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Regulation of circadian behaviour and metabolism by REV-ERB-α and REV-ERB-β.

Cho H, Zhao X, Hatori M, Yu RT, Barish GD, Lam MT, Chong LW, DiTacchio L, Atkins AR, Glass CK, Liddle C, Auwerx J, Downes M, Panda S, Evans RM - Nature (2012)

Bottom Line: Although REV-ERB-α has been shown to regulate Bmal1 expression directly, our cistromic analysis reveals a more profound connection between BMAL1 and the REV-ERB-α and REV-ERB-β genomic regulatory circuits than was previously suspected.Genes within the intersection of the BMAL1, REV-ERB-α and REV-ERB-β cistromes are highly enriched for both clock and metabolic functions.These data now unite REV-ERB-α and REV-ERB-β with PER, CRY and other components of the principal feedback loop that drives circadian expression and indicate a more integral mechanism for the coordination of circadian rhythm and metabolism.

View Article: PubMed Central - PubMed

Affiliation: Gene Expression Laboratory, Salk Institute for Biological Studies, La Jolla, California 92037, USA.

ABSTRACT
The circadian clock acts at the genomic level to coordinate internal behavioural and physiological rhythms via the CLOCK-BMAL1 transcriptional heterodimer. Although the nuclear receptors REV-ERB-α and REV-ERB-β have been proposed to form an accessory feedback loop that contributes to clock function, their precise roles and importance remain unresolved. To establish their regulatory potential, we determined the genome-wide cis-acting targets (cistromes) of both REV-ERB isoforms in murine liver, which revealed shared recognition at over 50% of their total DNA binding sites and extensive overlap with the master circadian regulator BMAL1. Although REV-ERB-α has been shown to regulate Bmal1 expression directly, our cistromic analysis reveals a more profound connection between BMAL1 and the REV-ERB-α and REV-ERB-β genomic regulatory circuits than was previously suspected. Genes within the intersection of the BMAL1, REV-ERB-α and REV-ERB-β cistromes are highly enriched for both clock and metabolic functions. As predicted by the cistromic analysis, dual depletion of Rev-erb-α and Rev-erb-β function by creating double-knockout mice profoundly disrupted circadian expression of core circadian clock and lipid homeostatic gene networks. As a result, double-knockout mice show markedly altered circadian wheel-running behaviour and deregulated lipid metabolism. These data now unite REV-ERB-α and REV-ERB-β with PER, CRY and other components of the principal feedback loop that drives circadian expression and indicate a more integral mechanism for the coordination of circadian rhythm and metabolism.

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Cistromic analyses of REV-ERBα and REV-ERBβ in livera, De novo HOMER motif analysis of in vivo REV-ERBα and REV-ERBβ binding. b, Venn diagram depicting the unique and common REV- ERBα and REV-ERBβ bound peaks. c, Commonly bound REV-ERBα and REV-ERBβ peaks are enriched for genes involved in lipid metabolism and associated with PPARs. d, REV-ERBα, REV-ERBβ and BMAL1binding at canonical circadian clock genes. Left axis indicates tag counts. e, BMAL1 cistrome significantly overlaps with REV-ERBα and REV-ERBβ. Examples of Clock related genes in overlap are listed and selected peaks shown in Supplementary Figure 4.
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Figure 1: Cistromic analyses of REV-ERBα and REV-ERBβ in livera, De novo HOMER motif analysis of in vivo REV-ERBα and REV-ERBβ binding. b, Venn diagram depicting the unique and common REV- ERBα and REV-ERBβ bound peaks. c, Commonly bound REV-ERBα and REV-ERBβ peaks are enriched for genes involved in lipid metabolism and associated with PPARs. d, REV-ERBα, REV-ERBβ and BMAL1binding at canonical circadian clock genes. Left axis indicates tag counts. e, BMAL1 cistrome significantly overlaps with REV-ERBα and REV-ERBβ. Examples of Clock related genes in overlap are listed and selected peaks shown in Supplementary Figure 4.

