5' RACE. Total RNA from C57BL/6J female mouse brain was isolated at zeitgeber time (ZT) 12. A commercially available 5' RACE kit (5' RACE System, Version 2.0; Invitrogen) was used according to the manufacturer’s protocols to identify the transcription start site. Primers GSP1 5'-ACTGGGTATTTCTCTGTT-3' and GSP2 5'-CTCTGGACCCATCCTAGGAACA-3' were used to make first-strand cDNA.

Vector Constructs. A 5' UTR and exon 1-containing 3.3-kb EcoRI/XbaI fragment was isolated from a positive bacterial artificial chromosome clone (Research Genetics, Huntsville, AL) (1) and subcloned into the pGL3-Basic luciferase reporter vector (Promega). The following primers were used to generate truncated promoter constructs pGL-5, pGL-4, pGL-3, pGL-2, pGL1, and E2 enhancer-pGL reporter:

(–1,128) 5'-GCCGAGCTCCAAGCTTGCCTTCTCCAT -3'

(2,064) 5'-CCGCTCGAGGGGCCACATCCCCAGTGGGA-3'

(1,536) 5'-CCGCTCGAGGTCTCAGATGAGGAAACTGGAG-3'

(1,060) 5'-CCGCTCGAGGAGAAACAGATCCTGAGACTGC 3'

(386) 5'-CCGCTCGAGTCGGACAAGATGCCTAACTAAT-3'

(–141) 5'-CCGCTCGAGCTTTACATAAGACGCACATGGA-3'

(–112) 5'-CGCGGATCCGAGCGTAGCTCTCAGGTTCCG-3'

(98) 5'-TGCTCTAGATCCCTTGCTCGGCCCGTCACT-3'.

The QuikChange site-directed mutagenesis kit (Stratagene) was used with the following primers for the E-box enhancer mutagenesis:

E2mut, 5'- GGGCGGGCTCAGCGCGCGCGGTGCTAGTTTCCACTATGTGACAGCGGAGG-3' (forward) and 5'-CCTCCGCTGTCACATAGTGGAAACTAGCACCGCGCGCGCTGAGCCCGCCC-3' (reverse).

To generate pGL3-promoters pE1, pE2a, pE2, pE3, pE4, and pE5, the following primers were used:

E1up 5'-CGCGGATCCGAAGCTGCTTAGCACCAGACT-3',

E1dw 5'-TGCTCTAGAAGTCAATTGTGTGGCTCTCTC-3',

E2aup 5'-CGCGGATCCATCAGCCAGCTGGCAGCTACT-3',

E2adw 5'-TGCTCTAGATAGTCCATTTAAGCACAGCAG-3',

E2up 5'-CGCGGATCCGAGCGTAGCTCTCAGGTTCCG-3',

E2dw 5'-TGCTCTAGATCCCTTGCTCGGCCCGTCACT-3',

E3up 5'-GAAGACATTCCAGAGCCAAA-3',

E3dw 5'-TGTGGTTCCCTCAGAGGTTC-3',

E4up 5'-CCTCAAATATGGAGAATCACCTC-3',

E4dw 5'-GCCCTTGCTATAGACCAAAAA-3',

E5up 5'-CGCGGATCCGATTCCTGCCACATTGAGATT-3', and

E5dw 5'-TGCTCTAGATAACCCTGGGTGACACAGAAG-3'.

Cell Culture. HepG2 cells (American Type Culture Collection) were grown in DMEM (Mediatech, Washington, DC) supplemented with 10% FBS containing penicillin/streptomycin (Invitrogen). Cells were plated the day before transfection at 2 × 10⁵ cells per well in six-well plates. Cells were transfected (OPTI-MEM medium; Invitrogen) with 50 ng of reporter, 100 ng of pCMV-b -galactosidase (Promega) for normalization, and pcDNA3.1 mClock and mBmal1 constructs (2) by using Lipofectamine Plus (Invitrogen) according to the manufacturer’s protocols. The total mass of transfected DNA was normalized to 1 m g using pcDNA3.1 plasmid. At 48 hours after transfection, cells were lysed and luminescence was measured from 20 m l of lysate in the Luciferase Assay System (Promega) by using a luminometer (AutoLumet Plus; Berthold, Nashua, NH).

Transgenic Mice. Transgenic mice were generated as described (3) by using 1 ng/m l of SalI/KpnI-linearized, gel-purified E2 enhancer luciferase fragment. Transgenic animals were identified by genotyping tail genomic DNA samples in PCRs by using the following luciferase primers:

5'-TATGAAGAGATACGCCCTGGTT-3' (forward) and

5'-TAAAACCGGGAGGTAGATGAGA-3' (reverse).

