II. Input: Various Inputs to the Core
Clock Allow Phase Entrainment to
the External Environment
One of the tenets of circadian rhythms is that
their period is buffered against different constant conditions but is still able to incorporate
signals from the environment, allowing the
clock phase to align, or “entrain,” to changes
in the external environment. The most obvious
of these signals is the light from the sun, a cue
to which circadian rhythms were adapted (Hut
and Beersma 2011). However, in recent years,
the circadian field has discovered that the clock
is responsive to many other environmental
cues, such as temperature and glucose (Garceau
et al. 1997; Liu et al. 1998; Sancar et al. 2012).
For this review, we have grouped these signals,
and the pathways that respond to them, into
three basic categories: light, temperature, and
nutrients.
A. Neurospora crassa, the Circadian Clock, and
Photoreception
While fungi are not photosynthetic organisms,
the ability to sense different colors and intensities of light still contributes to their overall
fitness (Idnurm et al. 2010). N. crassa has conserved proteins that are essential for the detection of red light, green light, and blue light,
though not all of these proteins are functional
photoreceptors (for more complete reviews, see
Fuller et al. 2015; Yu and Fischer 2019). The
primary external timing cue for the clock in N.
crassa is blue light, which is sensed through the
positive arm protein WC-1 (Fig. 4.2a). WC-1 is
a transcription factor that utilizes Per-ArntSim (PAS) domains for transcriptional activation. The PAS domain located toward the protein’s N-terminal end is of a special class called
a “light oxygen voltage” (LOV) domain (Ballario and Macino 1997; Ballario et al. 1998). The
LOV domain is able to form a transient cysteinyl adduct with a blue light-absorbing chromophore flavin adenine dinucleotide (FAD) when
exposed to blue light (Ballario et al. 1998; Linden et al. 1997). Along with its partner protein,
WC-2, WC-1 hetero-dimerizes to form the
white collar complex (WCC) (Cheng et al.
2002). When the WCC is exposed to blue light,
a conformational change occurs which creates a
transcriptionally active WCC (Froehlich et al.
2002; He et al. 2002; Zoltowski et al. 2007; Malzahn et al. 2010). The WCC transcriptionally
activates genes that have a light-responsive element (LRE) in their promoters, with hundreds
of genes affected, either directly or indirectly,
by WCC binding (Chen et al. 2009; Collett et al.
2002; Froehlich et al. 2003; Hurley et al. 2014;
Smith et al. 2010; Wu et al. 2014). As the WCC
contains both a light-sensitive element and
forms the positive arm of the circadian clock
(see Sect. III.A), research in N. crassa was foun⁄
ä
Fig. 4.2 (continued) numerous genes, including frequency (frq). In order to do this, the promoter regions of these
genes must be accessible and numerous chromatin remodeling factors (dark blue) work with the WCC to effect
remodeling at these promoters. The transcriptional activation of frq leads to the transcription and translation of
FRQ (light orange), which then dimerizes and associates with Frequency-interacting RNA helicase (FRH, dark
orange), forming the FFC. FRQ is then phosphorylated (cyan stars) by numerous kinases (light blue) until the FFC
can interact with the WCC, repressing WCC activation. The WCC is phosphorylated by several kinases, decreasing
transcription at the frq locus and other loci regulated by the WCC, and then potentially removed from the nucleus.
FRQ undergoes further phosphorylation until it can no longer interact with the WCC and it is recognized by FWD1 (pink) for degradation. Without the FFC, the WCC is dephosphorylated by phosphatases (blue) and returns to
the nucleus to restart the cycle. The genes under the influence of the WCC that do not play a role in the core clock
are called clock-controlled genes (ccgs, red) and are the drivers of circadian behavior. Some of these ccgs can act
back on the core circadian loop. In addition, inputs from the environment (yellow) are able to entrain the clock,
such as light, temperature, and nutrients (e.g., glucose). (b) Negative arm turnover via phosphorylation triggered
degradation was previously believed to relieve positive arm (WCC, green) repression. However, new evidence
suggests that the negative arm (FFC, orange) undergoes two types of phosphorylation, a clock-specific phosphorylation, which alters the ability of the FFC to suppress WCC activity, and degradation-specific phosphorylation,
which targets the negative arm proteins for degradation. This degradation occurs only after the FFC has lost its
ability to repress the WCC, and this highly phosphorylated FFC does not impact the clock
4 From Genetics to Molecular Oscillations: The Circadian Clock in Neurospora crassa
81
Clock Allow Phase Entrainment to
the External Environment
One of the tenets of circadian rhythms is that
their period is buffered against different constant conditions but is still able to incorporate
signals from the environment, allowing the
clock phase to align, or “entrain,” to changes
in the external environment. The most obvious
of these signals is the light from the sun, a cue
to which circadian rhythms were adapted (Hut
and Beersma 2011). However, in recent years,
the circadian field has discovered that the clock
is responsive to many other environmental
cues, such as temperature and glucose (Garceau
et al. 1997; Liu et al. 1998; Sancar et al. 2012).