Mentions: To clarify the regulatory potential of REV-ERBα and REV-ERBβ in circadian regulation, we generated isoform-specific antibodies (Supplementary Materials and Methods and Supplementary Fig. 1) and determined their genome-wide binding sites (cistromes) in the liver at Zeitgeber Time (ZT) 8, the peak of their protein expression (data not shown and 9). De novo motif analysis (Fig. 1a) revealed that in vivo, in addition to the classic REV-ERB DR2 motifs, other nuclear receptor binding sites (particularly DR1) are predominant in the peaks bound by both REV-ERBα and REV-ERBβ10. Though not surprising, extensive overlap was observed between the REV-ERB cistromes, with commonly bound peaks accounting for 54.8% and 60.7% of the total REV-ERBα and REV-ERBβ peaks, respectively (Fig. 1b). The somewhat limited overlap of our REV-ERBα cistrome with that published from Feng et al. 11 (< 50%) can most likely be attributed to differences in the antibody specificities (Supplementary Fig. 2, 3). Pathway analyses of our REV-ERBα and β overlapping peaks 12 revealed an enrichment in lipid metabolism genes (Fig. 1c), consistent with the hyperlipidemic phenotype previously observed in Rev-erbα animals 13. Notably, loci encoding circadian clock genes (Clk, Bmal1, Cry1/2, Per1/2; see Fig. 1d) were also enriched in the REV-ERBα/β cistromic overlap, suggesting that the coordinated actions of both REV-ERBs are directly linked with clock function. A comparison of the REV-ERBα/β cistrome with published BMAL1 binding sites 14 revealed that 28% of BMAL1 peaks (at ZT6 and ZT10) were shared with the REV-ERBα/β (ZT8) cistrome and 68% of these peaks (781) were occupied by all three transcription factors. Clear binding sites for each of these transcription factors were found on “core clock” gene loci as well as on many clock controlled target genes (Rev-erb α/β, ROR, Dbp, Hlf, Tef, Nfil3; see Fig. 1d,e). In addition to circadian annotated loci, the BMAL1/REV-ERBα/REV-ERBβ triple intersection is highly enriched for genes in the receptor tyrosine kinase signaling pathway as well as those known for energy homeostasis (Supplementary Table 1). Their confluence at hundreds of clock and clock output genes suggest that beyond a simple ‘binary relationship’, Rev-erbα/β and Bmal1 cooperate to coordinately regulate clock and clock output genes.


Regulation of circadian behaviour and metabolism by REV-ERB-α and REV-ERB-β.

Cho H, Zhao X, Hatori M, Yu RT, Barish GD, Lam MT, Chong LW, DiTacchio L, Atkins AR, Glass CK, Liddle C, Auwerx J, Downes M, Panda S, Evans RM - Nature (2012)

Cistromic analyses of REV-ERBα and REV-ERBβ in livera, De novo HOMER motif analysis of in vivo REV-ERBα and REV-ERBβ binding. b, Venn diagram depicting the unique and common REV- ERBα and REV-ERBβ bound peaks. c, Commonly bound REV-ERBα and REV-ERBβ peaks are enriched for genes involved in lipid metabolism and associated with PPARs. d, REV-ERBα, REV-ERBβ and BMAL1binding at canonical circadian clock genes. Left axis indicates tag counts. e, BMAL1 cistrome significantly overlaps with REV-ERBα and REV-ERBβ. Examples of Clock related genes in overlap are listed and selected peaks shown in Supplementary Figure 4.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3367514&req=5

