Usf1, a suppressor of the circadian Clock mutant, reveals the nature of the DNA-binding of the CLOCK:BMAL1 complex in mice
- Northwestern University, United States
- Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, United States
- University of Texas Southwestern Medical Center, United States
- Northwestern University Feinberg School of Medicine, United States
- Genomics Institute of the Novartis Research Foundation, United States
- University of Pennsylvania, United States
Figures
Figure 1
Suppression of ClockΔ19/+ circadian phenotype on the BALB genetic background.
(A) Representative locomotor activity records of wild-type (left) and ClockΔ19/+ (right) animals. The top two records are from B6 coisogenic mice, the middle two are from (BALB x B6)F1 hybrids, and …
Figure 2
High-resolution mapping of the Soc locus using interval-specific SNP haplotype analysis.
(A) Identification of ClockΔ19 suppressor and non-suppressor strains. B6 ClockΔ19/+ mice were crossed to 15 different inbred mouse strains. We used a two-way ANOVA to distinguish the effects of the …
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Figure 2—source data 1
Single nucleotide polymorphisms (SNP) in the Suppressor of Clock (Soc) candidate region among 16 mouse strains.
- https://doi.org/10.7554/eLife.00426.005
Figure 3
Usf1 is a candidate for the Soc locus.
(A) Gene expression profiling of 22 Soc candidates (gray and light blue) and nine clock genes (yellow) in 10 different tissues from Soc congenic ClockΔ19/+ mice. All tissues were collected at ZT6. …
Figure 4
Transgenic expression of Usf1 suppresses ClockΔ19/+ mice and mimics Soc.
(A) Representative locomotor activity records of Usf1 transgenic ClockΔ19/+ mice. Records on the left panel are from ClockΔ19/+ mice carrying a Usf1 transgene while those on the right are from ClockΔ…
Figure 5
Usf1 promoter analysis.
(A) Allele-specific expression difference between B6 and BALB Usf1 mRNA revealed by DNA sequencing. We chose an SNP at the 3′-UTR of Usf1 as a marker to distinguish between the B6 and BALB alleles. …
Figure 6
CLOCK:BMAL1 and USF1 binding to the E-box motif.
(A) CLOCK and USF1 binding at Dbp, Per2 and Per1 E-boxes revealed by ChIP-qPCR. E-box occupancy demonstrated by CLOCK (top panels) and USF1 (bottom panels) is shown. At all E-boxes examined, CLOCK …
Figure 7
Native CLOCK:BMAL1 and USF1 binding affinity.
(A) Saturation binding kinetics of CLOCK:BMAL1 and USF at E-boxes. EMSA analysis for the Dbp EP, Dbp EI2, Per2 E′, and Per1 EP1 E-boxes, top to bottom, respectively, with different probe …
Figure 8
Genome-wide location analysis (Chip-Seq) reveals extensive overlap between USF1 and CLOCK or BMAL1 binding sites.
(A) Venn diagram showing a dramatic increase in sites bound by both USF1 and CLOCK:BMAL1 in ClockΔ19 mutant animals (bottom) as compared to wild-type animals (top). (B) Heat map analysis of the 1916 …
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Figure 8—source data 1
ChIP-seq peak list for USF1.
- https://doi.org/10.7554/eLife.00426.015
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Figure 8—source data 2
ChIP-seq peak list for CLOCK.
- https://doi.org/10.7554/eLife.00426.016
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Figure 8—source data 3
ChIP-seq peak list for BMAL1.
- https://doi.org/10.7554/eLife.00426.017
Figure 9
Model for the USF1 suppression of the ClockΔ19 mutation within the circadian clock mechanism.
(A) SNP polymorphisms between B6 and BALB in the Usf1 promoter lead to a cis-mediated increase in Usf1 expression levels in suppressor mouse strains. (B) In the wild-type state, CLOCK:BMAL1 tandem …
Tables
Table 1
Two-way ANOVA for identification of Clock suppressor or non suppressor strains in Figure 2A
| Suppressor strain | |||
| Strain name | F-ratio (Clock × strain) | p value | df |
| BALB/cJ | 14.698 | <0.001 | df(1,61) |
| A/J | 13.13 | <0.001 | df(1,44) |
| AKR/J | 9.41 | 0.004 | df(1,44) |
| C3H/HeJ | 56.82 | <0.001 | df(1,52) |
| DBA/2J | 34.92 | <0.001 | df(1,46) |
| FVB/NJ | 7.24 | 0.010 | df(1,47) |
| SJL/J | 12.03 | 0.001 | df(1,55) |
| 129S1/SvImJ | 7.094 | 0.010 | df(1,67) |
| Non-suppressor strain | |||
| C58/J | 0.65 | 0.423 | df(1,46) |
| BTBR_T+_tf/J | 0.01 | 0.911 | df(1,53) |
| I/LnJ | 2.72 | 0.107 | df(1,40) |
| MA/MyJ | 0.02 | 0.891 | df(1,52) |
| JF1/Ms | 0.49 | 0.490 | df(1,37) |
| MOLF/EiJ | 0.90 | 0.348 | df(1,37) |
| CZECHII/EiJ | 0.08 | 0.780 | df(1,46) |
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Only the F-ratio for interaction is shown. Two-way ANOVA analyses for circadian period were performed with four groups; C57BL/6J WT, F1 WT, C57BL/6J Clock/+ and F1 Clock/+. Suppressor strains show a significant interaction between Clock genotype and strain background (Clock × strain).
