Age-matched male C57BL/6 mice (the Jackson Laboratory), male Bmal1mice, and male liver-specific Tfeb(Tfeb LiKO) mice were housed under a temperature-controlled, 12hr light/ 12hr dark schedule. Bmal1mice and Tfeb LiKO mice were previously described (). All animal experiments were performed in accordance with guidelines of the Institutional Animal Care and Use Committee at the University of California, Irvine.

Method Details

Fasting Schedule Male C57BL/6 mice, male Bmal1−/− mice, and male Tfeb LiKO mice were placed on a normal chow diet (2020X Teklad Global Extruded Rodent Diet). For the 24-hr fasting experiments, C57BL/6 mice at 8 weeks of age, Bmal1−/− mice at an average of 14.4 weeks of age, and Tfeb LiKO mice at an average of 18.5 weeks of age were randomly divided into an ad libitum fed group, or a fasting group. The fasted mice were placed in new cages without food 24-hr before sacrifice to prevent eating remnants of chow diet on the cage floor. For the 48-hr fasting and refeeding experiment, male wild-type mice to Bmal1−/− mice at an average of 14.9 weeks of age were randomly divided into ad libitum fed group, a fasting group, or a fasting with subsequent refeeding group. All mice were individually housed 1 week prior to harvesting tissue.

Metabolic Cage Analysis Indirect calorimetry was carried out by negative-flow system cages Oxymax/CLAMS (Columbus Instruments). Feeding and lighting conditions in metabolic cages were the same as those in the normal cages. Feeder counts less than 0 g or more than 0.8 g per 10 min during ad libitum fed experiment were excluded from the study. Any feeder counts during the fasting experiment were regarded as noise and treated as null since mice were restricted from any access of food.

Locomotor Activity Analysis Mice were individually housed under 12hr light/ 12hr dark schedule. Locomotor activity was measured using optical beam motion detection (Philips Respironics). Data were collected using the Minimitter VitalView 5.0 data acquisition software. Actograms and activity profiles were computed using Clocklab software (Actimetrics).

RNA Extraction and Reverse Transcription Total RNA was extracted from liver and skeletal muscle with TRIzol (Invitrogen), followed by precipitation with isopropanol and ethanol. Complimentary DNA (cDNA) was obtained by reverse transcription of 1 μg RNA using iScript cDNA Synthesis Kit (Bio-Rad Laboratories).

Protein Extraction and Western Blot Whole cell extracts of liver and skeletal muscle were obtained as follows: frozen liver and skeletal muscle tissues were homogenized in modified RIPA buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 1% NP-40, 0.5% Sodium Deoxycholate, 0.1% SDS, 5 mM MgCl 2 , and 1 mM PMSF) supplemented with Protease Inhibitor Cocktail (Roche), 1 mM DTT, 20 mM NaF, 1 mM Sodium Orthovanadate, 10 mM Nicotinamide, 330 nM Trichostatin A (T8552, Sigma). Samples were sonicated briefly, and centrifuged at 13200 rpm for 15 min at 4°C. Supernatants were stored as whole cell protein lysates. Nuclear extraction was carried out as follows: frozen liver tissues were homogenized in 1.5 mL of buffer A (10 mM HEPES [pH 7.8], 25 mM KCl, 0.5 mM spermidine, 1 mM EGTA, 1 mM EDTA, 320 mM sucrose, 0.3% Triton X-100, and 0.5 mM PMSF) supplemented with the same inhibitors for homogenization of whole cell extracts. Samples were centrifuged at 3300 rpm for 10 min at 4°C. Supernatants were stored as a cytoplasmic fraction. Pellets were resuspended in 1.5 mL of buffer A with the inhibitors. Samples were then spun down at 2000 rpm for 10 min at 4°C, and pellets were resuspended in 1 mL of low salt buffer (10 mM HEPES [pH 7.8], 25 mM KCl, 0.5 mM spermidine, 1 mM EGTA, 1 mM EDTA, 20% Glycerol, and 0.5 mM PMSF) with the same inhibitors for homogenization. Samples were centrifuged at 2000 rpm for 10 min at 4°C, and pellets were resuspended in 1 mL of low salt buffer with the inhibitors. Samples were further centrifuged at 2000 rpm for 5 min at 4°C, and pellets were resuspended in one volume (equal to the pellet size) of low salt buffer with the inhibitors. After measuring the total volume, two volumes of high salt buffer (10 mM HEPES [pH 7.8], 525 mM KCl, 0.5 mM spermidine, 1 mM EGTA, 1 mM EDTA, 20% Glycerol, and 0.5 mM PMSF) with the same inhibitors for homogenization were added to samples, followed by mixture and incubation for 1 h at 4°C. Samples were centrifuged at 12000 rpm for 20 min at 4°C, and supernatants were stored as nuclear fraction. 5-60 μg of protein lysates were separated on 6%–10% gels by SDS-PAGE, and transferred to nitrocellulose membranes. The membranes were incubated with primary antibodies and peroxidase-conjugated secondary antibodies at 4°C, and visualized by chemiluminescent HRP substrate (WBKLS0500, EMD Millipore). The blots were developed with autoradiography films (HyBlot CL, Denville Scientific). The films were scanned and densitometry was analyzed through ImageJ software.

