Circadian clock compensation

Just like humans and other Earthlings, fungi experience large fluctuations in their environment on a daily and seasonal basis. For the circadian clock to be useful to an organism, it must keep accurate time across all physiological environmental conditions.

Four Seasons: Wikimedia Commons

In the Kelliher lab, we have identified a group of Neurospora crassa mutant strains where compensation is no longer functional, and the ~24-hour circadian period length is no longer buffered against changes in temperature or nutrients in the environment. We seek to characterize the mechanisms of these compensation pathways and how they normally work to buffer the clock from the environment. We and others have found evidence that circadian compensation also functions in our human cells, too! By defining pathways that shield the circadian clock from external nutrient levels, we aspire to find new ways to increase circadian and sleep/wake cycle robustness in patients suffering from metabolic diseases and diabetes, where external nutrient levels can be pushed outside the healthy physiological range.

Source: S1 Movie from our 2023 publication in PLoS Biology

Neurospora crassa grows reasonably well without any carbon source or sugar supplied, and the circadian clock is functional with a signal reporter peaking approximately every 24 hours (A, blue quantification line). However, feed Neurospora its favorite carbon source of glucose sugar, and a much brighter biomass signal is produced (B, yellow quantification line)! Fascinatingly, whether completely carbon-starved or consuming an “all-candy” diet, the circadian clock remains relatively constant due to circadian compensation. The Kelliher lab seeks to understand this phenomenon!

Circadian-specific pool of a key kinase

Fungal, insect, and mammalian circadian clocks exist at the level of individual cells. Within each cell, daily time is kept through a transcription-translation feedback loop (TTFL). The Neurospora crassa TTFL is composed of the White Collar Complex (WCC) and FREQUENCY (FRQ). WCC acts as a transcription factor to turn on the frq gene once per day. FRQ protein is made after a time delay, and FRQ physically interacts with a kinase called Casein Kinase I (CKI). Unlike your typical kinase-substrate interaction, FRQ and CKI seem to bind together very stably throughout the repressive phase of the TTFL. FRQ, CKI, and a third complex member FRH then feed back to inhibit WCC activity in the nucleus and to stop new frq synthesis.

The casein kinase I (ck-1a, NCU00685) gene in Neurospora crassa is extremely complex– it’s 3’ Untranslated Region (UTR) is among the top 1% longest annotated among all genes in the Neurospora genome! The CKI kinase is involved in many cellular pathways in addition to its key role in the circadian clock. In the Kelliher lab, we ask how the clock-relevant pool of CKI mRNAs and/or proteins is specified for FRQ binding and clock function. We have uncovered multiple functional domains in the 5’ and 3’ UTRs of ck-1a and seek to define the circadian functions of each of the 3 annotated protein isoforms of the CKI kinase.


Circadian functional genomics

Neurospora crassa is unique among the eukaryotic model systems used to study the circadian clock. In a highly successful NIH-funded and multi-university effort, a whole genome knockout collection was built for each individual gene in Neurospora (2004 – 2014; NIGMS grant # P01 GM068087). 7,167 out of 10,082 total genes (71.1%) are not essential for fungal viability. It is the long term goal over the lifetime of the Kelliher lab to screen through functional groups of genes to identify new and unexpected regulators of the circadian clock. This process is well underway, with dozens of new mutants found with long period, short period, or less robust / arrhythmic circadian clock phenotypes. Some of the genes we have identified so far with new circadian defects are of completely UNKNOWN biological function!

Interested in learning more about our research projects?

PI Contact Information: Tina Kelliher
Telephone: 617-287-6683
Office Location: ISC-5-5700