REV-ERBα and REV-ERBβ function as key factors regulating Mammalian Circadian Output

To investigate the role of REV-ERBs in the cell-autonomous circadian gene expression, we employed the ES cell-differentiation assay that enables reproducible formation of the circadian rhythms of clock gene expression^17,18. We utilized Per2::Luciferase (Per2^Luc) knock-in mES cells^19–21 and introduced the targeted deletions of exons of Rev-erbα and Rev-erbβ genes by using CRISPR/Cas9 systems (Fig. 1a). In knockout cells, translational frameshifts were confirmed by sequencing of cDNA (Supplementary Fig. S1) and mRNAs including the CRISPR-targeted exon were not detected by quantitative PCR (Fig. 1b), indicating successful establishment of Rev-erbα/Rev-erbβ double knockout mES cells.

To evaluate the impact of REV-ERBs deficiency on global gene expression, the cells were differentiated with the embryoid body (EB) formation method and temporal RNA sequencing (RNA-seq) analysis with RNA samples at 4-h intervals over 2 days were performed. RNA samples from control Per2^Luc cells (WT) and Rev-erbα/β deficient cells (KO) were analyzed by polyA-selected RNA-seq. The number of expressed genes in WT and KO cells were comparable and most of them overlapped (Fig. 2a). Expression levels of several differentiation marker genes for ectoderm, endoderm, and mesoderm were not drastically changed between WT and KO cells, suggesting that overall differentiation of Rev-erbα/β deficient cells is likely to be comparable to that of WT cells (Fig. 2b). We evaluated the mRNA expression of core clock genes and found that expression levels of Per1, Per2, Per3, Cry1, Cry2, and Clock in Rev-erbα/β deficient cells were comparable to those in WT (Fig. 2c). In contrast, the expression levels of Bmal1 and Npas2, the direct targets of REV-ERBα/β, were significantly upregulated (Fig. 2c). Furthermore, previously reported REV-ERB-target genes such as E4bp4 (Nfil3), Dec1 (bhlhe40), and p21 (Cdkn1a)^22–24 were also upregulated (Fig. 2d). These results indicate that REV-ERBα/β deficiency in differentiated mES cells affects their target gene expression in a manner consistent with REV-ERBs function as transcriptional repressors.

To evaluate the impact of REV-ERBs deficiency on global gene expression rhythms, we performed a periodicity analysis on the time-course RNA-seq dataset. The number of cycling genes were 173 (1.2% of expressed genes) and 235 (1.6% of expressed genes) in WT and KO, respectively, and only 15 genes exhibited circadian oscillation of expression in both WT and KO cells (Fig. 3a, Supplementary Table S1). To our surprise, well-known circadian clock and clock-controlled genes (Per2, Per3, Cry1, Rev-erbα, Rev-erbβ, Rorc, Dbp, Tef, Hlf) were cycling in KO cells as well as WT cells. Furthermore, most of cycling genes were not common between WT and KO cells. This result indicates diverse changes in regulation of circadian output gene expression rhythms in KO cells, although the diverse phase distribution of cycling genes are similar in WT and KO cells with peaking around circadian time (CT) 18 (Fig. 3b,c). Transcription factor-binding motifs enrichment analysis revealed that HNF1 and NF-Y were enriched in the promoter of cycling genes in WT but not in KO (Fig. 3d, Supplementary Table S2), suggesting that HNF1 and NF-Y might be involved in the regulation of REV-ERB-dependent circadian output. This was supported by the results that Hnf1b was identified as a cycling gene only in WT cells (Supplementary Fig. S2), and that REV-ERBs bind to NF-Y and regulate target gene expression via the NF-Y-binding CCAAT motif in differentiating myoblasts²⁵. Intriguingly, expression pattern of core clock and clock-controlled genes including Per2, Per3, Cry1, Tef, and Hlf were very similar between WT and KO cells (Fig. 3e). Peak expression levels of Rev-erbα, Rev-erbβ, and Dbp expression rhythms were elevated, while the basal expression of Rorc was reduced (Fig. 3e). In contrast to these sustained rhythms, Bmal1 and Npas2 expression rhythms are abrogated and constantly upregulated (Fig. 3f). The sustained rhythm of Per2 and Cry1 and constant upregulation of Bmal1 and Npas2 were confirmed by quantitative PCR analysis (Fig. 3g). Collectively, these results demonstrate that REV-ERBs repress Bmal1 and Npas2 mRNA expression but have only minor influence on mRNA expression rhythms of other core clock genes including Per2. These data suggest that REV-ERBs are not necessary for the circadian core clock gene expression rhythms but play an important role in regulation of circadian genetic program linking the circadian clock and output gene expression. Further analysis of the temporal expression profile of PER2^Luc bioluminescence in differentiated mES cells revealed that PER2^Luc bioluminescence rhythm in KO cells was as robust as that in WT cells (Fig. 4a). Both period length and amplitude were comparable between WT and KO cells (Fig. 4b). These results emphasize the importance of REV-ERBs as circadian clock mediators that regulate diverse output gene expression rhythms (Fig. 4c).

Mouse ES cells derived from PER2^Luc knock-in mice are described previously^19–21. ES cells were maintained on MEF feeder cells in an ES medium (Glasgow Minimum essential Medium supplemented with 15% FBS (Hyclone), 0.1 mM MEM nonessential amino acids, 0.1 mM 2-mercaptoethanol, 1,000 U/mL of Leukemia inhibitory factor (LIF), and 100 U/mL penicillin-streptomycin).

