Rhythmic Expression of BMAL1 mRNA Is Altered in Clock Mutant Mice: Differential Regulation in the Suprachiasmatic Nucleus and Peripheral Tissues Katsutaka Oishi,* , † Hiromi Fukui,* and Norio Ishida* , ‡ ,1 *Ishida Group of Clock Gene, National Institute of Bioscience and Human Technology, Agency of Industrial Science and Technology, MITI, Higashi 1-1, Tsukuba, Ibaraki 305-8566, Japan; †Institute of Applied Biochemistry, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan; and ‡Department of Biomolecular Engineering, Tokyo Institute of Technology, 4259 Nagatsuda, Yokohama 226-8501, Japan Received December 3, 1999 BMAL1 is a putative clock gene which encodes a basic helix-loop-helix (bHLH)-PAS transcription fac- tor. To examine whether the CLOCK protein is re- quired for the circadian expression of BMAL1 mRNA, in situ hybridization and Northern blot analysis were performed in the suprachiasmatic nucleus (SCN) and peripheral tissues of homozygous Clock mutant mice. In the SCN of Clock mutants, BMAL1 mRNA did not oscillate significantly but apparently expressed with low levels, while in wild-type mice the mRNA was ro- bustly oscillated in a circadian manner. The peak– trough amplitudes of BMAL1 mRNA levels were 6.5-, 8.6-, and 6.7-fold in liver, heart, and kidney of wild- type mice, respectively. In Clock mutants, the ampli- tudes were extremely damped to 1.2-, 2.1-, and 1.4-fold, respectively. Furthermore, expressions of BMAL1 mRNA in the peripheral of Clock mutant mice were close to the peak level in wild-type mice, whereas mPer2 mRNA levels were severely blunted at trough values. Daily expression of albumin site D-binding protein (DBP), a clock controlled output gene (CCG), was also abolished at trough values by the Clock mu- tation in all tissues examined. These observations sug- gest that the circadian expression of BMAL1 mRNA is affected by the CLOCK-induced transcriptional feed- back loop in the SCN and peripheral tissues in a dif- ferent way and that the regulation mechanism ap- peared to be different from those in mPer2 and DBP expressions in vivo. © 2000 Academic Press Many organisms display rhythms of physiology and behavior that are entrained to the 24-h cycle of light and darkness prevailing on Earth. Some of these inter- nal biological rhythms are driven by endogenous oscil- lators called circadian clocks. Recently, the circadian oscillators are thought to be controlled by autoregula- tory feedback loops in clock genes’ expression in mam- mals as well as in Drosophila (1). In vitro studies imply the mechanisms about the transcriptional feedback loop at the level of protein-protein interactions (2– 6), transcriptional activation/inhibition (2–5, 7), and translocation to the nuclear (2, 5). However, little is known about in vivo mechanisms of the circadian os- cillators. Clock is the first clock gene identified in vertebrate by N-ethyl-N-nitrosourea mutagenesis screening (8). When transferred from a light– dark cycle (LD) to a constant darkness (DD), homozygous Clock mutants exhibit abnormally long periodicity of behavior for ini- tial 5 to 15 cycles, and consequently show a complete loss of circadian rhythmicity (8). The Clock gene en- codes a basic helix-loop-helix (bHLH)-PAS transcrip- tion factor (9). Clock allele possesses a truncation and deletion of 51 amino acids in its transcriptional activa- tion domain (9). BMAL1 is a putative clock gene which also encodes a bHLH-PAS transcription factor (1). BMAL1 has been identified as a potential dimerization partner for mam- malian CLOCK (4, 10). In Drosophila, cycle (cyc) is a homologue of mammalian BMAL1, and its homozygous mutants are completely arrhythmic (11). Homozygous dClock mutants (Jrk) are also arrhythmic, and express low levels of period ( per) and timeless (tim) transcripts (12), which are essential elements of the circadian tim- ing system in Drosophila. dCLOCK and dBMAL1 (CYC) form a heterodimer that drives per and tim transcriptions via the E-box (CACGTG motif) in their 1 To whom correspondence should be addressed at Ishida Group of Clock Gene, National Institute of Bioscience and Human Technol- ogy, Agency of Industrial Science and Technology, MITI, Higashi 1-1, Tsukuba, Ibaraki 305-8566, Japan. Fax: +81-298-54-6095. E-mail: nishida@nibh.go.jp. Biochemical and Biophysical Research Communications 268, 164 –171 (2000) doi:10.1006/bbrc.1999.2054, available online at http://www.idealibrary.com on 164 0006-291X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.