Saturday, February 12, 2011

Circadian-Clocks tick without DNA

                            Intricate machinary of a clock. Is there a master-tuner? [image]

This idea would forever change the way we would look at Circadain-Rythms, simply because it trashed a dogma - one that required DNA to run biological-clocks!

Circadian Rythms: All forms of life undergo circadian (roughly 24-hour) fluctuations in energy availability that are tied to alternating cycles of light and darkness. These self-sustained rhythms could be biochemical, physiological, or behavioural  processes. Our biological clocks organize such internal energetic cycles through 'transcription–translation feedback loops'. 

What is known: There are specific "clock-genes" that comprise of a 'forward limb' involving a set of transcriptional activators that induce the transcription of a set of repressors which comprise the 'negative limb' and duly feeds back to inhibit the forward limb [1]. This modulatory cycle repeats itself every 24 hours. Energy-cycles show transcription-dependent circadian periodicity; such cycles include the alternating oxygenic and nitrogen-fixing phases of photosynthesis, and the glycolytic and oxidative cycles in eukaryotes. 

BIG Questions:

1.Is the nucleus [actually the DNA contained in it] necessary for clock-maintainance in mammals?
Actually, several classical model organisms that are genetically tractable (for example, yeast and C. elegans) have not been found to express any known ‘clock genes’, but do exhibit circadian rhythms [2,3].

2.Are the transcriptional [DNA-based] and nontranscriptional [non-DNA based] cycles interrelated?

** How do you check for the necessity [or not] of DNA in a given life process? Easy, check for the process in a cell type that lacks a nucleus.
Neill and Reddy did exactly that! They established human red blood cells (RBCs, enucleated-mammalian cells) as an appropriate model system to test if they might have a rythmic-clock operating. Crucially, these cells lack both a nucleus and the energy-producing mitochondria.

RBC's : non-nucleated and just perfect! [image]
These cells therefore function mainly as oxygen shuttles, utilizing haemoglobin. Interestingly, RBCs possess the evolutionarily conserved enzymes of the peroxiredoxin family [4], which control intarcellular peroxide leves and react to rising intracellular reactive oxygen species (ROS) levels by forming oligomers. Importantly, they had been previously showed to exhibit circadian-periodicity in hepatic-cells [5]

1] Do Peroxiredoxins exhibit circadian redox rhythms in RBC's?
O’Neill and Reddy monitored the monomer–dimer transition of peroxiredoxin proteins in RBCs from three human volunteers. The oligomerization-pattern was self-sustained over several cycles within an approximate 24-hour period.
Next, they had to prove that RBC's should show the property of 'Entrainment', i.e to be a useful timing mechanism, oscillations (of oscillators) should be tuneable by external cues so that they can be reset when misaligned. Here, they used Temperature as a cue. And indeed, Peroxiredoxin oxidation cycles were synchronized in response to temperature cycles.
* To rule out the presence of contaminating nucleated cells [WBC etc.], inhibitors of translation (Cycloheximide) and transcription (a-Amanitin) were added, which could not perturb the peroxiredoxin oxidation rhythm, proving that this clock could run efficiently in the absence of transcription and was totally independant of DNA.
As it turns out, in RBC's, Haemoglobin [Hb] is a major source of Peroxides via autooxidation [Heme structure, here on Wiki]. So, did Hb also show periodicity? Yes, it did! They used intrinsic front-face fluorescence as a real-time assay of rhythmicity [6] and indicated reversible low amplitude oxidation of haemoglobin in RBCs.
* Now, what drives the rhythmic cycles of oligomerization for peroxiredoxin? Are they related to othe Biochemical cycles on RBCs'?
Given that red blood cells are dependant on glycolysis for ATP synthesis, and this contributes significantly to the NADH flux in RBCs'. Could ATP and NADPH oscillate with cycle? Yes, Indeed! The researchers reported weak oscillations in the levels of ATP (and also NADPH)!

2] Could they be related to Nuclear events? Is there a connection between the nucleus's interior and exterior?
they assayed mouse embryonic fibroblasts [MEF] from Cry1/Cry2 double-knockout mice, which lack cyclical expression of known clock genes/proteins [7]. Rhythms in peroxiredoxin oxidation were altered relative to those seen in wild-type MEFs. Therefore, in nucleated cells, peroxiredoxin rhythms are influenced by the transcription–translation feedback loop.
* Could the reverse be true? That is, if levels of Peroredoxin in cells fall, could it effect levels of transcription?
Indeed, as knockdown of PRX2 and PRX4 in human U2OS-cells resulted in a long-period phenotype, whereas si-RNAs directed against PRX3 and PRX5 depressed the amplitude of circadian oscillations!
Therefore, in nucleated cells, there is likely to be an intricate interplay between transcription-dependent processes and non-nuclear events, which seem to be reciprocally regulating each other.

 Take Home message:
Circadian Rythms are NOT maintained  exclusively by the Nucleus, but are also influenced by events occuring in the cytosol, AND adequate modulation is brought about by an essential interplay interplay of both processes.
* This paper
O’Neill, J. S. & Reddy, A. B. Circadian clocks in human red blood cells. Nature doi:10.1038/nature09702
** the other paper [in the same issue of Nature by same group]
O’Neill, J. S. et al. Circadian rhythms persist without transcription in a eukaryote.Nature doi:10.1038/nature09654
1. Reddy, A. B. & O’Neill, J. S. Healthy clocks, healthy body, healthy mind. Trends Cell Biol. 20, 36–44 (2009).
2. Eelderink-Chen, Z. et al. A circadian clock in Saccharomyces cerevisiae. Proc. Natl Acad. Sci. USA 107, 2043–2047 (2010).
3. Kippert, F., Saunders, D. S. & Blaxter, M. L. Caenorhabditis elegans has a circadian clock. Curr. Biol. 12, R47–R49 (2002).
4. Hall, A., Karplus, P. A. & Poole, L. B. Typical 2-Cys peroxiredoxins–structures, mechanisms and functions. FEBS J. 276, 2469–2477 (2009).
5. Reddy, A. B. et al. Circadian orchestration of the hepatic proteome. Curr. Biol. 16, 1107–1115 (2006).
6. Kennett, E. C. et al. Investigation of methaemoglobin reduction by extracellular NADH in mammalian erythrocytes. Int. J.Biochem.Cell Biol.37,1438–1445(2005).
7. Yagita, K., Tamanini, F., van der Horst, G. T. J.&Okamura, H. Molecular mechanisms of the biological clock in cultured fibroblasts. Science 292, 278–281 (2001).

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