2016). The ability of the WCC to bind DNA is
also dependent on its phosphorylation state, as
has been shown for Drosophila and mouse positive arm CLOCK protein, with daily rhythms in
phosphorylation occurring (He et al. 2005;
Baker et al. 2009; Yoshitane et al. 2009; Lee
et al. 2014; Wang et al. 2019). There are 80
phosphorylation sites on WC-1, with phosphorylations near its zinc-finger domain playing an important role in closing the negative
feedback loop (He et al. 2005; Baker et al. 2009;
Sancar et al. 2009; Wang et al. 2019). WC2 phosphorylation peaks around late subjective
day, with 15 phosphorylation sites detected in
2 different clusters, with 1 cluster near its zinc
finger domain, as in WC-1 (Schafmeier et al.
2005; Wang et al. 2019). While earlier work
suggested that the mutation of 5 phosphorylation sites in WC-1 leads to arrhythmicity, it is
now evident that phosphorylations on both WC1 and WC-2 are needed to close the clock’s
negative feedback loop and support circadian
rhythmicity (He et al. 2005; Wang et al. 2019).
Plainly, both the light-dependent change in
WCC structure and the overall phosphorylation
state of the WCC play an important role in delineating the WCC’s function in the light response
pathway from its role in the positive arm of the
circadian clock (Wang et al. 2016, 2019).
While the WCC is the primary transcriptional activator of the negative arm gene frq,
there are other proteins that influence frq transcription through modifications of the chromatin structure at the frq locus. The frq promoter
has two unique elements, the proximal LRE
(pLRE) and the Clock Box (c-box) (Froehlich
et al. 2003). The heterotrimeric, light-activated
WCC binds to the pLRE in response to light,
but the heterodimeric, dark-active WCC binds
to the c-box to regulate the rhythmic expression of frq that is necessary for proper clock
function in continual darkness (Froehlich et al.
2003; Gooch et al. 2014). This binding is facilitated by chromatin remodelers, including the
positive regulators Clock ATPase (CATP),
SWItch/SucroseNonFermentable (SWI-SNF),
Chromodomain helicase DNA-binding (CHD1), and Defective in methylation (DIM-2), and
the negative regulators Clockswitch (CSW-1)
and Su(var)3–9-enhancer-of-zeste-trithorax-1
and 2 (SET-1 and SET-2) (Fig. 4.2a). CATP
positively regulates frq expression via its promotion of the removal of histones at the frq
locus, allowing for WCC binding (Cha et al.
2013). Once the WCC binds to the c-box, it
recruits the SWI-SNF complex, which removes
a nucleosome that partially covers the c-box
and likely loops the DNA, bringing the WCC
closer to the transcription start site (Zhang
et al. 2006; Wang et al. 2014). CHD-1 also contributes to necessary changes in frq chromatin
structure to aid transcription, and DIM-2catalyzes the removal of transient DNA methylation that helps set clock phase timing
(Belden et al. 2011). The protein CSW-1 is
involved in the final stages of closing the clock’s
feedback loop, compacting the chromatin
structure at the c-box to down-regulate frq
expression (Belden et al. 2007b). SET-1 and
SET-2 further this process by methylating histones H3K4 and H3K36, respectively, making
the chromatin less accessible (Raduwan et al.
2013; Sun et al. 2016). Only when these supporting proteins properly maintain the chromatin
architecture of the frq locus can the WCC complete its positive activation of frq transcription
to maintain rhythmicity. Circadian rhythms in
histone modifications have also been detected
in mammal systems, and chromatin-modifying
proteins are important for the repression of
positive arm clock proteins (Koike et al. 2012;
Takahashi 2017).
