Circadian enhancers coordinate multiple phases of rhythmic gene transcription in vivo - PubMed (original) (raw)

Circadian enhancers coordinate multiple phases of rhythmic gene transcription in vivo

Bin Fang et al. Cell. 2014.

Abstract

Mammalian transcriptomes display complex circadian rhythms with multiple phases of gene expression that cannot be accounted for by current models of the molecular clock. We have determined the underlying mechanisms by measuring nascent RNA transcription around the clock in mouse liver. Unbiased examination of enhancer RNAs (eRNAs) that cluster in specific circadian phases identified functional enhancers driven by distinct transcription factors (TFs). We further identify on a global scale the components of the TF cistromes that function to orchestrate circadian gene expression. Integrated genomic analyses also revealed mechanisms by which a single circadian factor controls opposing transcriptional phases. These findings shed light on the diversity and specificity of TF function in the generation of multiple phases of circadian gene transcription in a mammalian organ.

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Figures

Figure 1

Figure 1. Circadian transcription in mouse liver

(A) Genome browser view of nascent transcripts at Bmal1/Arntl and Rev-erbα/Nr1d1 loci at 8 time points. GRO-seq signals on the + and- strand are illustrated in blue and red, respectively. Y-axis scale refers to the normalized tag count per 106 reads. (B) Heat map of the relative transcription of 1,261 oscillating genes sorted by oscillation phase. (C) Relative expression of pre-mRNA (green) and mRNA (black) determined by GRO-seq and RT-qPCR, respectively, throughout the day. Data are double plotted for better visualization. RT-qPCR data are expressed as the ±SEM (n=3–4 per time point) and normalized to the maximal expression of the day. See also Figure S1 and Table S1.

Figure 2

Figure 2. De novo identification of circadian liver enhancer RNAs

(A) Genome browser view of intergenic (upper panel) and intragenic (lower panel) eRNAs (yellow boxes). (B) GRO-seq tag densities in 4kb windows surrounding de novo intergenic (upper panel) and intragenic (lower panel) eRNA loci are shown for the plus (blue) and minus (red) strand. Y-axis shows average reads per 10 million reads (RPTM) per 10bp bin. (C) Average ChIP-seq tag densities of epigenetic marks in 2kb window surrounding all de novo eRNA loci (prior to the selection of high confidence eRNAs) and matched control regions. (D) Heat map of the relative transcription of oscillating eRNAs throughout the day. Color coding of eRNA population in 8 phase groups (from ZT0 to ZT24, at 3 hour intervals) is shown on the right. (E) Rose diagram showing the prevalence of eRNA loci in each phase group. For each wedge, the color corresponds to that in (D) and the area is proportional to the number of eRNAs in that group. (F) Genome browser view of oscillating eRNAs at Cry1 locus. (G) RT-qPCR validation of circadian transcription for intergenic, intragenic, and non-cyclic eRNAs at indicated gene loci. Data are expressed as mean±SEM (n=3–4 per time point) and normalized to the first time point. See also Figure S2 and Table S2.

Figure 3

Figure 3. Phase-specific transcription factors at circadian enhancers

(A) Relative transcription of genes closest to oscillating eRNAs (within 200kb of TSS). (B) Motifs specifically enriched in each eRNA group are labeled in the clock diagram on the left. Position weight matrix (PWM) of each motif and its best enrichment p-value in assigned groups are shown in the table on the right. (C) Correlation of motif occurrence and TF binding in 8 eRNA phase groups. In each plot, the red dots represent the fraction of eRNA loci bound by the indicated TF (top 3,000 ChIP-seq peaks), and black bars represent the fraction of eRNA loci containing the corresponding motif. Correlation coefficient r is shown for phase-specific motifs. TFs recognizing different types of motifs are grouped in colored boxes corresponding to those used for eRNA phases. See also Figure S3 and Table S3.

Figure 4

Figure 4. Phase-correlation between eRNA and gene body transcription marks functional enhancers of circadian genes

(A) Heat map of the relative transcription of 325 circadian genes in phase ZT18–24 (left panel) and their neighboring eRNAs (right panel). 539 eRNAs in correlated phase are shown in the red box while 857 non-correlated eRNAs are in the blue box. (B) Enrichment of RevDR2 and RORE motif in correlated eRNA loci relative to non-correlated eRNA loci (hypergeometric test, ***p<0.001). (C) ChIP-seq tag density of Rev-erbα (left) and HDAC3 (right) in 2kb windows surrounding correlated (red) and non-correlated eRNA loci (blue). y-axis shows the average tag count per 10bp bin normalized to 10 million total reads. (D) Comparison of GRO-seq tag density (RPTM per 10bp bin in 2kb window) surrounding correlated (left) and non-correlated (right) eRNA loci in WT and Rev-erbα −/− livers at ZT10. (E) Heat map of transcriptional changes between WT and Rev-erbα −/− livers at ZT10, for the 325 circadian genes in phase ZT18–24 (right column), compared to the same number of random genes (left column). Data are expressed as log2 fold change. See also Figure S4.

