The identification and analysis of clock genes in the fruit fly, Drosophila melanogaster, revealed that the circadian timekeeping mechanism is based on autoregulatory feedback loops in gene expression. Similar feedback loops serve to keep circadian time in essentially all eukaryotes, and the genes that drive feedback loop function are well conserved from insects to mammals. These feedback loops operate in many tissues, where they drive rhythmic transcription of output genes that impart tissue specific rhythms in physiology and metabolism. Despite remarkable progress in defining the molecular mechanisms that govern feedback loop progression, we do not understand how these feedback loops are activated in different cell types. Feedback loop function is marked by rhythmic period (per) and timeless (tim) expression in Drosophila. However, per and tim are activated by CLOCK-CYCLE (CLK-CYC) heterodimers, thus Clk and/or cyc expression represent the initial steps in feedback loop activation. Given that the mammalian orthologs of Clk and cyc (e.g. Clock and Bmal1, respectively) are highly conserved, what we learn about processes that initiate feedback loop function in Drosophila may apply to mammals. Clk is unique among Drosophila clock genes in that it can generate ectopic clocks when expressed in non-clock cells, though cyc is required for Clk to activate clock function in both ectopic and normal locations. We recently showed that CYC protein is present only in clock cells, which implies that Clk promotes cyc mRNA and/or protein expression in non-clock cells. However, cyc mRNA is not enriched in clock cells and is present at similar levels in Clkout null mutants, indicating that Clk controls the synthesis or accumulation of CYC protein. This possibility is consistent with data in mammals showing that Clock null mutants express Bmal1 mRNA at high levels, but accumulate low levels of BMAL1 protein. Given that CLK binds directly to CYC, we hypothesize that Clk promotes CYC expression by stabilizing CYC protein.
Two aims are proposed to test this hypothesis.
In Specific Aim 1, we will determine if CYC is broadly expressed and rapidly degraded by defining the pattern of cyc transcription and identifying the proteolytic pathway that degrades CYC in cultured S2 cells and Drosophila brains.
In Specific Aim 2, we will determine if CLK binding stabilizes CYC by measuring CYC half-life in the presence or absence of CLK in S2 cells and by monitoring CYC accumulation upon conditional expression of CLK in both clock and non-clock brain neurons. Successful completion of these aims will define the first molecular events required to initiate clock function within and outside the normal clock cell pattern. In addition, these studies will reveal cyc-expressing cell types that have the potential to generate clocks, enable experiments to determine if non-clock cells that express cyc retain a program for activating tissue-specific CLK-CYC output genes, and will encourage the development of Drosophila cell lines that initiate circadian clock function upon conditional Clk expression.
Essentially all organisms use circadian clocks to control daily rhythms in physiology, metabolism and behavior. Since animals use conserved activator proteins to initiate clock function in many brain neurons and peripheral tissues, our goal is to understand how these proteins initiate clock function in only certain cell types using a simple model organism - the fruit fly Drosophila melanogaster. Circadian clocks are of great clinical importance because their dysfunction can lead to sleep problems, metabolic disorders, and even cancer. Understanding how clock function is initiated will provide the molecular foundation necessary for developing novel treatments for clock disorders.
| Liu, Tianxin; Mahesh, Guruswamy; Yu, Wangjie et al. (2017) CLOCK stabilizes CYCLE to initiate clock function in Drosophila. Proc Natl Acad Sci U S A 114:10972-10977 |
