Alternative titles; symbols
HGNC Approved Gene Symbol: TIMELESS
Cytogenetic location: 12q13.3 Genomic coordinates (GRCh38) : 12:56,416,363-56,449,426 (from NCBI)
| Location | Phenotype |
Phenotype MIM number |
Inheritance |
Phenotype mapping key |
|---|---|---|---|---|
| 12q13.3 | ?Advance sleep phase syndrome, familial, 4 | 620015 | Autosomal dominant | 3 |
Cellular pacemakers located in the suprachiasmatic nucleus (SCN) of the anterior hypothalamus control circadian rhythms. In Drosophila, a central clock mechanism involves the dynamic regulation of 2 genes, 'period' (per; see 602260) and 'timeless' (tim), which physically interact and participate in an intracellular transcriptional/translational feedback loop. The transcription of per and tim is positively regulated by the Clock (601851) and BMAL1 (602550) proteins, which form heterodimers. By searching EST databases, Sangoram et al. (1998), Zylka et al. (1998), and Koike et al. (1998) identified cDNAs corresponding to human (TIM) and mouse (Tim) homologs of Drosophila timeless. Sangoram et al. (1998) reported that the predicted 1,208-amino acid human protein is 84% identical to mouse Tim. The mammalian proteins share 4 regions of homology with Drosophila tim, including regions involved in nuclear localization, protein-protein interaction with PER, and cytoplasmic localization. Northern blot analysis revealed that TIM was expressed as a 4.5-kb mRNA in all human tissues tested, with the highest levels in placenta, pancreas, thymus, and testis. In situ hybridization indicated that unlike those of Drosophila, mouse Tim transcript levels do not oscillate in the SCN or in the retina.
Sangoram et al. (1998) demonstrated that human TIM interacts with Drosophila per, mouse Per1, and mouse Per2 (see 603426) in vitro. When expressed in Drosophila cells, TIM mimicked a Drosophila tim cellular function by interacting with Drosophila per and translocating into the nucleus. In addition, when expressed in mammalian cells, human TIM and mouse PER1 specifically inhibited CLOCK-BMAL1-induced transactivation of the mouse PER1 promoter. These authors concluded that TIM and Tim are the mammalian orthologs of Drosophila tim. In contrast, Zylka et al. (1998) were unable to detect mouse Per-Tim interactions in yeast 2-hybrid assays. They found an array of interactions between the various mouse Per proteins, and suggested that Per-Per interactions have replaced the function of Per-Tim dimers in the molecular workings of the mammalian circadian clock.
Chan et al. (2003) found that C. elegans TIM1 participates in the regulation of chromosome cohesion. Their biochemical experiments defined the C. elegans cohesin complex and revealed its physical association with TIM1. Functional relevance of the interaction was demonstrated by aberrant mitotic chromosome behavior, embryonic lethality, and defective meiotic chromosome cohesion caused by the disruption of either TIM1 or cohesin. TIM1 depletion prevented the assembly of non-SMC (structural maintenance of chromosome) cohesin subunits onto meiotic chromosomes; however, unexpectedly, a partial cohesin complex composed of SMC components still loaded. Further disruption of cohesin activity in meiosis by the simultaneous depletion of TIM1 and an SMC subunit decreased homologous chromosome pairing before synapsis, revealing a novel role for cohesin in metazoans. On the basis of comparisons between TIMELESS homologs in worms, flies, and mice, Chan et al. (2003) proposed that chromosome cohesion, rather than circadian clock regulation, is the ancient and conserved function for TIMELESS-like proteins.
Barnes et al. (2003) demonstrated that conditional knockdown of TIM protein expression in the rat SCN disrupted SCN neuronal activity rhythms and altered levels of core clock elements. Full-length rat TIM protein exhibited a 24-hour oscillation, whereas a truncated isoform was constitutively expressed. Full-length rat TIM protein was associated with the mammalian clock Period proteins (PER1, PER2, and PER3 (603427)). The data suggested that murine Timeless is required for rhythmicity and is a functional homolog of Drosophila timeless on the negative feedback arm of the mammalian molecular clockwork.
