Transcript to get the file - Chair of Computational Biology

V6 Circadian clocks in Arabidopsis thaliana
Review of lecture V5 ...
Biological Sequence Analysis
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Circadian rhythms
Period : time to complete one cycle.
Phase : time of day for any given event. E.g. if the peak in a rhythm occurred at
dawn, the phase of the peak would be defined as 0 h. Phase is often defined in
zeitgeber time (ZT). Zeitgeber is German for time giver, and any stimulus that
imparts time information to the clock is a zeitgeber. The onset of light is a powerful
zeitgeber, and dawn is defined as ZT0.
Amplitude of the rhythm : one-half the peak-to-trough distance.
McClung Plant Cell 18, 792 (2006)
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Circadian rhythms
(1) Circadian rhythms are the subset of biological rhythms with period of 24 h.
The term circadian combines the Latin words ‘‘circa’’ (about) and ‘‘dies’’ (day).
(2) Circadian rhythms are endogenously generated and self-sustaining, so they
persist under constant environmental conditions, typically constant light (or dark)
and constant temperature.
Under these controlled conditions, the free-running period of 24 h is observed.
(3) For all circadian rhythms the period remains relatively constant over a range of
ambient temperatures. This is thought to be one facet of a general mechanism
that buffers the clock against changes in cellular metabolism.
What effect does temperature usually have on chemical reactions?
McClung Plant Cell 18, 792 (2006)
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Circadian clocks in Arabidopsis thaliana
Plants were the first organisms for which the observation of a circadian rhythm
was published (de Mairan, 1729).
The molecular study of plant clocks began in 1985 with the observation that the
mRNA abundance of the light-harvesting chlorophyll a/b-binding protein
genes (LHCB) of peas oscillated with a circadian rhythm.
This is still the most extensively studied clock-regulated gene in Arabidopsis.
Salomé et al. J. Biol. Rhythms 19, 425 (2004)
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Key players in Arabidopsis thaliana
LHCB transcription is induced by light and shows a circadian pattern of
expression with a peak in the middle of the subjective day.
The red-light photoreceptors, the phytochromes, mediate the light induction
ofLHCB through a motif in the LHCB promoter.
Minimal promoter fragments necessary and sufficient for light and circadian
regulation of LHCB were identified.
Tobin’s group identified a protein with affinity to this promoter fragment, CCA1
for CIRCADIAN CLOCK ASSOCIATED 1.
LATE ELONGATED HYPOCOTYL (LHY) is another gene encoding a single
Mybdomain protein closely related to CCA1.
Salomé et al. J. Biol. Rhythms 19, 425 (2004)
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Model of the Arabidopsis thaliana oscillator
Light perceived by the PHYs and CRYs induces the
expression of 2 transcription factors, CCA1 and LHY.
CCA1 and LHY mRNA abundance peaks shortly after
dawn. CCA1 requires phosphorylation by CK2 prior to
binding to DNA. PRR9, PRR7, PRR5, andPRR3 show
clock-regulated mRNA abundances, peaking in that
sequence at 2-h intervals throughout the day.
One known target of the repressive activity of CCA1and
LHY is TOC1, with the result that TOC1 (RRR1) mRNA
abundance peaks around dusk, following the turnover of
CCA1andLHY proteins.TOC1 then feeds back onto
CCA1 and LHY and induces their expression for the
next cycle. TOC1 may require a DNA-binding protein as
a cofactor, as it is not predicted to directly bind to DNA.
TOC1 degradation is mediated by the F-box protein
ZTL, whose activity is negatively regulated by light.
CCA1 and LHY also negatively regulate their own
promoters, possibly directly but possibly indirectly via
TOC1.
Light resetting may involve induction of CCA1 and LHY,
possibly mediated through phytochrome and
cryptochrome photoreceptors and PIFand PIF-like (PIL)
transcription factors.
