clock nutritional compensation are still to be
determined.
III. Core Oscillator: The N. crassa Core
Circadian Timekeeping
Mechanism
The circadian inputs described above feed into
the architecturally-conserved circadian transcriptional-translational negative feedback
loop (TTFL) (Hurley et al. 2016a; Dunlap and
Loros 2017). In addition to the conservation of
the underlying architecture of the TTFL across
higher eukaryotes, there is sequence homology
between WC-1 and WC-2, the positive arm
transcription factors in N. crassa, and transcription factors in other fungal and mammalian systems (Dunlap and Loros 2017).
Furthermore, conformational heterogeneity
and post-translational regulation of the clock
proteins by phosphorylation, especially of negative arm proteins such as FRQ, are also conserved (Lee et al. 2009; Chiu et al. 2011; Hurley
et al. 2016a; Pelham et al. 2018). Finally,
phosphorylation-dependent
ubiquitination
and degradation of the negative components
supports the functioning of most eukaryotic
clocks (Grima et al. 2002; Ko et al. 2002; He
et al. 2003; Eide et al. 2005). All of these conserved features of the clock were, at least in
part, discovered in N. crassa. In this section,
we review the abovementioned key elements
of the N. crassa core clock mechanism, which
have contributed greatly to the understanding
of the TTFL in other fungi and higher eukaryotes.
A. The Transcriptionally Active Positive Arm
of the Clock
In N. crassa, the circadian cycle is initiated by
the positive arm of the clock, a heterodimeric
GATA-like transcription factor complex
termed the White Collar Complex (WCC) comprising two proteins, WC-1 and WC-2 (Belden
et al. 2007b; Hurley et al. 2014). WCC transcriptional activity peaks around subjective dawn,
commencing the circadian cycle (Belden et al.
2007b; Hurley et al. 2014). WC-1 is a 1167
amino acid (aa) long protein that contains a
circadian transactivation domain, three PerArnt-Sim (PAS) domains (including the lightresponsive LOV domain and a “defective in
binding” (DBD) region involved in DNA binding and mediating interactions with the negative arm), and a single zinc finger domain
involved in DNA binding (Ballario et al. 1996;
Lee et al. 2000; Cheng et al. 2002; Wang et al.
2014, 2016). As mentioned previously in Sect.
II.A, WC-1 is the primary blue-light photoreceptor in N. crassa and is responsible for the
integration of light signaling into the circadian
clock.
WC-2 is a 530 aa-long protein that contains
a single zinc finger domain, a single PAS
domain and, unlike WC-1, is unable to sense
light (Linden and Macino 1997; Collett et al.
2002). WC-1 and WC-2 exhibit strong oscillations at the transcriptional level, but neither
show strong oscillations at the protein level
(Hurley et al. 2018). Interestingly, the negative
arm protein, FRQ, is needed for proper levels of
WC-1 to accumulate (Schafmeier et al. 2006).
WC-1 and WC-2 interact with each other
through their PAS domains to form the
White-Collar Complex (WCC), which promotes
the expression of genes in the negative arm as
well as other genes not involved in maintaining
the core clock, termed clock-controlled genes
(ccgs) (Fig. 4.2a) (Linden and Macino 1997;
Crosthwaite et al. 1997; Cheng et al. 2002; Hurley et al. 2014; Wu et al. 2014).
The WCC exists in two conformations, a
light-activated conformation and a dark conformation, which serve to control the DNA
binding of the WCC. The light-activated complex is composed of two WC-1s and one WC-2,
binding to a subset of genes that are considered
to be light-responsive (Froehlich et al. 2002;
Cheng et al. 2003). In contrast, the dark conformation comprises a single WC-1 and WC-2 and
is essential for the expression of frq and other
ccgs under “free-running” or constant dark
conditions (Froehlich et al. 2002; Cheng et al.
2003). The zinc fingers of both WC-1 and WC2 are essential for binding DNA in the dark,
aided by the DBD motif on WC-1 (Wang et al.
