Intragenic regulatory sites are important for high-level expression of Dbp in mouse liver. (A) Schematic representations of the Dbp wild-type and Dbp–lacZ fusion alleles. The position of the antisense RNA probe used in the ribonuclease protection experiments shown in B is indicated. Upon hybridization with Dbp mRNA or Dbp–lacZ mRNA 124 nucleotides or 235 nucleotides, respectively, are protected from ribonuclease digestion. (B) Ribonuclease protection experiments were performed with whole-cell liver RNA from Dbp^+/− mice extracted at the indicated time points from animals kept under 12:12 hr light/dark conditions (ZT) or animals kept for 10 days under constant conditions (CT). The RNA samples were hybridized to specific probes for Dbp–lacZ mRNA and tbp mRNA. tbp mRNA served as an internal control for a transcript that is constitutively expressed throughout the day. Autoradiographs were exposed for 15 hr (bottom) or 105 hr (top, see bar at left), and the resulting bands were quantified with a scanner. (MW) Molecular weight marker; (Dbp_pr, tbp_pr) undigested probes for Dbp–lacZ and tbp, respectively; (C) negative control with an equivalent amount of yeast tRNA; (+/+, −/−) whole-cell liver RNAs from wild-type or Dbp^−/− mice, respectively, at ZT9.

DNase I hypersensitive sites within the Dbp locus. (A) Schematic representation of the Dbp gene with its four exons (E1–E4) and three introns. The positions of the DNA hybridization probes and the restriction fragments used in the indirect end-labeling experiments are indicated. The approximate positions of the seven DNase I hypersensitive regions detected in B and C are depicted on top of the cartoon. (B) Mapping of DNase I hypersensitive sites starting from exon 4. Equal aliquots of liver nuclei harvested at the indicated times were treated with DNase I (ZT23 to ZT19) or without DNase I (lane C, derived from ZT7). After exhaustive digestion with EcoRV and BglII, the fragments were visualized using probe A. (C) Fine mapping of DNase I hypersensitive sites in the 5′ moiety of the Dbp locus. The DNA was digested with HindIII and probed with probe B. (D) Mapping of DNase I hypersensitive sites in the 3′ moiety of the Dbp locus. The same DNA was digested to completion with BamHI and processed as described in C. A hypersensitive site, mapping to about +500 from the polyadenylation site of Dbp, was not reproducibly observed in other experiments. (E) DNase I hypersensitive sites within the constitutive C/ebpα locus. DNase I/HindIII-digested DNAs were probed with a NcoI/HindIII fragment encompassing the ORF of the intron-less C/ebpα gene. (U) Full-length genomic fragment. (F) Comparison of DNase I hypersensitive sites within the Dbp wild-type allele (panels 1 and 2) and the Dbp–lacZ knockout allele (panel 3). Panel 4 shows DNase I hypersensitive sites within the C/ebp locus as a control. (+/+) Dbp wild-type mice; (+/−) Dbp heterozygous mice. The asterisk marks the position of the hypersensitive region present in the Dbp–lacZ allele.

An intronic E-box motif that binds CLOCK in vitro. (A) Two-dimensional gel EMSA. Nuclear extracts from mouse livers harvested at CT19 were incubated with an oligonucleotide containing the intronic E-box motif (+857) of Dbp. After separation on a 4% polyacrylamide/0.5% agarose composite gel (1st dimension) the protein–DNA complexes were UV cross-linked, size-fractionated by electrophoresis through a 7% SDS–polyacrylamide gel (2nd dimension), blotted onto a membrane, and visualized by autoradiography. In one reaction (+ antiCLOCK ab), the liver nuclear extract was incubated with a CLOCK-specific antiserum before the two-dimensional EMSA analysis was performed. The contours of the spots corresponding to radiolabeled cross-linked protein–DNA complexes are depicted in the drawings at the right-hand site of the autoradiographs. Two spots are lacking in the experiments with CLOCK-specific antiserum. Their vertical alignment in the second dimension indicates that the two complexes were part of a heterodimeric complex in the first dimension. (B) Daily accumulation of CLOCK and BMAL1 in liver nuclei. Nuclear proteins harvested at the indicated time points were separated on a 7% SDS-polyacrylamide gel and probed with CLOCK- or BMAL1-specific antibodies. The migrations of standard proteins according to their molecular mass are indicated on the left (in kD).

CLOCK activates transcription from the intronic Dbp E box. (A) Comparison of basal reporter gene activities of luciferase reporter gene constructs containing four different E-box motifs identified in Dbp. (B) Mouse LTK⁻ fibroblasts were cotransfected with either pGL3+857.luc or pGL3mut.luc and increasing amounts of expression vectors for CLOCK and BMAL1 or CLOCKΔ19 and BMAL1 (80 ng, 400 ng, or 2 μg each). Mean ± s.d. of six experiments. The transfection experiment with pGL3+857.luc and no expression vectors was set to onefold. An asterisk marks highly significant differences (P < 0.001) within the pGL3+857.luc series. There were no such differences within the pGL3mut.luc series.

Dbp mRNA levels are reduced in the SCN of Clock/Clock mice. Representative autoradiographs of in situ hybridizations with coronal brain sections containing the SCN from wild-type mice at ZT3 (A), wild-type mice at ZT15 (B), and Clock/Clock mutant mice at ZT3 (C). A Dbp antisense cRNA was used as a hybridization probe to detect Dbp RNA. (D) A section adjacent to that of the one shown in A hybridized with a Dbp sense cRNA probe (negative control). (E) The temporal accumulation profiles of Dbp mRNA in the SCN of wild-type mice (solid line) and Clock/Clock mutant mice (broken line) in a 12-hr light/dark cycle. Each value is the mean ± s.e.m. of 5–6 animals.

Circadian Dbp expression is obliterated in the liver of Clock mutant mice. (A) RNase protection assays with an antisense Dbp cRNA probe and whole-cell liver RNAs prepared from wild-type and Clock/Clock mutant mice at the indicated times (ZT0–ZT21). A tbp antisense cRNA was included as an internal control for a constitutively expressed mRNA (see Fig. 1). (Lane C) Negative control in which liver RNA was substituted with yeast tRNA. (B) RNase protection assays with an antisense Dbp cRNA probe and whole-cell liver RNAs prepared from mice of different genotypes. The ZT at which the animals were sacrificed and the genotypes of the mice are indicated at top. (Lane C) Negative control in which liver RNA was substituted with yeast tRNA.

Dbp expression may be hardwired to the circadian feedback loop circuitry. The cartoon depicts a speculative model according to which circadian oscillations are generated by a negative-feedback loop of gene expression. The CLOCK/BMAL1 heterodimer activates transcription of essential pacemaker genes such as mPers and mCrys through an E-box motif. The protein products of these pacemaker genes then form complexes that, once they reach a critical threshold level, attenuate CLOCK/BMAL1-mediated activation. The same mechanism may be employed to regulate circadian transcription of Dbp. The cyclic accumulation of DBP may then drive circadian transcription of downstream target genes.