap-tTA and TRE-Rev-erbα in order to maximize the expression of the transgene, whereas all mice used for in vitro liver explants were heterozygous for the mPER2::LUC allele. The experiments presented in Figure S2 were performed with Lap-tTA/TRE-Rev-erbα/mPER2::LUC triple heterozygous mice for reasons outlined in the legend to this figure.

RNA expression levels were determined using whole-cell RNA essentially as described in [16]. Liver whole-cell RNA was extracted according to reference [47], and Northern blot experiments were performed using 5 μg of whole-cell RNA and hybridization to radiolabeled DNA probes according to the Church protocol [48]. Bmal1 and mPer2 specific probes were generated using sequences encompassing the entire open reading frames as templates. For the Northern blot experiments displayed in Figure 5B, hybridization probes were generated from cloned PCR products encompassing the following sequences: bp 1,144 to 1,946 of Nocturnin, bp 429 to 1,583 of Fus, bp 2,325 to 2,991 of Hsp105/110, bp 1,032 to 1,784 of Hspca/Hsp90, and bp 552 to 1,139 of Cirbp. Real-time TaqMan RT-PCR was performed as described [16]. The primers and probes used in this study are all listed in Table S1. A primer-probe set for the Tata binding protein (TBP) transcript was used for normalization. The same TaqMan probe was used for mouse and rat Rev-erbα, because the mouse and rat sequences are identical in the region encompassing this DNA segment.

 

Nuclear extracts were prepared by the NUN procedure as described [49], and Western blotting was performed according to standard protocols using affinity-purified rabbit polyclonal antibodies. αCry1, αCry2, αPer1, αPer2, αBmal1, and αREV-ERBα antibodies were kindly provided by S. Brown and J. Ripperger.

 

Bioluminescence measurements of liver slices were performed essentially as described [2]. Mice were sacrificed by decapitation. The inferior vena cava was cut, and ice-cold Hank's Balanced Salt Solution (HBSS, Sigma Cat no. H1641; St. Louis, Missouri, United States) was perfused through the spleen in order to remove blood and refrigerate the liver. Tissue pieces were removed, placed into ice-cold HBSS, and sliced into smaller fragments (volume approximately 10 mm³). These tissue pieces were then placed on glass fiber filters in 35-mm tissue culture dishes containing 1.2–1.5 ml of HEPES-buffered phenol red-free DMEM (GIBCO Cat no. 1741; San Diego, California, United States) supplemented with 5% fetal calf serum, 2 mM glutamine, 100-U/ml penicillin,100-μg/ml streptomycin, and 0.1 mM luciferin. Only distal edges of the liver lobes were used, since they gave more reproducible and persistent cycles, perhaps owing to their favorable surface/volume ratio. When relevant, Dox was added to a final concentration of 10 μg/ml. Cultures were maintained at 37 °C in a light-tight incubator, and bioluminescence was monitored continuously using Hamamatsu photomultiplier tubes (PMT; Hamamatsu, Hamamatsu City, Japan) [31]. Photon counts were integrated over 1-min intervals. Dox pretreatment of animals (Figure 3) consisted of two intraperitoneal injections of 2-mg Dox in PBS. Temperature variations in tissue explant cultures were generated using homemade programmable heating chambers.

 

Thirty-six male mice double homozygous for the two transgenes fed with normal chow and an equal number of mice fed with Dox-supplemented chow were maintained on an LD12:12 light cycle and were used for these experiments. Six animals each for Dox-treated and untreated mice were sacrificed at ZT00, ZT04, ZT08, ZT12, ZT16, and ZT20, and whole-cell liver RNA was extracted from each animal. RNA pools of three male animals were assembled by mixing equal amounts of RNA. This resulted in 12 temporally staged RNA pools representing two entire days. A total of 5 μg of pooled RNA was used for the synthesis of biotinylated cRNA; 8.75 μg of biotinylated cRNA was then hybridized to 24 mouse Affymetrix 430 2.0 chips containing 45,000 feature sets and representing 39,000 genes, using standard Affymetrix protocols. The chips were washed and scanned, and the fluorescence signals were analyzed with the RMAexpress software using Robust Multi-array Analysis (RMA) [50].

The data thus obtained were used for a Fourier transform analysis, and the ratio of the F₂₄ spectral power to the sum of the other Fourier components (i.e., ∞, 48, 12, 9.6, and 8 h) was calculated for each feature set [51]. The time points were then permuted 50,000 times, and for each of the permutations, the Fourier transform was calculated together with the ratio of the F₂₄ Fourier component to the other components [52]. Expression data were fitted to a cosine curve. Temporal mRNA accumulation was considered as circadian if its amplitude was higher than 2-fold and if the ratio of the unscrambled Fourier transform was within the top 5% of the scrambled ratios. In order to find circadian genes unaffected by Dox treatment, we calculated a divergence (D) coefficient as follows:

where ZT0:ZT44 represents the hybridization signal for the specified time points. We did not apply any filter for “presence” or “absence” flags (as determined by the MAS Affymetrix software), since 95% of the hybridization signals obtained for the 432 feature sets that we qualified as circadian by Fourier transform analysis scored as “present” in more than 12 experiments out of 24 by the MAS software. By comparison, only 35% of total (circadian and non-circadian) features sets were considered as present in at least 12 experiments.