Salomé et al. J. Biol. Rhythms 19, 425 (2004)
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Probe gene expression by microarrays
Harmer et al. used oligonucleotide-based arrays to determine steady-state
mRNA levels in Arabidopsis at 4-hour intervals during the subjective day and
night.
 identify temporal patterns of gene expression in Arabidopsis plants under
constant light conditions using GeneChip arrays representing about 8200
different genes.
This is done by scoring genes with a greater than 95% probable correlation with
a cosine test wave with a period between 20 and 28 hours were as circadianregulated.
How is this done? Give formula ...
 453 genes (6% of the genes on the chip) were classified as cycling.
Harmer et al. Science 290, 2110 (2000)
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Photosynthesis genes peak near the middle of the day
Results after normalization of peak maximum.
(A) LHCA genes are in blue;. LHCB genes are in pink;.
(B) Photosystem I genes are in red;. Photosystem II genes are in green;.
(C) Model for function of photosynthesis gene products in photosystems II (left)
and I (right). Colors of proteins match colors of corresponding gene traces.
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Harmer et al. Science
290, 2110 (2000)
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Synchronized production of photoprotective pigments
„Phenolic sunscreen“
Substances absorb light in
the visible and UV range.
Harmer et al. Science 290, 2110 (2000)
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Circadian regulation of sugar metabolism
Genes encoding starch-mobilizing enzymes peak during the subjective night.
(A) Cycling genes encode a putative starch kinase that is related to potato R1
protein (dark blue); a b-amylase (gold); putative fructose-bisphosphate aldolase,
plastidic form, and putative fructose-bisphosphate aldolase, predicted to be
plastidic (red); a putative sugar transporter (light blue); and a sucrosephosphate synthase homolog (green).
(B) Model for the enzymatic functions of these gene products in the mobilization
of starch. Colored arrows indicate the function of the corresponding gene
indicated in (A). The chloroplast is bounded by a green box and the cytoplasm
by a black box.
Harmer et al. Science 290, 2110 (2000)
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Chilling resistance
Chilling resistance is an important trait in plants.
We found that a number of enzymes involved in lipid modification, including two
desaturases, were under clock regulation and peaked near subjective dusk.
This is consistent with previously observed rhythms in membrane lipid
desaturation levels that correlate with increased resistance to cold treatments
during the subjective night.
Speculate about mechanism ...
Gallego et al. Nat.Rev.Mol.Cell.Biol. 8, 140 (2007)
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Genes implicated in cell elongation are circadian-regulated
The rigid plant cell wall normally prevents cell expansion, but a simultaneous
loosing of cell wall components, uptake of water, and synthesis of cell wall
components seems allowed.
(A) Genes encoding the auxin efflux carriers PIN3 and PIN7 (red), a putative
expansin (green), a putative polygalacturonase (light blue), and aquaporin d-TIP
(dark blue) all peak toward the end of the subjective day. 3 enzymes implicated
in cell wall synthesis, all in gold, peak toward the end of the subjective night.
(B) Proposed mode of action of the products of these clock-controlled genes in
cell wall remodeling.
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Harmer et al. Science 290, 2110 (2000)
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Master regulator sequence of circadian-regulated genes?
Survey of genomic DNA regions upstream of
cycling genes for overrepresented promoter
elements  absolutely conserved motif,
AAAATATCT “evening element,” that
occurs 46 times in the promoters of 31
cycling genes. All genes demonstrated
impressive coregulation. All but one peak
toward the end of the subjective day.
Mutation of the conserved AAAATATCT, but not a closely
related motif, greatly reduced the ability of a promoter to
confer circadian rhythmicity on a luciferase reporter gene in
plants. Fusions to the luciferase gene consisted of 450 bp
(WT450), 130 bp ( WT130, mut130_1, mut130_2,
mut130_1,2), and 85 bp ( WT85) of the CCR2 promoter
upstream of the putative transcriptional
start site. Site 1 was replaced by gagcagctgc in constructs
mut130_1 and mut130_1,2; site 2 was replaced by
gagcagctgc in constructs mut130_2 and mut130_1,2.
