Oscillatory Mechanisms and Functional Differentiation of Circadian Clocks

Abstract

Circadian rhythms govern behavior, physiology and metabolism of living organisms. Recent studies have revealed the role of several genes in the clock mechanism both in Drosophila and mammals. The core molecular mechanism underlying circadian oscillation in Drosophila is comprised of two interlocked feedback transcriptional loops: a period (per)/ timeless (tim) loop as a negative loop, and a Clock (Clk)/cycle (cyc) loop as a positive one. The per/tim loop is initiated by CLK-CYC heterodimer which binds to the E-box sequences (CACGTG) in per and tim promoters. The per and tim gene are activated, and their products form a heterodimer (PER-TIM) in cytoplasm. The heterodimer then moves into the nucleus and inhibits CLK-CYC activity, there by repressing per/tim own transcriptions. The Clk/cyc positive loop is also initiated by CLK-CYC. CLK-CYC activates the expression of vrille (vri) and PAR domain protein 1 (Pdp1) both of which also have the E-box in the promoter region. While VRI acts as a negative regulator of Clk, Pdp1, which accumulates in a delayed fasion compared to VRI, activates the Clk transcription. Although the framework of molecular mechanism of circadian oscillation is extensively elucidated, there are many issues unsolved concerning to the basic features of circadian rhythm. For example, so far we don’t have the definitive explanation why the period of circadian rhythm is about 24 hours. Additionally, the molecular mechanism of temperature compensation of period, which is one of the most remarkable features of circadian rhythm, is still remained as absolute mystery. Also, we still don’t know the exact number of output genes whose expressions are controled by the feedback loops. To study how gene expression is globally regulated by the clock mechanism, we used a high-density oligonucleotide probe array (GeneChip) to profile gene expression patterns in Drosophila under light-dark and constant dark conditions. We found 712 genes showing a daily fluctuation in mRNA levels under light-dark conditions and among them expression of 115 genes was still cycling in constant darkness, i.e. under a free-running condition. Unexpectedly, expression of a large number of genes cycled only under constant darkness. We are now doing behavior screenings for core clock genes by RNAi method. This project is the collaborate work with Hiroki Ueda (RIKEN CDB), Ryu Ueda, Kuniaki Takahashi (NIG), Kaoru Saigo (Univ. Tokyo) and Teiichi Tanimura (Kyushu Univ.).