Chromatin Immunoprecipitation Approximately 250 mg of frozen liver tissues were homogenized in 4 mL PBS containing Protease Inhibitor Cocktail (Roche), 20 mM NaF, 0.5 mM PMSF, 10 mM Nicotinamide, and 330 nM Trichostatin A (T8552, Sigma). Liver homogenates were first cross-linked with 2 mM disuccinimidyl glutarate and 1 mM MgCl 2 for 30 min, and further cross-linked with 1% formaldehyde for 5 min at room temperature. Cross-linking reaction was stopped by incubation with 125 mM glycine for 10 min. Samples were centrifuged at 1000 g for 5 min at 4°C, and pellets were resuspended in 4 mL PBS with the inhibitors. Samples were then centrifuged at 1000 g for 10 min at 4°C, and pellets were resuspended in 1 mL of cell lysis buffer (5 mM HEPES [pH 8.0], 85 mM KCl, 0.5% NP-40, and the same inhibitors for homogenization) and incubated on ice for 10 min. Samples were spun down at 3000 rpm for 5 min at 4°C, and pellets were resuspended in 1 mL of SDS lysis buffer (50 mM Tris-HCl [pH 8.0], 10 mM EDTA [pH 8.0], 1% SDS, and the same inhibitors for homogenization) and incubated on ice for 20 min. Samples were sonicated with Bioruptor (Diagenode), and centrifuged at 13200 rpm for 10 min at 4°C. Supernatants were diluted 1:4 in dilution buffer (50 mM Tris-HCl [pH 8.0], 167mM NaCl, 1.1% Triton X-100, 0.11% Sodium Deoxycholate, and the same inhibitors for homogenization). 1 mL of chromatin samples were first pre-cleared with 50 μL of 50% slurry, salmon sperm DNA-blocked Protein G Sepharose (P3296, Sigma-Aldrich) for 2 hr at 4°C. After removal of Protein G Sepharose by quick centrifugation, samples were immunoprecipitated with antibodies overnight at 4°C. 50 μL of 50% slurry Protein G Sepharose beads were then added to samples, followed by incubation for 3 hr at 4°C. The beads were washed with 1ml of low-salt RIPA (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 1 mM EDTA [pH 8.0], 1% Triton X-100, 0.1% Sodium Deoxycholate, and 0.1% SDS), high-salt RIPA (50 mM Tris-HCl [pH 8.0], 500 mM NaCl, 1 mM EDTA [pH 8.0], 1% Triton X-100, 0.1% Sodium Deoxycholate, and 0.1% SDS), LiCl wash (10 mM Tris-HCl [pH 8.0], 250 mM LiCl, 1 mM EDTA [pH 8.0], 0.5% NP-40, and 0.5% Sodium Deoxycholate), and twice with TE wash (10 mM Tris-HCl [pH 8.0] and 1 mM EDTA [pH 8.0]) buffers. Samples were reverse cross-linked with 200 μL of elution buffer (10 mM Tris-HCl [pH 8.0], 300 mM NaCl, 5 mM EDTA [pH 8.0], and 0.5% SDS) overnight at 65°C, and subsequently treated with 2 μL of 2 mg/ml RNase A for 30 min at 37°C, and 1 μL of 10 mg/ml proteinase K for 1 hr at 55°C. After adding 2 μL of 20 mg/ml glycogen, samples were extracted twice with 210 μL of phenol/chloroform/isoamyl alcohol. Finally, DNA was precipitated with ethanol, and resuspended in TE wash buffer.