Harmer et al. Science 290, 2110 (2000)
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Essential elements of biological clocks
Our biological clocks contain three essential elements:
- a central oscillator that keeps time;
- the ability to sense time cues in the environment and to reset the clock as the
seasons change; and
- a series of outputs tied to distinct phases of the oscillator that regulate activity
and physiology.
In mammals, the central clock resides in the suprachiasmatic nucleus (SCN),
which produces a rhythmic output that consists of a multitude of neural and
hormonal signals that influence sleep and activity.
Most importantly, these signals set the peripheral clocks present throughout the
body. The SCN clock is reset by external light, which is sensed by the ganglion
cells of the retina.
Remarkably, circadian oscillators are also present in all tissues of the body,
where they are synchronized by unidentified signals to regulate, in a tissuespecific manner, transcriptional activity throughout the day.
Gallego et al. Nat.Rev.Mol.Cell.Biol. 8, 140 (2007)
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Feedback loops control the mammalian circadian core
clock
Gallego et al.
Nat.Rev.Mol.Cell.Biol.
8, 140 (2007)
The mammalian circadian rhythms core clock is a transcription–translation negative-feedback loop with a delay
between transcription and the negative feedback. It is initiated by a heterodimeric transcription factor that
consists of CLOCK and BMAL1. CLOCK and BMAL1 drive expression of their own negative regulators, the
period proteins PER1 and PER2 and the cryptochromes CRY1 and CRY2. Over the course of the day, the
PER and CRY proteins accumulate and multimerize in the cytoplasm, where they are phosphorylated by casein
kinase Iε (CKIε) and glycogen synthase kinase-3 (GSK3). They then translocate to the nucleus in a
phosphorylation-regulated manner where they interact with the CLOCK–BMAL1 complex to repress their own
activator. At the end of the circadian cycle, the PER and CRY proteins are degraded in a CKI-dependent manner,
which releases the repression of the transcription and allows the next cycle to start. An additional stabilizing
feedback loop, which involves the activator Rora and the inhibitor Rev-Erbα, controls BMAL1 expression and
reinforces the oscillations. RRE, R-response element.
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Conservation of Circadian clock mechanisms
Conservation of mechanism in the
Drosophila melanogaster and
Neurospora crassa circadian clocks.
Homologous genes regulate the
Drosophila melanogaster and
vertebrate clocks, although some
details might differ.
Clock (CLK) and cycle (CYC) activate the
transcription of the circadian genes in D.
melanogaster.
Period (PER) and timeless (TIM) form
heterodimers in the cytoplasm where they
are phosphorylated by double-time (DBT)
and shaggy (SGG).
They then translocate to the nucleus where
PER inhibits the transcriptional activity of the
CLK–CYC complex. Similarly to the
mammalian clock, a number of kinases
regulate PER and TIM.
In the stabilizing loop, the protein vrille (VRI)
inhibits, whereas PAR-domain protein-1
(PDP1) activates the transcription of Clk.
Gallego et al. Nat.Rev.Mol.Cell.Biol. 8, 140 (2007)
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The clock mechanism in Neurospora crassa
Gallego et al.
Nat.Rev.Mol.Cell.Biol.
8, 140 (2007)
The white collar complex (WCC)
activates the transcription of the
frequency (frq) gene.
The FRQ protein positively and
negatively regulates the WCC.
In the morning, FRQ interacting RNA helicase (FRH) and casein kinases I (CKI)
and CKII promote the FRQ dependent phosphorylation and inactivation of the WCC,
which results in the inhibition of frq transcription.
In the evening, high amounts of hyperphosphorylated FRQ in the cytoplasm support
the accumulation of WCC.
At night, hyperphosphorylated FRQ is degraded, the repression on WCC is relieved
and transcription of frq is activated.
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Why add phosphorylation to the clock?
Why are post-transcriptional modifications of crucial importance?
Transcription–translation feedback cycles generally operate on a timescale of up
to a few hours. If, following synthesis, the repressor proteins PER and CRY
translocated to the nucleus to repress CLOCK and BMAL1, the whole cycle
would take just a few hours rather than one day.