By yeast 2-hybrid analysis, Gotter (2003) identified mouse Tipin (610716) as a Tim-interacting protein. Coimmunoprecipitation analysis showed that Tipin interacted with Tim in vitro and in cultured cells. In transiently transfected cells, Tim promoted nuclear localization of Tipin. Immunoprecipitation experiments showed that Tipin disrupted the ability of Tim to form homomultimeric complexes, suggesting a mechanism through which Tipin may modulate Tim function.
Chou and Elledge (2006) showed that TIPIN and TIM formed a complex that maintained the level of both proteins in human cells, and that loss of either one led to loss of the other.
Busza et al. (2004) showed that Drosophila CRY (601933) binding to TIM is light-dependent in flies and irreversibly commits TIM to proteasomal degradation. In contrast, CRY degradation is dependent on continuous light exposure, indicating that the CRY-TIM interaction is transient. A novel CRY mutation reveals that CRY's photolyase homology domain is sufficient for light detection and phototransduction, whereas the carboxyl-terminal domain regulates CRY stability, CRY-TIM interaction, and circadian photosensitivity.
By fluorescence resonance energy transfer measurements using a single-cell imaging assay with fluorescent forms of PER (602260) and TIM, Meyer et al. (2006) showed that these proteins bind rapidly and persist in the cytoplasm while gradually accumulating in discrete foci. After approximately 6 hours, complexes abruptly dissociated, as PER and TIM independently moved to the nucleus in a narrow time frame. The per(l) mutation, which produces a delayed nuclear translocation phenotype in pacemaker cells of the Drosophila brain, delayed nuclear accumulation in vivo and in a cultured cell system, but without affecting rates of PER/TIM assembly or dissociation. Meyer et al. (2006) concluded that their finding points to a previously unrecognized form of temporal regulation that underlies the periodicity of the circadian clock.
Koh et al. (2006) identified mutations in Drosophila jetlag (FBXL15; see 610287), a gene coding for an F-box protein with leucine-rich repeats, that resulted in reduced light sensitivity of the circadian clock. Mutant flies showed rhythmic behavior in constant light, reduced phase shifts in response to light pulses, and reduced light-dependent degradation of TIM. Expression of Jet along with CRY in cultured cells conferred light-dependent degradation onto TIM, thereby reconstituting the acute response of the circadian clock to light in a cell culture system. Koh et al. (2006) concluded that their results suggest that JET is essential for resetting the clock by transmitting light signals from CRY to TIM.
Tauber et al. (2007) reported that a mutation, ls-tim, in the circadian clock gene timeless in D. melanogaster arose and spread by natural selection relatively recently in Europe, approximately 8,000 to 10,000 years ago, coinciding with the postglacial period and subsequent colonization of the Eurasian continent by D. melanogaster. The authors found that, when introduced into different genetic backgrounds, natural and artificial alleles of the timeless gene affected the incidence of diapause in response to changes in light and temperature. The natural mutant allele alters an important life history trait that may enhance the fly's adaptation to seasonal conditions.
Diapause is a protective response to unfavorable environments that results in a suspension of insect development and is most often associated with the onset of winter. The ls-tim mutation in the D. melanogaster clock gene timeless has spread in Europe over the past 10,000 years, possibly because it enhances diapause. Sandrelli et al. (2007) showed that the mutant allele attenuates the photosensitivity of the circadian clock and causes decreased dimerization of the mutant timeless protein isoform to cryptochrome (601933), the circadian photoreceptor. This interaction results in a more stable timeless product. Sandrelli et al. (2007) concluded that their findings revealed a molecular link between diapause and circadian photoreception.
Using HeLa and U2OS human cell lines, Yoshizawa-Sugata and Masai (2007) found that TIM and TIPIN stabilized each other. Knockdown of either protein reduced total protein content and caused cytoplasmic redistribution of the other protein. TIPIN was required for intra-S-phase checkpoint. Both TIM and TIPIN facilitated nuclear accumulation of claspin (CLSPN; 605434) and activation of CHK1 (CHEK1; 603078) following replication stress.
