1. Volume 135, Issue 5, Pages 1636-1644.e3 (November 2008)
Circadian Clock-Controlled Intestinal Expression of the Multidrug-Resistance Gene mdr1a in Mice 
Yuichi Murakami, Yuko Higashi, Naoya Matsunaga, Satoru Koyanagi, Shigehiro Ohdo 
Gastroenterology 
Volume 135, Issue 5, Pages e3 (November 2008)
DOI: /j.gastro
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2. Figure 1
Transcriptional regulation of the mdr1a gene by clock gene products. (A) Closed boxes indicate the sites that match the consensus PAR bZIP response element (PARRE) or E-box sequences in the mouse mdr1a gene. The sequence of those sites and the consensus PARRE sequence (R, purine; Y, pyrimidine) are noted (identical bases are shown by asterisks). The numbers of nucleotide residues indicate the distance from the transcription start site. (B) NIH3T3 cells were transfected with mdr1a reporter constructs (mdr1a (−912)-Luc) in the absence or presence of expression plasmids, encoding CLOCK, BMAL1, DBP, TEF, or HLF. Each value represents the mean ± SD of 3 experiments. (C) NIH3T3 cells were transfected with luciferase reporter constructs containing various lengths of the 5′-flanking region of the mdr1a gene in the absence or presence of HLF expression plasmids. Each value represents the mean ± SD of 3 experiments. (D) Repression of HLF-induced mdr1a promoter activity by E4BP4. Schematic representation of wild-type (mdr1a (−194)-Luc) or mutated (mdr1a (−194)-Luc) reporters is shown on the left and their corresponding relative activities on the right. Each value represents the mean ± SD of 3 experiments.
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3. Figure 2
Analysis of HLF and E4BP4 binding to the mdr1a promoter. (A) Gel mobility shift assays were performed using in vitro translated HLF and E4BP4 proteins and biotin-labeled oligonucleotide probe containing the PAR bZIP response element (PARRE) derived from the sequence of mouse mdr1a promoter. (B) Binding experiments were also performed using biotin-labeled oligonucleotide probe containing the mutated PARRE-like motif (bases −150 to −141).
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4. Figure 3
Influence of down-regulation of HLF and E4BP4 proteins on the expression of mdr1a mRNA in colon 26 cells. (A) Temporal mRNA expression profile for mdr1a in colon 26 cells after serum treatment. Each value represents the mean ± SD of 3 experiments. (B) RNA interference with HLF and E4BP4. Colon 26 cells were transfected with scrambled siRNA (Control siRNA), specific siRNA for HLF (HLF siRNA), or specific siRNA for E4BP4 (E4BP4 siRNA). Nontransfected (NT) cells served as nontreated control cells. Transfected or nontransfected cells were treated with 50% FCS for 2 hours and subsequently incubated in serum-starved media. The protein abundance of HLF, E4BP4, and ACTIN was determined at 24 hours and 36 hours after serum treatment. Data shown were confirmed in 2 independent transfected or nontransfected cells, respectively. (C) Influence of down-regulation of HLF and E4BP4 proteins on oscillation in the expression of mdr1a mRNA. Colon 26 cells were transfected with siRNA (Control siRNA, HLF siRNA, or E4BP4 siRNA). Transfected cells were treated with 50% FCS for 2 hours and subsequently incubated in serum-starved media. The mRNA levels of mdr1a were determined at 24 hours and 36 hours after serum treatment. Each value represents the mean ± SD of 3 experiments. *P < .05 when compared between the 2 groups.
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5. Figure 4
Influence of Clock mutation on the expression of mdr1a in the small intestine. (A) Left panel shows temporal expression profile of mdr1a mRNA in the ileum of wild-type (open circle) and Clock/Clock mice (solid circle) under light/dark cycle conditions. Mean peak values of wild-type group are set at 100. Each value represents the mean ± SD of 3 mice. **P < .01, *P < .05 compared with the value for the wild-type mouse group at the corresponding times. There was a significant 24-hour variation in mRNA levels of mdr1a in the ileum of wild-type mice (P < .05, ANOVA). The horizontal bar at the bottom indicates light and dark cycles: open is the light phase, and shaded is the dark phase. Right panel shows temporal expression profile of mdr1a mRNA in the ileum of wild-type mice under constant dark conditions. Mean values of CT10 are set at 100. Each value represents the mean ± SD of 4 mice. **P < .01 compared between the 2 groups. (B) Temporal expression profiles of HLF and E4BP4 proteins in the ileum of wild-type and Clock/Clock mice under light/dark cycle (left panel) and constant dark (right panel) conditions. (C) Quantification of temporal changes in protein levels of HLF and E4BP4 in wild-type (open circle) and Clock/Clock mice (solid circle) under light/dark cycle conditions. Each value was normalized to ACTIN and converted to percentage of maximal level of wild-type mice for each protein. Each value represents the mean ± SD of 4 mice.