Figure 1: Cistromic analyses of REV-ERBα and REV-ERBβ in livera, De novo HOMER motif analysis of in vivo REV-ERBα and REV-ERBβ binding. b, Venn diagram depicting the unique and common REV- ERBα and REV-ERBβ bound peaks. c, Commonly bound REV-ERBα and REV-ERBβ peaks are enriched for genes involved in lipid metabolism and associated with PPARs. d, REV-ERBα, REV-ERBβ and BMAL1binding at canonical circadian clock genes. Left axis indicates tag counts. e, BMAL1 cistrome significantly overlaps with REV-ERBα and REV-ERBβ. Examples of Clock related genes in overlap are listed and selected peaks shown in Supplementary Figure 4.
Mentions: To clarify the regulatory potential of REV-ERBα and REV-ERBβ in circadian regulation, we generated isoform-specific antibodies (Supplementary Materials and Methods and Supplementary Fig. 1) and determined their genome-wide binding sites (cistromes) in the liver at Zeitgeber Time (ZT) 8, the peak of their protein expression (data not shown and 9). De novo motif analysis (Fig. 1a) revealed that in vivo, in addition to the classic REV-ERB DR2 motifs, other nuclear receptor binding sites (particularly DR1) are predominant in the peaks bound by both REV-ERBα and REV-ERBβ10. Though not surprising, extensive overlap was observed between the REV-ERB cistromes, with commonly bound peaks accounting for 54.8% and 60.7% of the total REV-ERBα and REV-ERBβ peaks, respectively (Fig. 1b). The somewhat limited overlap of our REV-ERBα cistrome with that published from Feng et al. 11 (< 50%) can most likely be attributed to differences in the antibody specificities (Supplementary Fig. 2, 3). Pathway analyses of our REV-ERBα and β overlapping peaks 12 revealed an enrichment in lipid metabolism genes (Fig. 1c), consistent with the hyperlipidemic phenotype previously observed in Rev-erbα animals 13. Notably, loci encoding circadian clock genes (Clk, Bmal1, Cry1/2, Per1/2; see Fig. 1d) were also enriched in the REV-ERBα/β cistromic overlap, suggesting that the coordinated actions of both REV-ERBs are directly linked with clock function. A comparison of the REV-ERBα/β cistrome with published BMAL1 binding sites 14 revealed that 28% of BMAL1 peaks (at ZT6 and ZT10) were shared with the REV-ERBα/β (ZT8) cistrome and 68% of these peaks (781) were occupied by all three transcription factors. Clear binding sites for each of these transcription factors were found on “core clock” gene loci as well as on many clock controlled target genes (Rev-erb α/β, ROR, Dbp, Hlf, Tef, Nfil3; see Fig. 1d,e). In addition to circadian annotated loci, the BMAL1/REV-ERBα/REV-ERBβ triple intersection is highly enriched for genes in the receptor tyrosine kinase signaling pathway as well as those known for energy homeostasis (Supplementary Table 1). Their confluence at hundreds of clock and clock output genes suggest that beyond a simple ‘binary relationship’, Rev-erbα/β and Bmal1 cooperate to coordinately regulate clock and clock output genes.

Bottom Line: Although REV-ERB-α has been shown to regulate Bmal1 expression directly, our cistromic analysis reveals a more profound connection between BMAL1 and the REV-ERB-α and REV-ERB-β genomic regulatory circuits than was previously suspected.Genes within the intersection of the BMAL1, REV-ERB-α and REV-ERB-β cistromes are highly enriched for both clock and metabolic functions.These data now unite REV-ERB-α and REV-ERB-β with PER, CRY and other components of the principal feedback loop that drives circadian expression and indicate a more integral mechanism for the coordination of circadian rhythm and metabolism.

View Article: PubMed Central - PubMed

Affiliation: Gene Expression Laboratory, Salk Institute for Biological Studies, La Jolla, California 92037, USA.

ABSTRACT
The circadian clock acts at the genomic level to coordinate internal behavioural and physiological rhythms via the CLOCK-BMAL1 transcriptional heterodimer. Although the nuclear receptors REV-ERB-α and REV-ERB-β have been proposed to form an accessory feedback loop that contributes to clock function, their precise roles and importance remain unresolved. To establish their regulatory potential, we determined the genome-wide cis-acting targets (cistromes) of both REV-ERB isoforms in murine liver, which revealed shared recognition at over 50% of their total DNA binding sites and extensive overlap with the master circadian regulator BMAL1. Although REV-ERB-α has been shown to regulate Bmal1 expression directly, our cistromic analysis reveals a more profound connection between BMAL1 and the REV-ERB-α and REV-ERB-β genomic regulatory circuits than was previously suspected. Genes within the intersection of the BMAL1, REV-ERB-α and REV-ERB-β cistromes are highly enriched for both clock and metabolic functions. As predicted by the cistromic analysis, dual depletion of Rev-erb-α and Rev-erb-β function by creating double-knockout mice profoundly disrupted circadian expression of core circadian clock and lipid homeostatic gene networks. As a result, double-knockout mice show markedly altered circadian wheel-running behaviour and deregulated lipid metabolism. These data now unite REV-ERB-α and REV-ERB-β with PER, CRY and other components of the principal feedback loop that drives circadian expression and indicate a more integral mechanism for the coordination of circadian rhythm and metabolism.

Show MeSH