Somyajit et al. (2017) found that perturbation of ribonucleotide reductase (see RRM1, 180410) in humans elevates reactive oxygen species (ROS) that are detected by peroxiredoxin-2 (PRDX2; 600538). In the oligomeric state, PRDX2 forms a replisome-associated ROS sensor, which binds the fork accelerator TIMELESS when exposed to low levels of ROS. Elevated ROS levels generated by RNR attenuation disrupt oligomerized PRDX2 to smaller subunits, whose dissociation from chromatin enforces the displacement of TIMELESS from the replisome. This process instantly slows replication fork progression, which mitigates pathologic consequences of replication stress. Thus, Somyajit et al. (2017) concluded that redox signaling couples fluctuations of deoxynucleotide triphosphate (dNTP) biogenesis with replisome activity to reduce stress during genome duplication. The authors proposed that cancer cells exploit this pathway to increase their adaptability to adverse metabolic conditions.
By analysis of an interspecific backcross, Sangoram et al. (1998) mapped the mouse Tim gene to chromosome 10. Using radiation hybrid analysis, these authors localized the human TIM gene to human chromosome 12, in a region sharing homology of synteny with mouse chromosome 10. Koike et al. (1998) refined the positions of the genes to human chromosome 12q12-q13 and mouse chromosome 10D3 by fluorescence in situ hybridization.
By screening 25 circadian clock-relevant candidate genes and performing whole-exome sequencing in a man and his mother with familial advanced sleep phase syndrome (FASPS4; 620015), Kurien et al. (2019) identified a heterozygous nonsense mutation in the TIMELESS gene (R1081X; 603887.0001). The mutation resulted in cytoplasmic accumulation of the protein. The mutant protein had altered affinity for CRY2 (603732), leading to destabilization of the PER2 (603426)-CRY2 heterodimer. The mutation segregated with the phenotype in the family and was not found in public variant databases.
Kurien et al. (2019) used CRISPR to generate mice with a heterozygous R1078X mutation in the Tim gene, corresponding to the human R1081X mutation (603887.0001). The mutant mice exhibited advanced sleep phase with altered sensitivity to light pulses but normal circadian period length.
By screening 25 circadian clock-relevant candidate genes and performing whole-exome sequencing in a male proband and his mother (kindred K5602) with familial advanced sleep phase syndrome-4 (FASP4; 620015), Kurien et al. (2019) identified a heterozygous C-T transition in exon 27 of the TIMELESS gene, resulting in an arg1081-to-ter (R1081X) substitution and the loss of 128 amino acids at the C-terminal end of the conserved C domain, which contains a nuclear localization signal (NLS4). The mutation resulted in cytoplasmic accumulation of the protein. The mutant protein had altered affinity for CRY2 (603732), leading to destabilization of the PER2 (603426)-CRY2 heterodimer. The mutation segregated with the phenotype in the family and was not found in public variant databases.
Barnes, J. W., Tischkau, S. A., Barnes, J. A., Mitchell, J. W., Burgoon, P. W., Hickok, J. R., Gillette, M. U. Requirement of mammalian timeless for circadian rhythmicity. Science 302: 439-442, 2003. Note: Erratum: Science 302: 1153 only, 2003. [PubMed: 14564007] [Full Text: https://doi.org/10.1126/science.1086593]
Busza, A., Emery-Le, M., Rosbash, M., Emery, P. Roles of the two Drosophila CRYPTOCHROME structural domains in circadian photoreception. Science 304: 1503-1506, 2004. [PubMed: 15178801] [Full Text: https://doi.org/10.1126/science.1096973]