To maintain the daily oscillations of clock proteins, a significant delay between
the activation and repression of transcription is required. This is ensured by
regulation through post-translational modifications.
Reversible phosphorylation regulates important processes such as nuclear entry,
formation of protein complexes and protein degradation. Each of these can
individually contribute to introduce the delay that keeps the period at ~24 hours.
Gallego et al.
Nat.Rev.Mol.Cell.Biol.
8, 140 (2007)
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Multiple roles of CK1 in the mammalian circadian clock
Casein kinase I (CKI) has many roles
in the circadian clock. a | It has a
confusing role in regulating the
nuclear localization of the circadian
repression protein period (in this
example, PER1). In some cell types,
CKI activity promotes the cytoplasmic
accumulation of PER1, whereas in
others it mediates the nuclear
translocation of PER1.
b | Time-course studies have shown that the phosphorylation of PER proteins increases over the
course of the circadian day, peaking when the repression of the positive transcription factors CLOCK
and BMAL1 is maximal. Mapping studies indicate that there are many CKI sites on PER proteins, but
the function of only a subset of these sites is known.
c | One clear function of the phosphorylation of PER proteins is the regulation of protein stability.
Phosphorylation of one or two distinct sites on PER1 and PER2 target these proteins for ubiquitinmediated degradation by the 26S proteasome. Degradation of PER proteins can reset the clock,
allowing the CLOCK–BMAL1 complex to become active.
d | PER and CRY proteins are not the only substrates of CKI in the clock. CKIε-mediated
phosphorylation of the circadian regulator BMAL1 increases its transcriptional activity.
Gallego et al. Nat.Rev.Mol.Cell.Biol. 8, 140 (2007)
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Circadian rhythm disorders
Delayed sleep-phase syndrome
(DSPS)
Opposite to FASPS, DSPS causes late
sleep-onset and the inability to wake
up at a conventional time. A
polymorphism in the human PER3
gene (V647G) has been linked to the
pathogenesis of DSPS111. Residue
647 locates in a region similar to the
CKIε-binding region of PER1 and
PER2, close to the serine residue in
PER2 that is disrupted by the FASPS
mutation. Therefore, this polymorphism
might alter the CKIε-dependent
phosphorylation of human PER3.
Familial advanced sleep-phase syndrome (FASPS)
FASPS is a autosomal dominant human behavioural disorder that causes early sleep times,
early morning awakening and a short circadian period. Genetic analysis in one family affected
by FASPS identified a single amino-acid missense mutation in the human period-2 (PER2)
gene as the cause of that sleep disorder variation. The mutation, an S662G change, is in the
casein kinase Iε (CKIε)-binding domain of PER2 and decreases PER2 phosphorylation in vitro.
Another Thr to Ala mutation in the human CKIδ gene was found in a family with FASPS. The
mutation decreases the enzymatic activity of the kinase in vitro.
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Gallego et al.
Nat.Rev.Mol.Cell.Biol. 8, 140 (2007)20
Summary
Most organisms enhance fitness by coordinating their development with daily
environmental changes through molecular timekeepers known as circadian
clocks.
Clocks are generated by a transcription-translation negative feedback loop with a
crucial delay between stimulus and response.
This system of multiple connected loops increases the clock’s robustness and
provides numerous points of input and output to the clock.
Many metabolic pathways are regulated by circadian clocks in plants and
animals.
Kay & Schroeder Science 318, 1730 (2007)
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But: is this all?
Kay & Schroeder Science 318, 1730 (2007)
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[cADPR] oscillates with a circadian rhythm and a cADPR signaling antagonist
inhibits circadian [Ca2+]cyt oscillations.
(A) [cADPR] during 48 hours of constant light in Col-0 wild type and arrhythmic
CCA1-ox.
Dodd et al. Science 318, 1789 (2007)
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(C and D) Circadian [Ca2+]cyt oscillations in 11-day-old seedlings dosed every 3
hours with
(C) nicotinamide, mannitol (osmotic control), or water;
(D) GdCl3 and U73122.
Dodd et al. Science 318, 1789 (2007)
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