IV. Output: Clock Output Regulates
Cellular Physiology at the
Transcriptional and Posttranscriptional Levels
The core circadian oscillator affects many processes within the cell via transcriptional and
post-transcriptional regulation, termed the
clock’s “output” (Vitalini et al. 2006; Hurley
et al. 2014, 2018; Sancar et al. 2015). Regulation
occurs at multiple levels, including the regulation of transcriptional cascades by the positive
arm of the clock and the more recent evidence
of post-transcriptional regulation of protein
levels (Smith et al. 2010; Hurley et al. 2014,
2018). For the purposes of this review, we will
discuss output in two sections: transcriptional
output and post-transcriptional output.
A. Transcriptional Regulation of Cellular
Output via the Circadian Clock
1. Circadian Transcriptional Activation of
Clock-Controlled Genes (ccgs)
The positive arm of the TTFL in animal and
fungal clocks acts as a “pioneer-like” transcription factor to remodel chromatin and activate
the transcription of the negative arm as well as a
host of other genes, including other transcription factors (Hurley et al. 2014; Menet et al.
2014; Smith et al. 2010). Chromatinimmunoprecipitation (ChIP) sequencing identified that the WCC directly targets ~300 genes
near circadian dawn (Smith et al. 2010; Hurley
et al. 2014). Included in these WCC targets are
additional transcription factors that regulate
further groups of genes, creating a circadian
transcription factor cascade. Among the
detected WCC targets, a total of 28 transcription factors were found, including ccg-9, sah-1,
nit-2, hsf-2, adv-1, bek-1, sub-1, sah-2, sre, mip1, and vad-2 (Smith et al. 2010). The binding
motif for the WCC is “GATCGA,” a sequence
that is also found in the c-box and LRE of the
frq promoter (Froehlich et al. 2002; Pavesi et al.
2004; He and Liu 2005b; Carlson et al. 2007;
Smith et al. 2010).
Deep sequencing has shown that the transcriptional regulation stemming from the positive arm leads to up to 40% of genes within N.
crassa undergoing rhythmic changes in transcript levels under constant conditions; these
genes are termed clock-controlled genes or ccgs
(Fig. 4.2a) (Hurley et al. 2014; Loros et al. 1989;
Sancar et al. 2015). When the promoter
sequences of all detected ccgs were analyzed,
four motifs were found to be significantly
enriched at certain times of day: STACASTA,
GCRCTAAC, GRCGGGA, and GVCAGCCA,
with each motif associated with essential cellular processes (Hurley et al. 2014). It is important to note that the set of ccgs varies to some
extent depending on what growth media is
used, showing that the clock can adjust its output to current metabolic conditions (Hurley
et al. 2014). The extent and diversity of ccgs
highlights the broad effect of circadian regulation in the cell and how the clock can fine-tune
cellular output based on its metabolic environment. The diversity of output is also seen in
higher eukaryotes, where as much as 80% of
genes fell under circadian regulation when a
wide variety of tissues were analyzed (Mure
et al. 2018).
2. Circadian Transcriptional Repression of ccgs
Is Vital to Clock Output
While the positive arm is generally considered a
transcriptional activator, some of its direct or
indirect targets are known transcriptional
repressors. We have already reviewed some of
these transcriptional repressors in the context
of input signals to the clock (see Sect. II for
discussions of VVD, CSP-1, RCO-1, and RCM1). In addition to being a clock nutrient sensor
and WC-1 transcriptional repressor, CSP-1 is
also a direct target of the WCC and is expressed
in a rhythmic fashion (Lambreghts et al. 2009;
Smith et al. 2010). Forming a complex with
RCO-1 and RCM-1, it modulates the expression
of ~800, mostly evening-specific, genes in N.
crassa that are predominantly involved in the
metabolism of lipids and glucose (Sancar et al.