Quantitative real-time PCR analysis cDNA and immunoprecipitated chromatin DNA were used for quantitative real-time PCR (RT-qPCR) using SsoAdvanced Universal SYBR Green Supermix and CFX96 Detection System (Bio-Rad Laboratories). All gene expression was normalized to 18S Ribosomal RNA. The sequences of primers used in RT-qPCR are listed in Table S6

Antibodies The antibodies used for western blots in this study are as follows. BMAL1 (ab93806, Abcam, 1:10000), Acetyl-BMAL1 (AB15396, EMD Millipore, 1:1000), REV-ERBα (#13418, Cell Signaling Technology, 1:1000), PER2 (PER21-A, Alpha Diagnostic International, 1:1000), CRY1 (A302-614A, Bethyl Laboratories, 1:2000), GR (#12041, Cell Signaling Technology, 1:2000), Phospho-CREB (#9198, Cell Signaling Technology, 1:1000), CREB (#9197, Cell Signaling Technology, 1:1000), FOXO1 (#2880, Cell Signaling Technology, 1:1000), PPARα (sc-9000, Santa Cruz Biotechnology, 1:1000), SIRT1 (07-131, EMD Millipore, 1:10000), PPARδ (PA1-823A, Thermo Fisher Scientific, 1:1000), TFEB (A303-673A, Bethyl Laboratories, 1:2000), ACTIN (ab3280, Abcam, 1:10000), α−TUBULIN (T5168, SIGMA-ALDRICH, 1:10000), p84 (GTX70220, GeneTex, 1:10000), secondary antibodies (12-348 and AP160P, EMD Millipore, 1:10000). The antibodies used for ChIP are as follows. BMAL1 (ab93806, Abcam, 2 μg), GR (#12041, Cell Signaling Technology, 5 μl), CREB (sc-186 X, Santa Cruz Biotechnology, 10 μg), PPARα (sc-9000 X, Santa Cruz Biotechnology, 8 μg), normal rabbit IgG (sc-2027, Santa Cruz Biotechnology).

Serum Corticosterone quantitation Serum levels of corticosterone was quantified using “Corticosterone ELISA Kit” (ADI-900-097, Enzo Life Sciences) according to manufacturer’s instructions.

Serum free fatty acid quantitation Serum levels of free fatty acid were determined using “Free Fatty Acid Quantitation Kit” (MAK044, SIGMA-ALDRICH) according to manufacturer’s instructions.

β-Hydroxybutyrate quantitation Serum levels of β-Hydroxybutyrate were measured using “β-Hydroxybutyrate LiquiColor Assay” (2440-058, EKF DIAGNOSTICS-STANBIO) according to manufacturer’s instructions.