Human codon-optimized Cas9 expression vectors and sgRNA vectors were described previously³⁴. A pair of oligos for the sgRNA targeting site was annealed and ligated into the sgRNA plasmid. The sgRNA target sites in introns adjacent to the targeted exon were determined by using CRISPRdirect³⁵. The CRISPR-targeted sequences are as follows: Rev-erbα-T1, 5′-CCCAGACGTAGTTGATAGAGTT-3′; Rev-erbα-T2, 5′-TGCAAGGTGAGGCGGGTTAGGG-3′; Rev-erbβ-T1, 5′-CTACTGATAGATACCAGTAAGG-3′; Rev-erbβ-T2, 5′-GGGTTTAAACTCACTATGGTGG-3′. Underlines indicate protospacer adjacent motif (PAM) sequences.

Mouse ESCs were co-transfected with an hCas9 expression vector, two sgRNA expression vectors targeting Rev-erbβ, and a plasmid with a puromycin selection marker using FuGENE HD (Promega). The cells were selected with 2 μg/mL of puromycin for two days and then passaged for mutant cloning. ESC colonies were picked and cultured to establish mutant cell lines. Rev-erbα was targeted in established Rev-erbβ-deficient mES cells. The genomic deletion and the exon skipping in mRNA were confirmed by sequencing analysis of genomic DNA and cDNA, respectively.

Genomic DNA samples were extracted from mouse ESCs grown under feeder-free conditions. cDNA samples were synthesized using Moloney murine leukemia virus (M-MLV) reverse transcriptase (Invitrogen) from total RNA extracted from mES cells using the RNeasy Mini kit (Qiagen) according to the manufacturer’s instructions. PCR was performed with PrimeSTAR MAX DNA Polymerase (TAKARA) under the following conditions: 98 °C for 1 min; 35 cycles of 98 °C for 10 sec, 60 °C for 5 sec, 72 °C for 10 sec; 72 °C for 20 sec; hold at 4 °C. PCR products were treated with Exonuclease I (New England Biolabs) and shrimp alkaline phosphatase (TAKARA) at 37 °C for 30 min followed by inactivation at 95 °C for 10 min and used for sequencing analysis. Sequencing was performed with the same primers as used for the PCR. The primer sequences are listed in Supplementary Table. S3

The in vitro differentiation of ESCs was performed as described previously^18,20. Briefly, in order to form embryoid bodies (EBs), 2 × 10³ of the dissociated ESCs were seeded in low-attachment 96-well plates in a differentiation medium (DMEM supplemented with 10% FBS, 1 mM sodium pyruvate, 0.1 mM non-essential amino acids, GlutaMax-I (Invitrogen), 0.1 mM 2-mercaptoethanol, and 100 U/mL penicillin-streptomycin). Two days later, the EBs were plated onto gelatin-coated 24-well plates and cultured for 26 days in a differentiation medium, which was exchanged every other day.

Total RNA was extracted from cells with the RNeasy Mini kit (Qiagen) according to the manufacturer’s instructions and subjected to cDNA synthesis with random hexamer primers and M-MLV reverse transcriptase (Invitrogen) according to the manufacturer’s instructions. Real-time quantitative PCR was performed with iTaq Universal SYBR Green Supermix (Bio-Rad Laboratories) and StepOnePlus real-time PCR system (Applied Biosystems). The PCR primers for quantitative PCR are listed in Supplementary Table S3. Of note, primers for Rev-erbα and Rev-erbβ were designed in the CRISPR-targeted exons to evaluate the successful deletion.

At differentiation day 28, cells were treated with 100 nM dexamethasone and frozen at the indicated time points. Total RNA was extracted from cells with the RNeasy Mini kit (Qiagen) according to the manufacturer’s instructions. Total RNA from differentiated cells from three embryoid bodies were pooled and used for the analysis. PolyA RNA selection, library construction using TruSeq RNA Sample Prep Kit v2, and sequencing were performed by Macrogen Japan (Kyoto) using Illumina NovaSeq6000 with 101-bp paired-end reads according to the manufacturer’s instructions (Supplementary Table S4). After adaptor sequence trimming using Trimmomatic³⁶, sequence reads were mapped to the mouse genome (GRCm38/mm10) using STAR³⁷. To obtain reliable alignments, the reads with a mapping quality of less than 10 were removed by SAMtools³⁸. The University of California, Santa Cruz (UCSC) known canonical gene set (32,989 genes) was used for annotation, and the reads mapped to the exons were quantified using Homer³⁹ as described previously⁴⁰. Among the UCSC known canonical genes, genes were considered to be expressed if there were more than 0.5 reads per million reads mapped on average of 12 time points in the exons of the genes. RNA cycling was determined using Metacycle⁴¹ with P < 0.05, rAMP > 0.17, and the following options: minper = 20, maxper = 28, cycMethod = c(“ARS”,“JTK”,“LS”), analysisStrategy = “auto”, outputFile = TRUE, outIntegration = “both”, adjustPhase = “predictedPer”, combinePvalue = “fisher”, weightedPerPha = TRUE, ARSmle = “auto”, and ARSde- faultPer = 24. For transcription factor-binding motif analysis, known motifs in a range from −500 to + 500 of transcription start site (TSS) of cycling genes were discovered by Homer.

For bioluminescence recording, the medium was replaced with a differentiation medium containing 0.2 mM luciferin and 100 nM dexamethasone. Bioluminescence was measured and integrated for one min at 20 min intervals with PMT-based equipment.

The bioluminescence data recorded by PMT were analyzed using a sine wave fitting. A linear baseline was subtracted from the raw data. The detrended data from between 36–108 hours was then used for analysis. Sine wave fitting was performed using the following equation:

where A = amplitude, k = damping constant, t = time, τ = period, and φ = phase.

For statistical analyses, two-tailed Student’s t tests were performed unless otherwise described.

The RNA-seq data has been deposited in the NCBI Gene Expression Omnibus (GEO) with the accession number GSE125696↗.