Explant Tissue Culture. Tissue explant cultures were performed as reported (1).

Bioluminescence Data Analysis. Period and phase measurements were calculated as in previous studies (1). Period measurements of the mPer2-E2::Luc animals crossed to Clock and Bmal1 mutants were calculated by using the LumiCycle data-analysis program (Actimetrics, Wilmette, IL).

DnaseI Hypersensitive Site Mapping. Nuclei from Hepa1-6 cells (C57BL/6J hepatoma cell line) and C57BL/6J female mice were isolated as described (4, 5) with modification. Briefly, mice were anesthetized with ketamine (ketamine (80 m g per g of body mass) and xylazine (0.4 m g per g of body mass), and perfused with 50 ml of 37°C PBS containing100 m g/ml heparin and 0.5 mg/ml collagenase (Sigma). After perfusion, whole liver was isolated and homogenized with a Dounce homogenizer (type B pestle; 5 strokes). Hepa1-6 cells or liver samples isolated from perfusion (4 × 10⁷ cells each) were washed twice in PBS and resuspended in 1 ml of buffer A (0.34 M sucrose/10 mM Hepes, pH 8/60 mM KCl/2 mM EDTA/0.5 mM EGTA/1.5 mM DTT/0.5 mM spermine/0.15 mM spermidine with 0.5% Nonidet P-40). Nuclei were pelleted at 2,040 × g for 5 min at 4°C in a microcentrifuge, and washed again in buffer A without Nonidet P-40. The nuclei were resuspended in 800 m l of buffer B (10 mM Hepes, pH 8/20 mM KCl/3 mM MgCl₂/6 mM CaCl₂). Isolated nuclei (200 m l) were treated with 0, 8, 16, and 24 units of of DNaseI (Promega) for 1 min at room temperature; reactions were stopped by adding EDTA (pH 8.0) to a final concentration of 12 mM. Isolated genomic DNA (20 m g) was digested to completion with XbaI and resolved on 1% agarose gels. Two primers described earlier (–1,128 and –141) were used to generate the 1.2-kb Probe A, whereas Probe B was a 626-bp KpnI/XbaI fragment derived from pGL-6 for Southern blot analysis.

Chromatin Immunoprecipitation Assays and Real-Time PCR. Chromatin immunoprecipitation was performed as described (6), without any modification (Fig. 3A) or with the modification of adding 0.5% SDS during sonication (Fig. 3 B and C). When SDS was added, the sonication was performed on ice for ten times, each with a 1-sec pulse. Before immunoprecipitation, SDS was removed by subjecting sonicated samples to two consecutive Bip-Spin 6 columns, and then 0.5% Triton X-100 (final) was added. A Bio-Rad iCycler real-time PCR machine was used for the quantitation experiments. Primer sequences for real-time PCR are as follows: E1, 5'-GTGGGTGCAGAGAGCCTCTGCTG-3' (forward) and 5'-CTCCCTGCTTCTAACTTGCTCTAGAC-3' (reverse); E2, 5'-GGTTCCGCCCCGCCAGTATGC-3' (forward) and 5'-CCGTCACTTGGTGCGCTCGGC-3' (reverse); and

E5, 5'-GATTCCTGCCACATTGAGATTTGGG-3' (forward) and 5'-CAGGAGGCTGAGACTGAAGGTTC-3' (reverse).

1. Yoo, S. H., Yamazaki, S., Lowrey, P. L., Shimomura, K., Ko, C. H., Buhr, E. D., Siepka, S. M., Hong, H. K., Oh, W. J., Yoo, O. J., et al. (2004) Proc. Natl. Acad. Sci. USA 101, 5339–5346

2. Wilsbacher, L. D., Yamazaki, S., Herzog, E. D., Song, E. J., Radcliffe, L. A., Abe, M., Block, G., Spitznagel, E., Menaker, M. & Takahashi, J. S. (2002) Proc. Natl. Acad. Sci. USA 99, 489–494.

3. Antoch, M. P., Song, E. J., Chang, A. M., Vitaterna, M. H., Zhao, Y., Wilsbacher, L.D., Sangoram, A. M., King, D. P., Pinto, L. H. & Takahashi, J. S. (1997) Cell 89, 655–667.

4. Boyes, J. & Felsenfeld, G. (1996) EMBO J. 15, 2496-2507.

5. Ripperger, J. A., Shearman, L. P., Reppert, S. M. & Schibler, U. (2000) Genes Dev. 14, 679–689.

6. Lee, C., Etchegaray, J. P., Cagampang, F. R., Loudon, A. S. & Reppert, S. M. (2001) Cell 107, 855–867.