84
M. S. Jankowski et al.
determined.
III. Core Oscillator: The N. crassa Core
Circadian Timekeeping
Mechanism
The circadian inputs described above feed into
the architecturally-conserved circadian transcriptional-translational negative feedback
loop (TTFL) (Hurley et al. 2016a; Dunlap and
Loros 2017). In addition to the conservation of
the underlying architecture of the TTFL across
higher eukaryotes, there is sequence homology
between WC-1 and WC-2, the positive arm
transcription factors in N. crassa, and transcription factors in other fungal and mammalian systems (Dunlap and Loros 2017).
Furthermore, conformational heterogeneity
and post-translational regulation of the clock
proteins by phosphorylation, especially of negative arm proteins such as FRQ, are also conserved (Lee et al. 2009; Chiu et al. 2011; Hurley
et al. 2016a; Pelham et al. 2018). Finally,
phosphorylation-dependent
ubiquitination
and degradation of the negative components
supports the functioning of most eukaryotic
clocks (Grima et al. 2002; Ko et al. 2002; He
et al. 2003; Eide et al. 2005). All of these conserved features of the clock were, at least in
part, discovered in N. crassa. In this section,
we review the abovementioned key elements
of the N. crassa core clock mechanism, which
have contributed greatly to the understanding
of the TTFL in other fungi and higher eukaryotes.
A. The Transcriptionally Active Positive Arm
of the Clock
In N. crassa, the circadian cycle is initiated by
the positive arm of the clock, a heterodimeric
GATA-like transcription factor complex
termed the White Collar Complex (WCC) comprising two proteins, WC-1 and WC-2 (Belden
et al. 2007b; Hurley et al. 2014). WCC transcriptional activity peaks around subjective dawn,
commencing the circadian cycle (Belden et al.
2007b; Hurley et al. 2014). WC-1 is a 1167
amino acid (aa) long protein that contains a
circadian transactivation domain, three PerArnt-Sim (PAS) domains (including the lightresponsive LOV domain and a “defective in
binding” (DBD) region involved in DNA binding and mediating interactions with the negative arm), and a single zinc finger domain
involved in DNA binding (Ballario et al. 1996;
Lee et al. 2000; Cheng et al. 2002; Wang et al.
2014, 2016). As mentioned previously in Sect.
II.A, WC-1 is the primary blue-light photoreceptor in N. crassa and is responsible for the
integration of light signaling into the circadian
clock.
WC-2 is a 530 aa-long protein that contains
a single zinc finger domain, a single PAS
domain and, unlike WC-1, is unable to sense
light (Linden and Macino 1997; Collett et al.
2002). WC-1 and WC-2 exhibit strong oscillations at the transcriptional level, but neither
show strong oscillations at the protein level
(Hurley et al. 2018). Interestingly, the negative
arm protein, FRQ, is needed for proper levels of
WC-1 to accumulate (Schafmeier et al. 2006).
WC-1 and WC-2 interact with each other
through their PAS domains to form the
White-Collar Complex (WCC), which promotes
the expression of genes in the negative arm as
well as other genes not involved in maintaining
the core clock, termed clock-controlled genes
(ccgs) (Fig. 4.2a) (Linden and Macino 1997;
Crosthwaite et al. 1997; Cheng et al. 2002; Hurley et al. 2014; Wu et al. 2014).
The WCC exists in two conformations, a
light-activated conformation and a dark conformation, which serve to control the DNA
binding of the WCC. The light-activated complex is composed of two WC-1s and one WC-2,
binding to a subset of genes that are considered
to be light-responsive (Froehlich et al. 2002;
Cheng et al. 2003). In contrast, the dark conformation comprises a single WC-1 and WC-2 and
is essential for the expression of frq and other
ccgs under “free-running” or constant dark
conditions (Froehlich et al. 2002; Cheng et al.
2003). The zinc fingers of both WC-1 and WC2 are essential for binding DNA in the dark,
aided by the DBD motif on WC-1 (Wang et al.
84
M. S. Jankowski et al.