2011; see Sect. II). It is predicted that CSP-1
regulates evening-specific gene expression by
4 From Genetics to Molecular Oscillations: The Circadian Clock in Neurospora crassa
91
Cellular Physiology at the
Transcriptional and Posttranscriptional Levels
The core circadian oscillator affects many processes within the cell via transcriptional and
post-transcriptional regulation, termed the
clock’s “output” (Vitalini et al. 2006; Hurley
et al. 2014, 2018; Sancar et al. 2015). Regulation
occurs at multiple levels, including the regulation of transcriptional cascades by the positive
arm of the clock and the more recent evidence
of post-transcriptional regulation of protein
levels (Smith et al. 2010; Hurley et al. 2014,
2018). For the purposes of this review, we will
discuss output in two sections: transcriptional
output and post-transcriptional output.
A. Transcriptional Regulation of Cellular
Output via the Circadian Clock
1. Circadian Transcriptional Activation of
Clock-Controlled Genes (ccgs)
The positive arm of the TTFL in animal and
fungal clocks acts as a “pioneer-like” transcription factor to remodel chromatin and activate
the transcription of the negative arm as well as a
host of other genes, including other transcription factors (Hurley et al. 2014; Menet et al.
2014; Smith et al. 2010). Chromatinimmunoprecipitation (ChIP) sequencing identified that the WCC directly targets ~300 genes
near circadian dawn (Smith et al. 2010; Hurley
et al. 2014). Included in these WCC targets are
additional transcription factors that regulate
further groups of genes, creating a circadian
transcription factor cascade. Among the
detected WCC targets, a total of 28 transcription factors were found, including ccg-9, sah-1,
nit-2, hsf-2, adv-1, bek-1, sub-1, sah-2, sre, mip1, and vad-2 (Smith et al. 2010). The binding
motif for the WCC is “GATCGA,” a sequence
that is also found in the c-box and LRE of the
frq promoter (Froehlich et al. 2002; Pavesi et al.
2004; He and Liu 2005b; Carlson et al. 2007;
Smith et al. 2010).
Deep sequencing has shown that the transcriptional regulation stemming from the positive arm leads to up to 40% of genes within N.
crassa undergoing rhythmic changes in transcript levels under constant conditions; these
genes are termed clock-controlled genes or ccgs
(Fig. 4.2a) (Hurley et al. 2014; Loros et al. 1989;
Sancar et al. 2015). When the promoter
sequences of all detected ccgs were analyzed,
four motifs were found to be significantly
enriched at certain times of day: STACASTA,
GCRCTAAC, GRCGGGA, and GVCAGCCA,
with each motif associated with essential cellular processes (Hurley et al. 2014). It is important to note that the set of ccgs varies to some
extent depending on what growth media is
used, showing that the clock can adjust its output to current metabolic conditions (Hurley
et al. 2014). The extent and diversity of ccgs
highlights the broad effect of circadian regulation in the cell and how the clock can fine-tune
cellular output based on its metabolic environment. The diversity of output is also seen in
higher eukaryotes, where as much as 80% of
genes fell under circadian regulation when a
wide variety of tissues were analyzed (Mure
et al. 2018).
2. Circadian Transcriptional Repression of ccgs
Is Vital to Clock Output
While the positive arm is generally considered a
transcriptional activator, some of its direct or
indirect targets are known transcriptional
repressors. We have already reviewed some of
these transcriptional repressors in the context
of input signals to the clock (see Sect. II for
discussions of VVD, CSP-1, RCO-1, and RCM1). In addition to being a clock nutrient sensor
and WC-1 transcriptional repressor, CSP-1 is
also a direct target of the WCC and is expressed
in a rhythmic fashion (Lambreghts et al. 2009;
Smith et al. 2010). Forming a complex with
RCO-1 and RCM-1, it modulates the expression
of ~800, mostly evening-specific, genes in N.
crassa that are predominantly involved in the
metabolism of lipids and glucose (Sancar et al.
2011; see Sect. II). It is predicted that CSP-1
regulates evening-specific gene expression by
4 From Genetics to Molecular Oscillations: The Circadian Clock in Neurospora crassa
91