Generation of a novel allelic series of cryptochrome mutants via mutagenesis reveals residues involved in protein-protein interaction and CRY2-specific repression.
CRYPTOCHOME proteins are necessary for mammalian circadian rhythms and have many well-established biochemical roles within the molecular clock. While studies examining the effect of null Cry alleles have been informative, they have failed to dissect out the relative importance of, and the molecular mechanisms behind, the many roles of the CRY1 and CRY2 proteins. To address this, we created an allelic series of Cry mutants through random mutagenesis, followed by a cell-based screen to isolate mutants with aberrant repression of CLOCK-BMAL1. We identified 22 mutants with mutations resulting in single amino acid substitutions which cause a variety of deficiencies in different CRY functions. To illustrate the breadth and value of these new tools, we present an in-depth analysis of two of these mutants, CRY2G354D and CRY2G351D; the former shows deficiency in clock protein binding and is required for repression by both CRYs, while in contrast, the latter displays normal binding function but exhibits a CRY2-specific repression phenotype. Further, while overexpression of CRY2 in NIH 3T3 cells caused a dose-dependent decrease in rhythm amplitude, overexpression of CRY2G351D abolished rhythmicity. In summary, characterization of these unique alleles provides new opportunities for more-sophisticated insight into the multifaceted functions of the CRY proteins in circadian rhythms.
McCarthy
Ellena V
EV
Department of Neuroscience, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., NB4.204G, Dallas, TX 75390-7433, USA.
Baggs
Julie E
JE
Geskes
Jeanne M
JM
Hogenesch
John B
JB
Green
Carla B
CB
D000483:Alleles; D019943:Amino Acid Substitution; D000818:Animals; D001683:Biological Clocks / Q000502:physiology; D019556:COS Cells; D002522:Chlorocebus aethiops; D002940:Circadian Rhythm; D056931:Cryptochromes; D005420:Flavoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D051379:Mice; D016296:Mutagenesis; D009154:Mutation / Q000235:genetics / Q000502:physiology; D041681:NIH 3T3 Cells; D011487:Protein Conformation
C538963:CRY1 protein, human; C538966:CRY2 protein, human; C538964:Cry1 protein, mouse; C538967:Cry2 protein, mouse; D056931:Cryptochromes; D005420:Flavoproteins
Nat Genet. 2006 Mar;38(3):369-74
16474407
Cell. 1999 Jul 23;98(2):193-205
10428031
Cell. 2007 Jun 1;129(5):1011-23
17462724
Electrophoresis. 1997 Dec;18(15):2714-23
9504803
Cell. 2007 May 4;129(3):605-16
17482552
Science. 2007 May 11;316(5826):897-900
17463252
Nat Genet. 2005 Feb;37(2):187-92
15665827
Curr Biol. 2003 Sep 16;13(18):1653-8
13678599
Mol Cell Biol. 2004 Jul;24(14):6278-87
15226430
Mol Cell Biol. 2006 Mar;26(5):1743-53
16478995
Nat Neurosci. 2004 Aug;7(8):834-40
15258584
Mol Cell Biol. 2001 Oct;21(19):6651-9
11533252
Proc Natl Acad Sci U S A. 1999 Oct 12;96(21):12114-9
10518585
Genes Dev. 2002 Oct 15;16(20):2633-8
12381662
Nature. 1999 Apr 15;398(6728):627-30
10217146
Nat Genet. 2006 Mar;38(3):312-9
16474406
Nature. 1999 Apr 15;398(6728):557-8
10217136
Science. 2000 May 12;288(5468):1013-9
10807566
EMBO J. 2002 Mar 15;21(6):1301-14
11889036
Nat Rev Genet. 2005 Jul;6(7):544-56
15951747
Brain Res Mol Brain Res. 1999 Aug 25;71(2):238-43
10521578
Nature. 2003 Jan 9;421(6919):177-82
12483227
Science. 2004 Jun 4;304(5676):1503-6
15178801
Science. 2007 May 11;316(5826):900-4
17463251
Trends Biochem Sci. 2002 Feb;27(2):107-9
11852249
Biochem J. 2007 Mar 15;402(3):525-36
17115977
Acta Crystallogr D Biol Crystallogr. 2004 Jul;60(Pt 7):1205-13
15213381
J Biol Chem. 2005 Apr 8;280(14):13272-8
15689618
Mol Cell Biol. 2007 Mar;27(6):2120-9
17210647
Nature. 2002 Aug 1;418(6897):534-9
12152080
Science. 1999 Oct 22;286(5440):768-71
10531061