Nuclear Localization and Transcriptional Repression Are Confined to Separable Domains in the Circadian Protein CRYPTOCHROME

Zhu, Haisun; Conte, Francesca; Green, Carla B.

doi:10.1016/j.cub.2003.08.033

Cited by 36 publications

(50 citation statements)

References 20 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…6E). Introduction of CLOCK on its own slightly increased Per1 promoter-dependent gene expression, whereas the combined expression of wild-type CLOCK and BMAL1 caused a marked increase that was abolished by CRY1, as described previously (22,52). In sharp contrast, when either component of the CLOCK/BMAL1 heterodimer was replaced with a mutant lacking an essential domain for transactivation, the dimeric transcription factor no longer elicited robust induction of reporter gene expression.…”

Section: Vol 26 2006 Bmal1 Shuttling Controls Circadian Gene Expressupporting

confidence: 71%

BMAL1 Shuttling Controls Transactivation and Degradation of the CLOCK/BMAL1 Heterodimer

Kwon

Lee

Chang

et al. 2006

Molecular and Cellular Biology

149

162

View full text Add to dashboard Cite

CLOCK and BMAL1 are bHLH-PAS-containing transcription factors that bind to E-box elements and are indispensable for expression of core circadian clock components such as the Per and Cry genes. A key step in expression is the heterodimerization of CLOCK and BMAL1 and their accumulation in the nucleus with an approximately 24-h periodicity. We show here that nucleocytoplasmic shuttling of BMAL1 is essential for transactivation and for degradation of the CLOCK/BMAL1 heterodimer. Using serial deletions and point mutants, we identified a functional nuclear localization signal and Crm1-dependent nuclear export signals in BMAL1. Transient-transfection experiments revealed that heterodimerization of CLOCK and BMAL1 accelerates their turnover, as well as E-box-dependent clock gene transcription. Moreover, in embryonic mouse fibroblasts, robust transcription of Per2 is tightly associated with massive degradation of the CLOCK/BMAL1 heterodimer. CRY proteins suppressed this process during the transcription-negative phase and led to nuclear accumulation of the CLOCK/BMAL1 heterodimer. Thus, these findings suggest that the decrease of BMAL1 abundance during the circadian cycle reflects robust transcriptional activation of clock genes rather than inhibition of BMAL1 synthesis.Almost all organisms from bacteria to mammals have developed circadian physiology and behavior to adapt to the environmental changes created by the rotation of our planet. Such daily rhythms are controlled by genetically determined and self-sustaining circadian clocks that are composed of networks of transcription-translation feedback loops involving sets of clock genes (15,30,37). In mammals, a network of feedback loops functions robustly not only in the master circadian pacemaker, the suprachiasmatic nucleus (SCN) of the hypothalamus (26), but also in most tissues (including liver, heart, lung, and muscle) and even in immortalized cell lines (1, 50, 51). These widely dispersed circadian systems are primarily synchronized by the SCN to coordinate circadian timing in vivo (29,36).One of the main questions in clock biology is how the timekeeping system is controlled at the molecular level. Intensive studies in the mouse using genetic and molecular approaches have largely clarified the structure of the central feedback loop (3,30). Two bHLH-PAS-containing transcription factors, CLOCK and BMAL1, form heterodimers that bind to E-box enhancer elements in the promoters of target genes driving the transcription of three period genes (Per1, Per2, and Per3) and two cryptochrome genes (Cry1 and Cry2) (11,14,17,18). After the PER and CRY proteins have been translated in the cytoplasm, they form heterocomplexes that translocate into the nucleus and inhibit their own transcription. CRY plays a crucial role in this negative feedback process by interacting directly with the CLOCK/BMAL1 heterodimers (12,22).Analysis of Bmal1 defective mice has revealed the indispensable role of BMAL1 as the mainspring of the molecular clockwork; thus, targeted disruption of Bmal1 results...

show abstract

Section: Vol 26 2006 Bmal1 Shuttling Controls Circadian Gene Expressupporting

confidence: 71%

BMAL1 Shuttling Controls Transactivation and Degradation of the CLOCK/BMAL1 Heterodimer

Kwon

Lee

Chang

et al. 2006

Molecular and Cellular Biology

149

162

View full text Add to dashboard Cite

show abstract

“…As a result of our random mutagenesis screen, we generated many novel mutant Cry alleles. The residue changes corresponding with defects in transcriptional repression were confined to the PHR, and no changes were observed in the Cterminal tail, which has been shown to be important for nuclear localization in mammals (4,20) and Xenopus (31). This is in line with data from Chaves et al (4), who reported that interaction of mCRY1 with BMAL1 via the coiled-coil domain in its PHR could facilitate CRY's nuclear localization, even in the event that its NLSs are mutated.…”

