ratios of the two isoforms of FRQ: long FRQ (LFRQ; 1–989 aa) and short FRQ (S-FRQ; 100–989
aa). At higher temperatures, L-FRQ levels
increase relative to S-FRQ (Liu et al. 1997;
Colot et al. 2005; Diernfellner et al. 2007).
L-FRQ-only strains have a shorter clock period
than S-FRQ-only strains (Liu et al. 1997; Diernfellner et al. 2007). Therefore, the temperaturebased ratio of L- to S-FRQ levels is believed to
contribute to the robustness of the clock and
the fine-tuning of the clock period at different
ambient temperatures (Liu et al. 1997; Diernfellner et al. 2005, 2007). Evidence also suggests
that post-translational mechanisms are
involved in temperature compensation, e.g.,
the phosphorylation of FRQ. The mutation of
phosphorylation sites that exist only on L-FRQ
yield a strain with a longer period, similar to the
period of a S-FRQ-only strain (Diernfellner
et al. 2007; Baker et al. 2009). In addition, mutations in kinases that phosphorylate FRQ (i.e.,
CK2) have been shown to impact temperature
compensation (Mehra et al. 2009b). While different FRQ isoform ratios and changes in FRQ
phosphorylation contribute to temperature regulation, further work is necessary to uncover
the underlying mechanisms that provide temperature compensation to the clock.
C. The Impact of Nutrient Sensing on the
Circadian Clock
Though a great deal of research has gone into
the understanding of how light and temperature align the molecular clock, recent data from
N. crassa has shown that levels of nutrients,
such as glucose and lipids, can also affect the
clock and must be compensated for, termed
metabolic or nutritional compensation (Hurley
et al. 2016b; Sancar et al. 2012; Starkey et al.
2013). The best studied type of nutritional compensation is glucose compensation, which
includes a known circadian-auxiliary loop that
utilizes Conidial Separation Protein 1 (CSP-1)
(Fig. 4.2a). When glucose levels are high, the
core clock proteins and many of their downstream targets, including CSP-1, are translated
at an increased rate (Sancar et al. 2012). To
compensate for the increased clock protein
levels, the resulting higher levels of CSP-1 interact with additional clock-regulated proteins,
Regulation of Conidiation 1 (RCO-1) and Regulation of Conidiation and Morphology-1
(RCM-1), to repress wc-1 expression and complete a negative feedback loop that provides
glucose compensation (Sancar et al. 2012;
Dovzhenok et al. 2015; Olivares-Yan ˜ez et al.
2016). Other proteins are also recognized for
their impact on glucose compensation, including Period-1 (PRD-1/DBP-2) and Ras-like2 (RAS-2). PRD-1 is an ATP-dependent
DEAD-box RNA helicase, and the clock in
prd-1 mutants is not compensated against glucose changes, leading to longer periods when
glucose is not limited (Starkey et al. 2013;
Emerson et al. 2015; Adhvaryu et al. 2016).
Like prd-1 mutants, the clock in a Dras2 strain
displays longer periods and is not glucosecompensated (Gyo ¨ngyo ¨si et al. 2017). Further
work is needed to fully understand how glucose
compensation is imparted, either by multiple
feedback loops including CSP-1, RCO-1, RCM1, PRD-1, and RAS-2, or through an underlying
mechanism yet to be discovered. Nutrient compensation has long been shown to be an important element of the mammalian circadian clock
and the research into the mechanisms that regulate nutrient compensation in N. crassa has
enabled an appreciation of how these might
work in higher eukaryotes (Peek et al. 2012).
Beyond glucose compensation, there is evidence that the clock can be affected by lipid
levels. Longevity assurance gene (LAG-1) is
thought to be part of a ceramide synthase complex and likely plays a role in metabolic clock
regulation through stress responses involving
sphingolipid metabolism (Case et al. 2014). In a
lag-1 mutant, the clock period lengthens to
41 h, suggesting that changes in sphingolipid
levels can affect clock period. Further evidence
that the clock is sensitive and responsive to
lipid levels includes the relationship between
high levels of DAG (sn-1,2-diacylglycerol) and
longer clock periods (Ramsdale and LakinThomas 2000; Case et al. 2014). With the extent
of reported metabolic effects on the clock
through glucose and lipid levels, it is likely
that other cell components that play a role in
4 From Genetics to Molecular Oscillations: The Circadian Clock in Neurospora crassa
83
aa). At higher temperatures, L-FRQ levels
increase relative to S-FRQ (Liu et al. 1997;
Colot et al. 2005; Diernfellner et al. 2007).