Chan, R. C., Chan, A., Jeon, M., Wu, T. F., Pasqualone, D., Rougvie, A. E., Meyer, B. J. Chromosome cohesion is regulated by a clock gene paralogue TIM-1. Nature 423: 1002-1009, 2003. [PubMed: 12827206] [Full Text: https://doi.org/10.1038/nature01697]
Chou, D. M., Elledge, S. J. Tipin and Timeless form a mutually protective complex required for genotoxic stress resistance and checkpoint function. Proc. Nat. Acad. Sci. 103: 18143-18147, 2006. [PubMed: 17116885] [Full Text: https://doi.org/10.1073/pnas.0609251103]
Gotter, A. L. Tipin, a novel Timeless-interacting protein, is developmentally co-expressed with Timeless and disrupts its self-association. J. Molec. Biol. 331: 167-176, 2003. [PubMed: 12875843] [Full Text: https://doi.org/10.1016/s0022-2836(03)00633-8]
Koh, K., Zheng, X., Sehgal, A. JETLAG resets the Drosophila circadian clock by promoting light-induced degradation of TIMELESS. Science 312: 1809-1812, 2006. [PubMed: 16794082] [Full Text: https://doi.org/10.1126/science.1124951]
Koike, N., Hida, A., Numano, R., Hirose, M., Sakaki, Y., Tei, H. Identification of the mammalian homologues of the Drosophila timeless gene, timeless1. FEBS Lett. 441: 427-431, 1998. [PubMed: 9891984] [Full Text: https://doi.org/10.1016/s0014-5793(98)01597-x]
Kurien, P., Hsu, P.-K., Leon, J., Wu, D., McMahon, T., Shi, G., Xu, Y., Lipzen, A., Pennacchio, L. A., Jones, C. R., Fu, Y.-H., Ptacek, L. J. TIMELESS mutation alters phase responsiveness and causes advanced sleep phase. Proc. Nat. Acad. Sci. 116: 12045-12053, 2019. [PubMed: 31138685] [Full Text: https://doi.org/10.1073/pnas.1819110116]
Meyer, P., Saez, L., Young, M. W. PER-TIM interactions in living Drosophila cells: an interval timer for the circadian clock. Science 311: 226-229, 2006. [PubMed: 16410523] [Full Text: https://doi.org/10.1126/science.1118126]
Sandrelli, F., Tauber, E., Pegoraro, M., Mazzotta, G., Cisotto, P., Landskron, J., Stanewsky, R., Piccin, A., Rosato, E., Zordan, M., Costa, R., Kyriacou, C. P. A molecular basis for natural selection at the timeless locus in Drosophila melanogaster. Science 316: 1898-1900, 2007. [PubMed: 17600216] [Full Text: https://doi.org/10.1126/science.1138426]
Sangoram, A. M., Saez, L., Antoch, M. P., Gekakis, N., Staknis, D., Whiteley, A., Fruechte, E. M., Vitaterna, M. H., Shimomura, K., King, D. P., Young, M. W., Weitz, C. J., Takahashi, J. S. Mammalian circadian autoregulatory loop: a timeless ortholog and mPer1 interact and negatively regulate CLOCK-BMAL1-induced transcription. Neuron 21: 1101-1113, 1998. [PubMed: 9856465] [Full Text: https://doi.org/10.1016/s0896-6273(00)80627-3]
Somyajit, K., Gupta, R., Sedlackova, H., Neelsen, K. J., Ochs, F., Rask, M.-B., Choudhary, C., Lukas, J. Redox-sensitive alteration of replisome architecture safeguards genome integrity. Science 358: 797-802, 2017. [PubMed: 29123070] [Full Text: https://doi.org/10.1126/science.aao3172]
Tauber, E., Zordan, M., Sandrelli, F., Pegoraro, M., Osterwalder, N., Breda, C., Daga, A., Selmin, A., Monger, K., Benna, C., Rosato, E., Kyriacou, C. P., Costa, R. Natural selection favors a newly derived timeless allele in Drosophila melanogaster. Science 316: 1895-1898, 2007. [PubMed: 17600215] [Full Text: https://doi.org/10.1126/science.1138412]
Yoshizawa-Sugata, N., Masai, H. Human Tim/Timeless-interacting protein, Tipin, is required for efficient progression of S phase and DNA replication checkpoint. J. Biol. Chem. 282: 2729-2740, 2007. [PubMed: 17102137] [Full Text: https://doi.org/10.1074/jbc.M605596200]
Zylka, M. J., Shearman, L. P., Levine, J. D., Jin, X., Weaver, D. R., Reppert, S. M. Molecular analysis of mammalian timeless. Neuron 21: 1115-1122, 1998. [PubMed: 9856466] [Full Text: https://doi.org/10.1016/s0896-6273(00)80628-5]