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Circadian rhythms and molecular clocks

The research group led by the biochemist Professor Michael Brunner at Heidelberg University is investigating the molecular mechanisms of the 24-h circadian rhythm of Neurospora crassa. The researchers have been able to show how the fungus is able to maintain the day-night rhythm even in the presence of disturbing light signals at night.

Neurospora crassa. Asci arranged in a rosette; the nuclei of the ascospores are stained with H1-GFP. © N.B.Raju, Stanford University, for NIH. gov.

Neurospora crassa became known to biologists through the work of a small group of mycologists (fungus researchers) including the American scientists George W. Beadle and Edward L. Tatum who came up with their "one gene - one enzyme" hypothesis based on their investigations on this inconspicuous mould fungus. The two researchers were awarded the 1958 Nobel Prize for their work on the biochemistry of the genetics of this fungus, which represented a milestone in the field of molecular biology. Subsequent work has led to further refinement of Beadle and Tatum's hypothesis and it is now known that many proteins are made up of more than one polypeptide chain. The small, filamentous fungus has become a popular model organism for genetic investigations as it can easily be kept in the laboratory and it forms its spores in neat lines inside sacs called asci (plural of ascus). Each ascus contains ascospores that are produced by meiotic- followed by mitotic cell divisions and are arranged in the sac as they are produced, in the same way as peas in a pod. All eight ascospores (they are haploid and are therefore excellently suited for genetic investigations) can be individually removed, cultured and investigated.

A fungus that is excellently suited for circadian clock investigations

Prof. Dr. Michael Brunner © BZH

Professor Dr. Michael Brunner chose N. crassa as a study object to investigate the molecular mechanisms of the circadian clock that synchronises the life rhythm of organisms with a 24-h day-night cycle related to the rotation of the earth on its axis. The majority of organisms have circadian clocks (Latin: circa - around; dies - day). In humans, the circadian rhythm controls our sleep-wake rhythm, behaviour and metabolic activities. Our circadian clock is especially noticeable when our internal clock is out of sync with the external environment. For example, some people suffer from jet lag after travelling by air across several time zones in a short period of time.

Circadian clocks are molecular oscillators that control the expression of a large number of genes in the cells of eukaryotic organisms in a roughly 24-hour cycle. This leads to numerous biochemical, physiological and behavioural processes in living entities that all occur in a time-of-day specific manner.

Circadian clocks consist of a network of interconnected positive and negative feedback loops that affect the periodic expression and modification of one or several clock proteins. These circadian oscillations are self-sustained (endogenous) and persist in a roughly 24-hour cycle when not affected by external signals. In nature, circadian rhythms are adjusted to the environment by external cues called zeitgebers (from German for ‘time giver') that synchronise the internal clocks of organisms with the 24-h period of earth rotation. The primary zeitgebers are light, temperature and nutrients. Brunner's group of researchers at the BZH at the University of Heidelberg uses the filamentous fungus N. crassa as a model organism in order to understand how the circadian clock works as a programme that coordinates complex expression profiles in a temporal fashion.

The molecular clock of Neurospora crassa

A key element of the circadian clock of Neurospora is the gene "frequency" (frq). The expression levels of frq RNA and FRQ protein oscillate in a circadian fashion. Expression of frq is controlled by the transcription factors "white collar-1" (WC-1) and WC-2 that regulate the expression of light-induced genes. WC-1 and WC-2, which belong to the GATA-type zinc finger transcription factors group, assemble into a hetero-oligomeric protein complex known as white collar complex (WCC). They bind to two specific elements of the frq promotor and trigger the clock-controlled transcription of frq RNA and the transcription of frq in response to light. The FRQ protein, a dimeric phosphoprotein, is part of a negative feedback loop that inhibits the synthesis of its own RNA. The phosphorylation level of the FRQ protein increases progressively upon exposure to light during the course of a day, producing a hyperphosphorylated form that is degraded. The negative feedback effect gradually fades as the levels of FRQ protein decrease; the frq RNA level begins to rise and a new circadian period starts. However, the FRQ protein does not just act as a repressor of its own RNA, it also activates (in a positive feedback loop) the formation of WC-1 and WC-2  (https://www.biooekonomie-bw.debzh.db-engine.de/default.asp?lfn=2573).

Molecular sunglasses stabilise the circadian clock

Neurospora crassa; hyphae © Dept. Mycology, University of Kaiserslautern

In Neurospora, the FRQ/WCC oscillator modulates the rhythmic expression of around 1,000 genes (which corresponds to about ten per cent of the entire Neurospora genome). However, only a fraction of these genes is directly controlled by the white collar complex on the transcription level. In future, it will be necessary to carry out intensive research to find out how the network of genes controlled by the internal clock is organised and regulated.

It was previously also not known how the circadian clock was able to synchronise the expression of genes in a 24-h rhythm with light as zeitgeber, even in cases where other light signals such as artificial illumination of moonlight affected the rhythm. Brunner's team is currently working in cooperation with the Hungarian scientist Dr. Krisztina Káldi from the Institute of Physiology at Semmelweis University in Budapest and the researchers have been able to clarify how this synchronisation works. Their results have recently been published in the journal "Cell".

The adaptation to light, which suppresses transcription at low light intensities (for example moonlight or lamplight), depends on the interaction of two antagonistic photoreceptors: the LOV (light-oxygen-voltage) domain of the WCC transcription factor and the VVD receptor that also has a LOV domain, which functions however as a negative regulator. The vvd gene is expressed upon the activation of WCC. Simplified, the control loop works as follows:

  • During darkness, the transcription factor and light receptor WCC is present in an inactive monomeric form. The VVD light receptor is not expressed.
  • At dawn, increasing light intensity induces the dimerisation and activation of WCC; the two LOV domains bind to each other. The activation of WCC leads to the expression of the negative regulator gene vvd and other light-induced genes.
  • In order to adapt to altered light intensities, the LOV domain of the light-activated regulator protein VVD binds to the LOV domain of WCC, thereby inhibiting the dimerisation of WCC. This in turn leads to the decrease in the transcription of vvd and other light-induced genes.
  • The following night, the VVD proteins produced during the daytime serve as a molecular memory of the brightness of the preceding day and suppress responses (dimerisation and activation of WCC is prevented) to light cues of lower intensity. When the light intensity once again increases the following morning, the largest proportion of VVD proteins has been degraded, and the WCC transcription factor is activated again.

This feedback loop is like a pair of "molecular sunglasses" that ensure that Neurospora crassa's internal clock does not mistake day for night as a result of disturbing light signals such as moonlight or artificial illumination.

Malzahn E, Ciprianidis S, Káldi K, Schafmeier T, Brunner M: Photo-Adaptation in Neurospora is Mediated by Competitive Interaction of Activating and Inhibitory Light-Oxygen-Voltage Domains. Cell 142(5), 762-772, 2010.
Further information:
Prof. Dr. Michael Brunner
Biochemisty Centre at the University of Heidelberg
Im Neuenheimer Feld 328
69120 Heidelberg
Tel.: +49 (0)6221-54 4207
E-mail: michael.brunner(at)bzh.uni-heidelberg.de
Website address: https://www.biooekonomie-bw.de/en/articles/news/circadian-rhythms-and-molecular-clocks/