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6. Figure 5
Temporal profiles of endogenous HLF and E4BP4 binding to the mdr1a gene in the ileum of wild-type and Clock/Clock (Clk/Clk) mice under light/dark cycle (A) and constant dark (B) conditions. Photographs show representative electrophoretic image of the PCR products of HLF, E4BP4 binding, and input DNA, respectively.
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7. Figure 6
Influence of Clock mutation on the function of mdr1a in the small intestine. (A) Left panel shows temporal expression profile of P-glycoprotein (P-gp) in the ileum of wild-type and Clock/Clock mice under light/dark cycle conditions. Right panel shows temporal expression profile of P-gp in the ileum of wild-type mice under constant dark conditions. Bottom panels are sections of an identical gel stained with Coomassie brilliant blue (CBB) to show approximately equal loading. (B) Left panel shows temporal profiles of intestinal accumulation [3H]-digoxin in wild-type (open circle) and Clock/Clock mice (solid circle) under light/dark cycle conditions. Each value represents the mean ± SD of 3 mice. **P < .01, *P < .05 compared with the value for the wild-type mouse group at the corresponding times. There was significant 24-hour variation in mRNA levels of mdr1a in the ileum of wild-type mice (P < .05, ANOVA). Right panel shows the temporal profile of intestinal accumulation [3H]-digoxin in the wild-type under constant dark conditions. *P < .05 compared between the 2 groups.
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8. Figure 7
Regulation of intestinal expression of the mdr1a gene by HLF and E4BP4. The expressions of HLF and E4BP4 are governed by central components of circadian oscillator; the expression of these genes fluctuated in almost the opposite phase. HLF activates the transcription of the mdr1a gene, whereas E4BP4 periodically suppresses transcription when E4BP4 is abundant. Thereby, HLF and E4BP4 control the amplitude of the rhythm in mdr1a expression.
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9. Supplementary Figure 1
(A) NIH3T3 cells were transfected with scrambled small interfering RNA (siRNA) (Control siRNA; 20 nmol/L) or specific siRNA for CLOCK (CLOCK siRNA; 20 nmol/L). Nontransfected cells served as nontreated control cells. The siRNA sequence of Clock gene was designed for targeting the sequence CAGTGTATCAACTTCAACA and synthesized by using the BLOCK-iTTM RNAi Designer (Invitrogen). The scrambled sequence of CLOCK siRNA was used as a control. At 24 hours after siRNA transfection, the CLOCK messenger RNA (mRNA) levels were determined by reverse-transcription polymerase chain reaction (RT-PCR) using the following primers: Mouse Clock (GenBank accession No. AB000998) forward: 5′-AAGATTCTG GGTCTGATAAA-3′, Clock reverse: 5′-TTGCAGCTTGAGACATCGCT-3′; mouse GAPDH (GenBank accession No. M32599) forward: 5′-AACGACCCCTTCATTGAC-3′, Gapdh reverse: 5′-TCCACGACATACTCAGCAC-3′. (B) At 24 hours after transfecting NIH3T3 cells with siRNA, mdr1a (−912)-Luc were cotransfected with HLF expression vector.
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10. Supplementary Figure 2
Colon 26 cells were transfected with scrambled siRNA (Control siRNA; 20 nmol/L) or specific siRNA for CLOCK (CLOCK siRNA; 20 nmol/L). The siRNA sequence of Clock gene was designed as described in the legend of Supplementary Figure 1. At 24 hours after siRNA transfection, cells were incubated in serum-starved medium for 12 hours. On the day of serum shock, 50% FCS was added for 2 hours and then changed back to starvation medium. Cells were harvested for nuclear protein and RNA extraction at 0, 24, and 36 hours after serum treatment. Protein levels of CLOCK, HLF, E4BP4, and ACTIN were determined by Western blotting using specific antibodies. The mRNA levels of mdr1a gene were measured by real-time PCR.
Gastroenterology  , e3DOI: ( /j.gastro )
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