Figure 5

Figure 5. Circadian eRNAs reveal the function of the Rev-erb

α cistrome at oscillating genes. (A) Distribution of Rev-erbα ChIP-seq peaks near circadian genes (upper panel) and subdistribution of eRNA-producing Rev-erbα peaks near circadian genes (lower panel). (B) Boxplot showing Rev-erbα and HDAC3 peak height at binding sites from panel A. Y-axis indicates normalized tag count in each peak (RPTM) (***p<0.001, one-way ANOVA and Tukey’s test). (C) Enrichment of derepressed genes in Rev-erbα −/− mice at circadian genes bound by different Rev-erbα peaks from panel A relative to a random set of Rev-erbα peaks (hypergeometric test, ***p<0.001). (D) Enrichment of derepressed genes in Rev-erbα −/− mice in 8 groups of circadian genes with indicated phases relative to randomly selected genes (hypergeometric test, ***p<0.001). See also Figure S5 and Table S4.

Figure 6

Figure 6. E4BP4 functions downstream of Rev-erbα

(A) Enrichment of oscillating eRNAs in each phase group near genes downregulated in Rev-erbα −/− livers relative to control genes. Significantly enriched phases are noted as corresponding to D-box/E4BP4-enriched phase group. (hypergeometric test, *p<0.05). (B) mRNA expression of E4BP4/Nfil3 in WT and Reverbα −/− livers measured by RT-qPCR throughout the day. Data are expressed as mean±SEM (n=2 per time point and genotype) normalized to the first WT time point. (C) Enrichment of E4BP4+eRNA bound genes among those downregulated (green) or upregulated (red) in Reverbα −/− livers relative to unchanged genes (grey) (hypergeometric test, *p<0.05). (D-F) Average circadian expression profiles in WT mouse livers (Hughes et al., 2009) and corresponding transcription profiles by GRO-seq for (D) genes downregulated in Rev-erbα −/− livers within 200kb of E4BP4 binding at ZT9–15 circadian eRNAs, (E) genes upregulated in Rev-erbα −/− livers within 200kb of Rev-erbα binding at ZT18–24 circadian eRNAs, and (F) non-regulated control genes (expressed in liver within 200kb of near circadian eRNA in any phase). (G) ChIP-qPCR of E4BP4 binding at genes downregulated in Rev-erbα −_/− livers at ZT10. Binding is shown at ZT10 (solid bars) and ZT22 (hashed bars) in WT (blue) and Rev-erbα −/_− (orange) livers. Data are expressed as mean±SEM (One-way ANOVA, *p<0.05, #p<0.1, n=3–4 per group). (H) mRNA expression measured by RT-qPCR in liver overexpressing Rev-erbα (mice injected with AAV-Tbg-Rev-erbα) or control liver (mice injected with AAV-Tbg-GFP) at ZT22. Data are expressed as mean±SEM (One-way ANOVA, *p<0.05, #p<0.1; n=6 per group). (I) ChIP-qPCR of E4BP4 binding at same sites as (G) in liver overexpressing Rev-erbα (blue) or control liver (orange). Data are expressed as mean±SEM (One-way ANOVA, *p<0.05, #p<0.1, n=5–6 per group). See also Figure S6.

Figure 7

Figure 7. Circadian eRNAs define functional cistromes that distinguish CLOCK and Reverbα target genes

(A) Average expression of genes within 200kb of CLOCK binding sites producing eRNA in phase with CLOCK binding and target gene expression in WT (yellow line) and Clock mutant (orange line) mouse livers from (Miller et al., 2007) (*Wilcoxon test of gene fold-change distribution versus matching time points in (B), *p<0.05). **(B)** Average expression of genes within 200kb of CLOCK binding sites lacking in phase eRNA in WT (dark grey line) and _Clock_ mutant (light grey line) mouse livers from (Miller et al., 2007). **(C)** Enrichment of circadian genes expressed in phase with CLOCK binding (ZT6–12) or anti-phase to CLOCK binding (ZT18–24) for the gene groups used in panel A (yellow) and panel B (grey) relative to random genes (hypergeometric test, *p<0.05). **(D)** Enrichment of genes downregulated in _Clock_ mutant livers among the gene groups used in panels A-C relative to random genes (hypergeometric test, *p<0.05). **(E)** Fraction of oscillating genes co-bound by CLOCK and Rev-erbα that are within 200kb of TF binding sites producing rhythmic eRNA in phase with CLOCK activation (blue), Rev-erbα repression (red), or both (green). Oscillating genes are divided according to their phases. Representative genes are noted in each group. **(F)** Enrichment of CLOCK and Rev-erbα regulatedgenes (expression fold change in mutant >95% of random genes) in those with eRNA predicted functional binding sites in panel E, relative to random genes (hypergeometric test, ***p<0.001, N.S. p>0.05). See also Figure S7 and Table S5.

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