Table 2
Subspecific origin of the Soc region of mouse chromosome 1
| Strain | Chromosome | Start position (bp) | End position (bp) | Subspecies | Sup or non-sup |
| BALB/cJ | 1 | 173300000 | 174200000 | Dom | S |
| A/J | 1 | 173300000 | 174200000 | Dom | S |
| AKR/J | 1 | 173300000 | 173302142 | Mus * | S |
| AKR/J | 1 | 173302461 | 174200000 | Dom | S |
| C3H/HeJ | 1 | 173300000 | 174200000 | Dom | S |
| DBA/2J | 1 | 173300000 | 174200000 | Dom | S |
| FVB/NJ | 1 | 173300000 | 174200000 | Dom | S |
| SJL/J | 1 | 173300000 | 174200000 | Dom | S |
| 129S1SvlmJ | 1 | 173300000 | 174200000 | Dom | S |
| C57BL/6J | 1 | 173300000 | 174200000 | Mus | NS |
| C58/J | 1 | 173300000 | 174028368 | Mus | NS |
| C58/J | 1 | 174187607 | 174200000 | Dom* | NS |
| BTBR T<+>tf/J | 1 | 173300000 | 174200000 | Mus | NS |
| I/LnJ | 1 | 173300000 | 174200000 | Mus | NS |
| JF1/Ms | 1 | 173300000 | 174200000 | Mus | NS |
| MOLF/EiJ | 1 | 173300000 | 174200000 | Mus | NS |
| CZECHII/EiJ | 1 | 173300000 | 174200000 | Mus | NS |
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From the Mouse Phylogeny Viewer (Yang et al., 2011): http://msub.csbio.unc.edu. The AKR/J and C58/J strains contain the proximal and distal breakpoints, respectively, for the Soc locus, and thus there are two entries for these two strains. Dom = M. domesticus, Mus = M. musculus, S = suppressor, NS = non-suppressor.
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*
does not include Soc locus.
Table 3
Apparent binding affinity (Kd) and maximal binding (Bmax) for CLOCK:BMAL1 and USF at four different E-boxes in Figure 7
| CLOCK:BMAL1 | CLOCKΔ19:BMAL1 | USF1 WT | USF1 ClockΔ19/ClockΔ19 | |
| Dbp EP-box | ||||
| Bmax | 0.97 ± 0.02 | 1.25 ± 0.05 * | 0.83 ± 0.06 | 0.87 ± 0.04 |
| Kd | 2.01 ± 0.22* | 32.94 ± 3.92 | 42.8 ± 9.30 | 49.56 ± 6.20 |
| Dbp EI2-box | ||||
| Bmax | 0.79 ± 0.02* | 2.38 ± 0.05 | 2.25 ± 0.05 | 2.10 ± 0.05 |
| Kd | 0.71 ± 0.10* | 6.18 ± 0.54 | 6.10 ± 0.52 | 9.64 ± 0.96 |
| Per2 E′-box | ||||
| Bmax | 0.26 ± 0.02* | 0.43 ± 0.02 | 0.36 ± 0.03 | 0.41 ± 0.02 |
| Kd | 2.00 ± 0.64* | 8.05 ± 1.57 | 17.81 ± 5.08 | 16.71 ± 2.46 |
| Per1 EP1-box | ||||
| Bmax | 0.74 ± 0.12* | 7.38 ± 1.18 | 3.06 ± 0.64 | 7.96 ± 2.01 |
| Kd | 16.55 ± 9.49 | 43.69 ± 19.40 | 33.36 ± 20.99 | 58.62 ± 37.82 |
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Values are the mean + SEM derived from four-parameter nonlinear curve-fitting.
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*
indicates that the Bmax or Kd is significantly different from other groups, p<0.0001.
Additional files
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Supplementary file 1
Oligonucleotide sequences.
- https://doi.org/10.7554/eLife.00426.019