For this review, we have grouped these signals,
and the pathways that respond to them, into
three basic categories: light, temperature, and
nutrients.
A. Neurospora crassa, the Circadian Clock, and
Photoreception
While fungi are not photosynthetic organisms,
the ability to sense different colors and intensities of light still contributes to their overall
fitness (Idnurm et al. 2010). N. crassa has conserved proteins that are essential for the detection of red light, green light, and blue light,
though not all of these proteins are functional
photoreceptors (for more complete reviews, see
Fuller et al. 2015; Yu and Fischer 2019). The
primary external timing cue for the clock in N.
crassa is blue light, which is sensed through the
positive arm protein WC-1 (Fig. 4.2a). WC-1 is
a transcription factor that utilizes Per-ArntSim (PAS) domains for transcriptional activation. The PAS domain located toward the protein’s N-terminal end is of a special class called
a “light oxygen voltage” (LOV) domain (Ballario and Macino 1997; Ballario et al. 1998). The
LOV domain is able to form a transient cysteinyl adduct with a blue light-absorbing chromophore flavin adenine dinucleotide (FAD) when
exposed to blue light (Ballario et al. 1998; Linden et al. 1997). Along with its partner protein,
WC-2, WC-1 hetero-dimerizes to form the
white collar complex (WCC) (Cheng et al.
2002). When the WCC is exposed to blue light,
a conformational change occurs which creates a
transcriptionally active WCC (Froehlich et al.
2002; He et al. 2002; Zoltowski et al. 2007; Malzahn et al. 2010). The WCC transcriptionally
activates genes that have a light-responsive element (LRE) in their promoters, with hundreds
of genes affected, either directly or indirectly,
by WCC binding (Chen et al. 2009; Collett et al.
2002; Froehlich et al. 2003; Hurley et al. 2014;
Smith et al. 2010; Wu et al. 2014). As the WCC
contains both a light-sensitive element and
forms the positive arm of the circadian clock
(see Sect. III.A), research in N. crassa was foun⁄
ä
Fig. 4.2 (continued) numerous genes, including frequency (frq). In order to do this, the promoter regions of these
genes must be accessible and numerous chromatin remodeling factors (dark blue) work with the WCC to effect
remodeling at these promoters. The transcriptional activation of frq leads to the transcription and translation of
FRQ (light orange), which then dimerizes and associates with Frequency-interacting RNA helicase (FRH, dark
orange), forming the FFC. FRQ is then phosphorylated (cyan stars) by numerous kinases (light blue) until the FFC
can interact with the WCC, repressing WCC activation. The WCC is phosphorylated by several kinases, decreasing
transcription at the frq locus and other loci regulated by the WCC, and then potentially removed from the nucleus.
FRQ undergoes further phosphorylation until it can no longer interact with the WCC and it is recognized by FWD1 (pink) for degradation. Without the FFC, the WCC is dephosphorylated by phosphatases (blue) and returns to
the nucleus to restart the cycle. The genes under the influence of the WCC that do not play a role in the core clock
are called clock-controlled genes (ccgs, red) and are the drivers of circadian behavior. Some of these ccgs can act
back on the core circadian loop. In addition, inputs from the environment (yellow) are able to entrain the clock,
such as light, temperature, and nutrients (e.g., glucose). (b) Negative arm turnover via phosphorylation triggered
degradation was previously believed to relieve positive arm (WCC, green) repression. However, new evidence
suggests that the negative arm (FFC, orange) undergoes two types of phosphorylation, a clock-specific phosphorylation, which alters the ability of the FFC to suppress WCC activity, and degradation-specific phosphorylation,
which targets the negative arm proteins for degradation. This degradation occurs only after the FFC has lost its
ability to repress the WCC, and this highly phosphorylated FFC does not impact the clock
4 From Genetics to Molecular Oscillations: The Circadian Clock in Neurospora crassa
81