L-FRQ-only strains have a shorter clock period
than S-FRQ-only strains (Liu et al. 1997; Diernfellner et al. 2007). Therefore, the temperaturebased ratio of L- to S-FRQ levels is believed to
contribute to the robustness of the clock and
the fine-tuning of the clock period at different
ambient temperatures (Liu et al. 1997; Diernfellner et al. 2005, 2007). Evidence also suggests
that post-translational mechanisms are
involved in temperature compensation, e.g.,
the phosphorylation of FRQ. The mutation of
phosphorylation sites that exist only on L-FRQ
yield a strain with a longer period, similar to the
period of a S-FRQ-only strain (Diernfellner
et al. 2007; Baker et al. 2009). In addition, mutations in kinases that phosphorylate FRQ (i.e.,
CK2) have been shown to impact temperature
compensation (Mehra et al. 2009b). While different FRQ isoform ratios and changes in FRQ
phosphorylation contribute to temperature regulation, further work is necessary to uncover
the underlying mechanisms that provide temperature compensation to the clock.
C. The Impact of Nutrient Sensing on the
Circadian Clock
Though a great deal of research has gone into
the understanding of how light and temperature align the molecular clock, recent data from
N. crassa has shown that levels of nutrients,
such as glucose and lipids, can also affect the
clock and must be compensated for, termed
metabolic or nutritional compensation (Hurley
et al. 2016b; Sancar et al. 2012; Starkey et al.
2013). The best studied type of nutritional compensation is glucose compensation, which
includes a known circadian-auxiliary loop that
utilizes Conidial Separation Protein 1 (CSP-1)
(Fig. 4.2a). When glucose levels are high, the
core clock proteins and many of their downstream targets, including CSP-1, are translated
at an increased rate (Sancar et al. 2012). To
compensate for the increased clock protein
levels, the resulting higher levels of CSP-1 interact with additional clock-regulated proteins,
Regulation of Conidiation 1 (RCO-1) and Regulation of Conidiation and Morphology-1
(RCM-1), to repress wc-1 expression and complete a negative feedback loop that provides
glucose compensation (Sancar et al. 2012;
Dovzhenok et al. 2015; Olivares-Yan ˜ez et al.
2016). Other proteins are also recognized for
their impact on glucose compensation, including Period-1 (PRD-1/DBP-2) and Ras-like2 (RAS-2). PRD-1 is an ATP-dependent
DEAD-box RNA helicase, and the clock in
prd-1 mutants is not compensated against glucose changes, leading to longer periods when
glucose is not limited (Starkey et al. 2013;
Emerson et al. 2015; Adhvaryu et al. 2016).
Like prd-1 mutants, the clock in a Dras2 strain
displays longer periods and is not glucosecompensated (Gyo ¨ngyo ¨si et al. 2017). Further
work is needed to fully understand how glucose
compensation is imparted, either by multiple
feedback loops including CSP-1, RCO-1, RCM1, PRD-1, and RAS-2, or through an underlying
mechanism yet to be discovered. Nutrient compensation has long been shown to be an important element of the mammalian circadian clock
and the research into the mechanisms that regulate nutrient compensation in N. crassa has
enabled an appreciation of how these might
work in higher eukaryotes (Peek et al. 2012).
Beyond glucose compensation, there is evidence that the clock can be affected by lipid
levels. Longevity assurance gene (LAG-1) is
thought to be part of a ceramide synthase complex and likely plays a role in metabolic clock
regulation through stress responses involving
sphingolipid metabolism (Case et al. 2014). In a
lag-1 mutant, the clock period lengthens to
41 h, suggesting that changes in sphingolipid
levels can affect clock period. Further evidence
that the clock is sensitive and responsive to
lipid levels includes the relationship between
high levels of DAG (sn-1,2-diacylglycerol) and
longer clock periods (Ramsdale and LakinThomas 2000; Case et al. 2014). With the extent
of reported metabolic effects on the clock
through glucose and lipid levels, it is likely
that other cell components that play a role in
4 From Genetics to Molecular Oscillations: The Circadian Clock in Neurospora crassa
83