RNA sequencing Total RNA extracted from liver and skeletal muscle as described previously was used for RNA sequencing (RNA-seq). Total RNA was monitored for quality control using the Agilent Bioanalyzer Nano RNA chip and Nanodrop absorbance ratios for 260/280 nm and 260/230 nm. Library construction was performed according to the Illumina TruSeq mRNA stranded protocol. The input quantity for total RNA was 0.75 ug and mRNA was enriched using oligo dT magnetic beads. The enriched mRNA was chemically fragmented for four minutes. First strand synthesis used random primers and reverse transcriptase to make cDNA. After second strand synthesis the ds cDNA was cleaned using AMPure XP beads and the cDNA was end repaired and then the 3′ ends were adenylated. Illumina barcoded adapters were ligated on the ends and the adaptor ligated fragments were enriched by nine cycles of PCR. The resulting libraries were validated by qPCR and sized by Agilent Bioanalyzer DNA high sensitivity chip. The concentrations for the libraries were normalized and then multiplexed together. The concentration for clustering on the flowcell was 12.75 pM. The 72 samples were multiplexed into three libraries of 24 samples each, and sequenced on an Illumina HiSeq 2500 instrument during two single-end 100 cycles sequencing runs by the Genomics High-Throughput Facility at the University of California, Irvine. The version of HiSeq control software was HCS 2.2.58 with real time analysis software, RTA 1.18.64. Each library of 24 samples was sequenced between four and five times on distinct flowcell lanes for a total of 13 flowcell lanes. The resulting sequencing data for each library were post-processed and demultiplexed to produce FastQ files using Illumina software CASAVA 1.8.2. Reads failing Illumina’s standard quality tests were not included in the FastQ files. The quality of the remaining sequences was further assessed using the PHRED quality scores produced in real time during the base-calling step of the sequencing runs.

Short reads alignment to reference genome and transcriptome The sequencing reads from each replicate in the experiment were separately aligned to the reference genome mm10 and corresponding known splice junctions extracted from the RefSeq database using the short-read aligner ELAND v2e (Illumina). Reads with a non-unique best match or with more than two mismatches with the reference sequences were discarded from the analyses. The remaining uniquely aligned reads were used to estimate both mature RNA and precursor mRNA relative abundance in the next steps of the analyses.

Read counting methods Gaidatzis et al., 2015 Gaidatzis D.

Burger L.

Florescu M.

Stadler M.B. Analysis of intronic and exonic reads in RNA-seq data characterizes transcriptional and post-transcriptional regulation. Gene expression results produced by standard RNA-seq analysis tools are obtained by normalizing the number of reads located inside exonic regions of known genes. To compare the rhythmicity of the pre-mRNAs and mRNAs in our datasets, we implemented the Exon Intron Split Analysis (EISA) method using a protocol similar to the one proposed by the authors of the EISA method (). All the components of this analysis were performed using programs specifically developed in-house. This section describes the method used in these programs to decide if a read is exonic or intronic, based on the RefSeq gene annotations available at the time of the analysis as well as the alignment results. Exonic reads are detected following a common protocol. In brief, all isoforms of a given gene are merged together to create a single meta-transcript for the gene, where each exonic position of the meta-transcript belongs to an exon of at least one gene isoform. Similarly, the union of all the splice junctions in all the gene isoforms is used to detect the exonic reads. The resulting meta-transcripts and splice junctions are further processed to discard any exonic position of a gene overlapped by any position of a different gene to remove the corresponding ambiguities. 23,989 gene meta-transcripts were extracted following this protocol. Sequencing reads located in the exons of a meta-transcript, or uniquely mapped to any of the splice junctions associated with the meta-transcript, were considered exonic and therefore contributed to the estimate of the mRNA relative abundance for the corresponding gene in each sample. Gaidatzis et al., 2015 Gaidatzis D.

Burger L.

Florescu M.

Stadler M.B. Analysis of intronic and exonic reads in RNA-seq data characterizes transcriptional and post-transcriptional regulation. Intronic reads were detected following a protocol equivalent to the protocol reported in the EISA method (). Similar to the protocol reported above to detect exonic reads, all isoforms of a given gene are merged together to create a single meta-transcript for the gene. A notable difference for the intronic positions of the curated meta-transcripts is that they belong to the intersection of the introns of all the gene isoforms, instead of the union which is used in the exonic case. This protocol ensures that there is no overlap between the exonic and intronic positions of the curated meta-transcripts. Intronic positions overlapped by any position of a different gene were discarded, as in the exonic case. Genes with no intron and genes with no intron longer than 100 bp in the corresponding meta-transcripts were discarded. This protocol resulted in 19,433 gene meta-transcripts suitable for the EISA analysis. Sequencing reads located in the introns of a meta-transcript were used to estimate the pre-mRNA relative abundance for the corresponding gene.