Three labs find a novel gene involved in mouse circadian rhythms
By Melissa Lee Phillips | April 26, 2007
Researchers from three different labs have identified a new circadian gene in the mouse, according to two papers in Science and one paper in Cell published online this week. Mutagenesis screens revealed that mutations in a protein called FBXL3 lengthen the mouse circadian period by several hours, and biochemical analyses showed that FBXL3 is necessary for degradation of key circadian clock proteins.
"I think this is a tremendously exciting discovery," said Steve Kay of the Scripps Research Institute in La Jolla, Calif., who was not involved in any of the work. "It shows that forward genetics is still a very powerful tool in identifying new clock genes."
Mammalian circadian rhythms are generated by feedback loops of gene transcription and translation. The transcription factors CLOCK and BMAL1 drive expression of clock genes Period (Per) and Cryptochrome (Cry), whose proteins interact with CLOCK and BMAL1 to inhibit their own transcription. When PER and CRY proteins degrade, this step releases the inhibition, and transcription begins again.
Delays are built into the circuit so that the entire cycle takes approximately 24 hours. One important delay consists of post-translational modifications of the PER and CRY proteins, which determine how long it takes for these proteins to degrade.
In the firstScience paper, researchers led by Sofia Godinho of the Medical Research Council (MRC) in Harwell, UK, and Elizabeth Maywood of MRC in Cambridge, UK, mutagenized mice to search for new circadian clock genes. They associated one allele with a long circadian period -- about 27 hours in mice homozygous for the mutant allele, which they named "after hours" (Afh). The mutation lies in an F-box gene called Fbxl3.
"They find a phenotype that's pretty remarkable in those mutant animals," said David Weaver of the University of Massachusetts Medical School in Worcester, who was not involved in any of the work. "That suggests that [FBXL3] is a pretty important regulator."
In an accompanying paper in Science, researchers led by Luca Busino of New York University School of Medicine show that FBXL3 protein binds to CRY proteins and drives their degradation. Without functional FBXL3, CRY degradation slows, which prolongs reactivation of CLOCK:BMAL1. The result is a lengthened circadian cycle in mice.
"Our findings provide direct in vivo evidence that proteasomal degradation, via F-box proteins, is an important regulatory mechanism in maintaining mammalian circadian homeostasis," Patrick Nolan of MRC Harwell, senior author of the first paper, told The Scientist in an Email.
"The two stories very nicely complement each other," said Michele Pagano of NYU, senior author on the second paper. "They have in vivo evidence and we have the explanation for their phenotype."
In a paper in Cell, researchers led by Sandra Siepka and Seung-Hee Yoo of Northwestern University in Evanston, Ill., also report a lengthened circadian period in an FBXL3 mutant, which they name Overtime (Ovtm). This mouse has a mutation in a different amino acid from the mouse in the Science paper. The researchers also show that loss of FBXL3 function helps stabilize the CRY proteins, subsequently repressing the transcription of several clock genes.
"Because the CRY proteins are not degraded, they repress transcription driven by CLOCK and BMAL," explained senior author Joseph Takahashi, also of Northwestern.
Future studies will likely reveal more genes that influence the circadian clock by controlling degradation of clock proteins, Takahashi told The Scientist. "This example with CRY suggests that there are going to be additional examples of specific regulation of protein degradation," he said.
Some genes that regulate clock protein degradation may not be specific to the circadian clock but instead may "be more general factors that regulate protein stability," said Weaver. "There are probably a lot of mechanisms for modifying these proteins and their stability."
Melissa Lee Phillips
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