Transcript Computational Biology

V11 Circadian clocks in mammals and plants
Most organisms (animals, plants, fungi and cyanobacteria) enhance their fitness by
coordinating their development with daily environmental changes through
molecular timekeepers (circadian clocks)
Mammals display circadian rhythms in behavioural and physiological processes,
such as
- sleep
- feeding
- blood pressure and
- metabolism
Roles in plants e.g.:
- opening of flowers in the morning and their closure at night
Circadian rhythms are guided by external light–dark signals that are integrated
through intrinsic central and peripheral molecular clocks
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.
They persist under constant environmental conditions, typically constant light (or
dark) and constant temperature. Under these controlled conditions, the freerunning 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 property 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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Essential elements of biological clocks
Our biological clocks contain 3 essential elements:
(1) a central oscillator that keeps time;
(2) the ability to sense time cues in the environment and to reset the clock as
the seasons change; and
(3) a series of outputs tied to distinct phases of the oscillator that regulate
activity and physiology.
Gallego et al. Nat.Rev.Mol.Cell.Biol. 8, 140 (2007)
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Parameters of Circadian clocks
Period : time to complete one
cycle.
Amplitude of the rhythm :
one-half the peak-to-trough
distance.
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.
McClung Plant Cell 18, 792 (2006)
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Suprachiasmatic nucleus (SCN)
In mammals, the central clock resides in the suprachiasmatic nucleus (SCN),
a small region of the brain that contains ca. 20,000 neurons.
The SCN produces a rhythmic output that consists of a multitude of neural
and hormonal signals that influence sleep and activity.
Most importantly, the SCN 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.
Gallego et al. Nat.Rev.Mol.Cell.Biol. 8, 140 (2007)
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Autonomous oscillators everywhere
Remarkably, autonomous circadian oscillators are also
present in all tissues of the body,
where they are synchronized by unidentified signals to
regulate, in a tissue-specific manner, transcriptional
activity throughout the day.
Paolo Sassone-Corsi,
UC Irvine
Eckel-Mahan & Sassone-Corsi,
Nat. Struct. Mol. Biol. 16, 462 (2009)
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Basic molecular elements of the mammalian clock
(a) 2 TFs CLOCK and BMAL1
heterodimerize.
(b) BMA1:CLOCK binds to the
E-boxes in the promoters of
the PER and CRY genes, as
well as in the clock-controlled
genes, activating their
transcription.
This is the minimal scheme for the
mammalian clock.
It requires several interconnecting
transcriptional, translational and posttranslational loops to achieve gene
expression with circadian periodicity
(c) The PER and CRY proteins
dimerize, enter the nucleus
and inhibit CLOCK-BMAL1–
activated transcription.
Sancar,
Nat. Struct. Mol. Biol. 15, 23 (2008)
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Full (?) circuit of circadian rhythms in mammals
PER: period
CRY: cryptochrome
CK1: casein kinase
Rev-erb, ROR: retinoic acidrelated orphan nuclear receptors
Cdg: clock-controlled gene(s)
Ko & Takahashi Hum Mol Genet 15, R271 (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 (PHY), mediate the light
induction of LHCB through a motif in the LHCB promoter.
Comment: LHs absorb maximally at 850 nm (red light).
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.
This TF was named CCA1 for CIRCADIAN CLOCK ASSOCIATED 1.
LATE ELONGATED HYPOCOTYL (LHY) is another gene encoding a 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, and PRR3
show clock-regulated mRNA
abundances, peaking in that
sequence at 2-h intervals throughout
the day.
Salomé et al. J. Biol. Rhythms 19, 425 (2004)
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Model of the Arabidopsis thaliana oscillator
One known target of the repressive
activity of CCA1 and LHY is TOC1
(Timing of Cab Expression 1), with
the result that TOC1 (RRR1) mRNA
abundance peaks around dusk,
following the turnover of CCA1 and
LHY 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.
Salomé et al. J. Biol. Rhythms 19, 425 (2004)
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Model of the Arabidopsis thaliana oscillator
TOC1 degradation is mediated by
the F-box protein ZTL (Zeitlupe =
slow motion), 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
PIF and PIF-like (PIL) transcription
factors.
Salomé et al. J. Biol. Rhythms 19, 425 (2004)
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Detect unknown control mechanisms:
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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Energy conversion: Photosynthetic Unit
Structure suggested by
force field based
molecular docking.
http://www.ks.uiuc.edu/Research/vmd/gallery
3. Lecture WS 2006/07
Bioinformatics III
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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. Why?
Plant stores starch in chloroplast for use during the night when the plant cannot do
photosynthesis.
(A) Cycling genes encode a putative starch kinase that is related to potato R1
protein (dark blue); a β-amylase (gold); fructose-bisphosphate aldolase, (red); a
putative sugar transporter (light blue); and a sucrose-phosphate 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.
A number of enzymes involved in lipid modification, including two
desaturases, were found to be 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
(B) Proposed mode
of action of the
products of these
clock-controlled
genes in cell wall
remodeling.
The rigid plant cell wall normally prevents cell expansion, but a simultaneous
loosening 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. Auxins are phytohormones – they
regulate cell extension.
3 enzymes implicated in cell wall synthesis (all in gold) peak toward the end of the
subjective night.
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Harmer et al. Science 290, 2110 (2000)
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Master regulator sequence of circadian-regulated genes?
Check 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.
Harmer et al. Science 290, 2110 (2000)
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Review: 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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Circadian clock in D. melanogaster
(1) Clock (CLK) and cycle (CYC) activate
the transcription of the circadian genes in D.
melanogaster.
(2) Period (PER) and timeless (TIM) form
heterodimers in the cytoplasm where they
are phosphorylated by double-time (DBT)
and shaggy (SGG).
(3) PER and TIM then translocate to the
nucleus where PER inhibits the
transcriptional activity of the CLK–CYC
complex.
(4) Similarly to the mammalian clock, a
number of kinases regulate PER and TIM.
(5) In the stabilizing loop, the protein vrille
(VRI) inhibits, whereas PAR-domain protein1 (PDP1) activates the transcription of Clk.
Gallego et al. Nat.Rev.Mol.Cell.Biol. 8, 140 (2007)
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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 (here
PER1). In some cell types, CKI
activity promotes the cytoplasmic
accumulation of PER1, whereas in
others it mediates the nuclear
translocation of PER1.
b | 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. 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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New: Dual roles of CLOCK acetyltransferase activity
CLOCK acetylates (Ac) histones H3 and H4 in nucleosomes (green) to confer
‘open’ chromatin structure and enable CLOCK-BMAL1 to bind to the E-boxes in
cognate promoters and turn on transcription.
CLOCK also acetylates BMAL1, making it a target for binding of the CRY
repressor, concomitant with deacetylation of histones by histone deacetylases
(HDAC). These dual effects of acetylation by CLOCK contribute to circadian
periodicity of gene expression.
Sancar,
Nat. Struct. Mol. Biol. 15, 23 (2008)
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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.
V12: Coupling of circadian clocks and metabolism!
Kay & Schroeder Science 318, 1730 (2007)
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additional slides (not used)
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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)29
The clock mechanism in Neurospora crassa
The white collar complex (WCC)
activates the transcription of the
frequency (frq) gene.
The FRQ protein positively and
negatively regulates the WCC.
Homologous genes regulate the Drosophila
melanogaster and vertebrate (Neurospora
crassa ) clocks, although some details
might differ.
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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Gallego et al.
Nat.Rev.Mol.Cell.Biol.
8, 140 (2007)
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