Cycling of CRYPTOCHROME proteins is not necessary for circadian-clock function in mammalian fibroblasts.
BACKGROUND
An interlocked transcriptional-translational feedback loop (TTFL) is thought to generate the mammalian circadian clockwork in both the central pacemaker residing in the hypothalamic suprachiasmatic nuclei and in peripheral tissues. The core circadian genes, including Period1 and Period2 (Per1 and Per2), Cryptochrome1 and Cryptochrome2 (Cry1 and Cry2), Bmal1, and Clock are indispensable components of this biological clockwork. The cycling of the PER and CRY clock proteins has been thought to be necessary to keep the mammalian clock ticking.


RESULTS
We provide a novel cell-permeant protein approach for manipulating cryptochrome protein levels to evaluate the current transcription and translation feedback model of the circadian clockwork. Cell-permeant cryptochrome proteins appear to be functional on the basis of several criteria, including the abilities to (1) rescue circadian properties in Cry1(-/-)Cry2(-/-) mouse fibroblasts, (2) act as transcriptional repressors, and (3) phase shift the circadian oscillator in Rat-1 fibroblasts. By using cell-permeant cryptochrome proteins, we demonstrate that cycling of CRY1, CRY2, and BMAL1 is not necessary for circadian-clock function in fibroblasts.


CONCLUSIONS
These results are not supportive of the current version of the transcription and translation feedback-loop model of the mammalian clock mechanism, in which cycling of the essential clock proteins CRY1 and CRY2 is thought to be necessary.
Fan
Yunzhen
Y
Department of Biological Sciences, Vanderbilt University, Nashville, TN 37235-1634, USA.
Hida
Akiko
A
Anderson
Daniel A
DA
Izumo
Mariko
M
Johnson
Carl Hirschie
CH
D056930:ARNTL Transcription Factors; D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins / Q000378:metabolism; D001683:Biological Clocks / Q000502:physiology*; D056926:CLOCK Proteins; D002460:Cell Line; D002463:Cell Membrane Permeability; D002940:Circadian Rhythm / Q000502:physiology*; D056931:Cryptochromes; D005347:Fibroblasts / Q000378:metabolism*; D005420:Flavoproteins / Q000378:metabolism*; D005786:Gene Expression Regulation; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D051381:Rats; D015534:Trans-Activators / Q000378:metabolism; D014158:Transcription, Genetic
D056930:ARNTL Transcription Factors; D051792:Basic Helix-Loop-Helix Proteins; D056926:CLOCK Proteins; D056931:Cryptochromes; D005420:Flavoproteins; D015534:Trans-Activators; C538960:BMAL1 protein, human; C538961:Bmal1 protein, mouse; C538963:CRY1 protein, human; C538966:CRY2 protein, human; C538964:Cry1 protein, mouse; C538965:Cry1 protein, rat; C538967:Cry2 protein, mouse; C538968:Cry2 protein, rat; C538943:CLOCK protein, human; C538944:Clock protein, mouse; C538945:Clock protein, rat