Timeless (gene)

Timeless (tim) is a gene in Drosophila which encodes TIM, an essential protein that regulates circadian rhythms. Timeless mRNA and protein oscillate rhythmically with time as part of a transcription-translation [negative feedback] loop involving the period gene and its protein.

Discovery
Timeless was discovered by Dr. Michael W. Young and colleagues in 1994. This gene was found when they noticed an arrhythmic tim01 mutant via a hybrid dysgenesis screen.

Function in the circadian clock
The timeless gene is an essential component of the molecular circadian clock in Drosophila. It acts as part of an autoregulatory feedback loop in conjunction with the period (per) gene product. Its circadian properties were noted in studies performed by Dr. Michael Rosbash's lab and Dr. Charles Weitz's lab. Both indicated that timeless protein (TIM) and period protein (PER) form a heterodimer that exhibits circadian rhythms in wild type Drosophila. Researchers in Rosbash's lab also showed that tim mRNA levels and TIM protein levels have circadian rhythms that are similar to those of the period(per) and its product.

The PER/TIM heterodimer regulates transcription of period (per) and timeless (tim) genes. First the PER/TIM heterodimers form in the cytoplasm, accumulate, and then translocate to the nucleus. The complex then blocks the positive transcription factor clock (CLK) and cycle (CYC).

As part of the circadian clock timeless is essential for entrainment to light-dark (LD) cycles. The typical period length of free-running Drosophila is 23.9 hours, requiring adaptations to the 24-hour environmental cycle. Adaptation first begins with exposure to light. This process leads to the rapid degradation of the TIM protein, allowing organisms to entrain at dawn to environmental cycles. In light-dark cycles, TIM protein level decreases rapidly in late night/early morning, followed by the similar but more gradual changes in PER protein level. TIM degradation is independent of per and its protein, and releases PER from the PER/TIM complex. This ends the PER/TIM repression of the CLK/CYC-mediated transcription of per and tim genes, allowing per and tim mRNA to be produced to restart the cycle. In some cell types, the photoreceptor protein cryptochrome (CRY) physically associates with TIM and helps regulate light-dependent degradation. CRY is activated by blue light, which binds to TIM and tags it for degradation.

This mechanism allows entrainment of flies to environmental light cues. When Drosophila receive light inputs in the early subjective night, light-induced TIM degradation causes a delay in TIM accumulation, which creates a phase delay. When light inputs are received in the late subjective night, a light pulse causes TIM degradation to occur earlier than under normal conditions, leading to a phase advance.

In Drosophila, the negative factors PER/TIM, as well as the positive factors CLK/CYC, are eventually degraded by a casein kinase-mediated phosphorylation cycle, allowing fluctuations in gene expression according to environmental cues. These proteins mediate the oscillating expression of the transcription factor VRILLE (VRI), which is required for behavioral rhythmicity, per and tim expression, and accumulation of PDF (pigment-dispersing factor).

Timeless in the cricket Gryllus bimaculatus
Timeless does not appear to be essential for oscillation of the circadian clock for all insects. In wild type crickets, tim mRNA shows rhythmic expression in both LD and DD (dark-dark cycles) similar to that of per, peaking during the (subjective) night. When injected with tim double-stranded RNA (dstim), tim mRNA levels were significantly reduced and its circadian expression rhythm was eliminated. After the dstim treatment, however, adult crickets showed a clear locomotor rhythm in constant darkness, with a free-running period significantly shorter than that of control crickets injected with Discosoma sp. Red2 (DsRed2) dsRNA. These results suggest that in the cricket, tim plays some role in fine-tuning of the free-running period but may not be essential for oscillation of the circadian clock.

Mammalian homologs for timeless
In 1998, researchers identified a mouse homolog and a human homolog of the Drosophila timeless gene. The exact role of TIM in mammals is still unclear, as Tim transcription does not oscillate rhythmically and the TIM protein remains in the nucleus. Moreover, mammalian tim is more orthologous the Tim-2 (Timeout) paralog of the Drosophila Timeless gene. The function of Timeout has yet to be identified.

The timeless protein is thought to directly connect the cell cycle with the circadian rhythm in mammals. In this model called a “direct coupling” the two cycles share a key protein whose expression exhibits a circadian pattern.

Mice
Recent work on the mammalian timeless (mTim) in mice has suggested that the genes identified may not play the same essential role in mammals as in Drosophila as an essential function of the circadian clock. mTim is expressed in the suprachiasmatic nucleus (SCN), but there is no oscillation of its accompanying RNA or protein products in constant conditions. The mammalian TIM protein levels do not shift with light signals, but there is reported interaction with the mammalian period protein PER1 and mammalian cryptochrome (CRY1 and CRY2). mTim is shown to be necessary for embryonic development in mice, indicating a different gene function than in Drosophila This suggests a divergence between mammalian clocks and the Drosophila clock.

Humans
The human timeless protein (hTIM) has been shown to be required for the production of electrical oscillations output by the suprachiasmatic nucleus (SCN), the major clock governing all tissue-specific circadian rhythms of the body. This protein also interacts with the products of major clock genes CLOCK, BMAL, PER1, PER2 and PER3.

Sancar and colleagues investigated whether hTIM played a similar role to orthologs in C. elegans and other types of yeast, which are known to play play important roles in the cell cycle. Their experiments suggested that hTIM plays an integral role in the G2/M and intra-S cell cycle checkpoints. With respect to the G2/M checkpoint, hTIM binds to the ATRIP subunit on ATR – a protein kinase sensitive to DNA damage. This binding between hTIM and ATR then leads to the phosphorylation of Chk1, resulting in cell cycle arrest or apoptosis. This process serves as an important control to stop the proliferation of cells with DNA damage prior to mitotic division. The role of hTIM in the intra-S checkpoint is less clear at the molecular level. However, down-regulation of hTIM leads to an increase in the rate of generation of replication forks – even in the presence of DNA damage and other regulatory responses.