B. The Negative Arm of the Clock
1. Oscillations in FRQ Protein and
Phosphorylation Levels Determine Clock
Period
Frequency’s role in determining clock period
was originally discovered through the isolation
of “banding” (bd) mutants with different period
lengths (Aronson et al. 1994; Feldman and
Hoyle 1973; Gardner and Feldman 1980; Loros
et al. 1986). In constant conditions (25
C and
constant darkness), the bd strain displays a
conidial band once every 21.6 (Æ 0.5)
h (Fig. 4.1). However, mutants in the bd strain
were discovered to have periods ranging from
4 From Genetics to Molecular Oscillations: The Circadian Clock in Neurospora crassa
85
also dependent on its phosphorylation state, as
has been shown for Drosophila and mouse positive arm CLOCK protein, with daily rhythms in
phosphorylation occurring (He et al. 2005;
Baker et al. 2009; Yoshitane et al. 2009; Lee
et al. 2014; Wang et al. 2019). There are 80
phosphorylation sites on WC-1, with phosphorylations near its zinc-finger domain playing an important role in closing the negative
feedback loop (He et al. 2005; Baker et al. 2009;
Sancar et al. 2009; Wang et al. 2019). WC2 phosphorylation peaks around late subjective
day, with 15 phosphorylation sites detected in
2 different clusters, with 1 cluster near its zinc
finger domain, as in WC-1 (Schafmeier et al.
2005; Wang et al. 2019). While earlier work
suggested that the mutation of 5 phosphorylation sites in WC-1 leads to arrhythmicity, it is
now evident that phosphorylations on both WC1 and WC-2 are needed to close the clock’s
negative feedback loop and support circadian
rhythmicity (He et al. 2005; Wang et al. 2019).
Plainly, both the light-dependent change in
WCC structure and the overall phosphorylation
state of the WCC play an important role in delineating the WCC’s function in the light response
pathway from its role in the positive arm of the
circadian clock (Wang et al. 2016, 2019).
While the WCC is the primary transcriptional activator of the negative arm gene frq,
there are other proteins that influence frq transcription through modifications of the chromatin structure at the frq locus. The frq promoter
has two unique elements, the proximal LRE
(pLRE) and the Clock Box (c-box) (Froehlich
et al. 2003). The heterotrimeric, light-activated
WCC binds to the pLRE in response to light,
but the heterodimeric, dark-active WCC binds
to the c-box to regulate the rhythmic expression of frq that is necessary for proper clock
function in continual darkness (Froehlich et al.
2003; Gooch et al. 2014). This binding is facilitated by chromatin remodelers, including the
positive regulators Clock ATPase (CATP),
SWItch/SucroseNonFermentable (SWI-SNF),
Chromodomain helicase DNA-binding (CHD1), and Defective in methylation (DIM-2), and
the negative regulators Clockswitch (CSW-1)
and Su(var)3–9-enhancer-of-zeste-trithorax-1
and 2 (SET-1 and SET-2) (Fig. 4.2a). CATP
positively regulates frq expression via its promotion of the removal of histones at the frq
locus, allowing for WCC binding (Cha et al.
2013). Once the WCC binds to the c-box, it
recruits the SWI-SNF complex, which removes
a nucleosome that partially covers the c-box
and likely loops the DNA, bringing the WCC
closer to the transcription start site (Zhang
et al. 2006; Wang et al. 2014). CHD-1 also contributes to necessary changes in frq chromatin
structure to aid transcription, and DIM-2catalyzes the removal of transient DNA methylation that helps set clock phase timing
(Belden et al. 2011). The protein CSW-1 is
involved in the final stages of closing the clock’s
feedback loop, compacting the chromatin
structure at the c-box to down-regulate frq
expression (Belden et al. 2007b). SET-1 and
SET-2 further this process by methylating histones H3K4 and H3K36, respectively, making
the chromatin less accessible (Raduwan et al.
2013; Sun et al. 2016). Only when these supporting proteins properly maintain the chromatin
architecture of the frq locus can the WCC complete its positive activation of frq transcription
to maintain rhythmicity. Circadian rhythms in
histone modifications have also been detected
in mammal systems, and chromatin-modifying
proteins are important for the repression of
positive arm clock proteins (Koike et al. 2012;
Takahashi 2017).
B. The Negative Arm of the Clock
1. Oscillations in FRQ Protein and
Phosphorylation Levels Determine Clock
Period
Frequency’s role in determining clock period
was originally discovered through the isolation
of “banding” (bd) mutants with different period
lengths (Aronson et al. 1994; Feldman and
Hoyle 1973; Gardner and Feldman 1980; Loros
et al. 1986). In constant conditions (25
C and
constant darkness), the bd strain displays a
conidial band once every 21.6 (Æ 0.5)
h (Fig. 4.1). However, mutants in the bd strain
were discovered to have periods ranging from
4 From Genetics to Molecular Oscillations: The Circadian Clock in Neurospora crassa
85