Section: Random Mutagenesis and Cell-based Functional Screen Ofmentioning

confidence: 98%

“…The main body of the protein, called the photolyase homology region (PHR), has been shown to be sufficient to repress CLOCK-BMAL1 in Xenopus laevis. (31) and is well conserved between CRY1 and CRY2. While it has been shown that CRY binding with CLOCK-BMAL1 is required for repression of their transcriptional activity (21), the mechanism by which the repression occurs is not known.…”

mentioning

confidence: 99%

Generation of a Novel Allelic Series of Cryptochrome Mutants via Mutagenesis Reveals Residues Involved in Protein-Protein Interaction and CRY2-Specific Repression

McCarthy

Baggs

Geskes

et al. 2009

Molecular and Cellular Biology

Self Cite

View full text Add to dashboard Cite

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.Organisms from cyanobacteria to humans have evolved circadian rhythms to anticipate the dramatic changes in environmental conditions that occur on a daily basis. The driving force behind the 24-h periodicity of these endogenous rhythms is a cell-autonomous molecular oscillator, composed of transcriptional/translational feedback loops (reviewed in reference 1).In mammals, the Cryptochrome (CRY) proteins CRY1 and CRY2 play an integral role in the circadian clock by closing the core negative-feedback loop (9,12,28,29). In 1999, two groups showed that mice lacking both Crys demonstrate a complete loss of rhythm in circadian wheel-running behavior when put in constant darkness (26,28), suggesting an essential role for murine CRY proteins (mCRYs) in the core molecular mechanism. This year, it was shown that both CRY1 and CRY2 repressed CLOCK-BMAL1-mediated transcription much more potently than the mammalian PERIOD (PER) proteins in transiently transfected cells (9, 12). While it is known that CRYs are essential for circadian rhythms at both the behavioral (28, 29) and molecular (21) levels, many questions concerning how the various known actions of the CRY proteins contribute to their essential roles in the molecular mechanism of the circadian clock remain unanswered.Mammalian CRY1 and CRY2 have several well-established biochemical roles in the molecular oscillator in addition to their role in repressing the CLOCK-BMAL1 heterodimer. Both CRY1 and CRY2 bind the PER proteins (9, 12), and this interaction affects PER's stability (30) and localization (12). While the CRY interaction domain on PER has been described, the regions important for PER binding on C...

show abstract

“…5, right lower alignment). The C-tail of amCRY does not contain a second putative NLS found on the C-tails of mCRY2 and xCRY2b (Zhu et al 2003;Sakakida et al 2005;Chaves et al 2006). These analyses show that amCRY does not contain sequences thought to be necessary for dCRY photoreceptor function.…”

Section: Genome Research 1355mentioning

confidence: 99%

Molecular and phylogenetic analyses reveal mammalian-like clockwork in the honey bee (Apis mellifera) and shed new light on the molecular evolution of the circadian clock

et al. 2006

View full text Add to dashboard Cite

The circadian clock of the honey bee is implicated in ecologically relevant complex behaviors. These include time sensing, time-compensated sun-compass navigation, and social behaviors such as coordination of activity, dance language communication, and division of labor. The molecular underpinnings of the bee circadian clock are largely unknown. We show that clock gene structure and expression pattern in the honey bee are more similar to the mouse than to Drosophila. The honey bee genome does not encode an ortholog of Drosophila Timeless (Tim1), has only the mammalian type Cryptochrome (Cry-m), and has a single ortholog for each of the other canonical "clock genes." In foragers that typically have strong circadian rhythms, brain mRNA levels of amCry, but not amTim as in Drosophila, consistently oscillate with strong amplitude and a phase similar to amPeriod (amPer) under both light-dark and constant darkness illumination regimes. In contrast to Drosophila, the honey bee amCYC protein contains a transactivation domain and its brain transcript levels oscillate at virtually an anti-phase to amPer, as it does in the mouse. Phylogenetic analyses indicate that the basal insect lineage had both the mammalian and Drosophila types of Cry and Tim. Our results suggest that during evolution, Drosophila diverged from the ancestral insect clock and specialized in using a set of clock gene orthologs that was lost by both mammals and bees, which in turn converged and specialized in the other set. These findings illustrate a previously unappreciated diversity of insect clockwork and raise critical questions concerning the evolution and functional significance of species-specific variation in molecular clockwork.

show abstract

Nuclear Localization and Transcriptional Repression Are Confined to Separable Domains in the Circadian Protein CRYPTOCHROME

Cited by 36 publications

References 20 publications

BMAL1 Shuttling Controls Transactivation and Degradation of the CLOCK/BMAL1 Heterodimer

BMAL1 Shuttling Controls Transactivation and Degradation of the CLOCK/BMAL1 Heterodimer

Generation of a Novel Allelic Series of Cryptochrome Mutants via Mutagenesis Reveals Residues Involved in Protein-Protein Interaction and CRY2-Specific Repression

Molecular and phylogenetic analyses reveal mammalian-like clockwork in the honey bee (Apis mellifera) and shed new light on the molecular evolution of the circadian clock

Contact Info

Product

Resources

About