V6 Circadian clocks in Arabidopsis thaliana

SS 2008 lecture 6

Biological Sequence Analysis1

V6 Circadian clocks in Arabidopsis thaliana

Review of lecture V5 ...

SS 2008 lecture 6


Circadian rhythms

McClung Plant Cell 18, 792 (2006)

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.

SS 2008 lecture 6


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)

SS 2008 lecture 6


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)

SS 2008 lecture 6


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.


SS 2008 lecture 6


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.


SS 2008 lecture 6


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 circadian-

regulated.

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)

SS 2008 lecture 6


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.


SS 2008 lecture 6


Synchronized production of photoprotective pigments


„Phenolic sunscreen“

Substances absorb light inthe visible and UV range.

SS 2008 lecture 6


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 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 geneindicated in (A). The chloroplast is bounded by a green box and the cytoplasm by a black box. Harmer et al. Science 290, 2110 (2000)

SS 2008 lecture 6


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)

SS 2008 lecture 6


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.Harmer et al. Science 290, 2110 (2000)

SS 2008 lecture 6


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,” thatoccurs 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 transcriptionalstart 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.

SS 2008 lecture 6


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 tissue-

specific manner, transcriptional activity throughout the day.


SS 2008 lecture 6


Feedback loops control the mammalian circadian core clock

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.


SS 2008 lecture 6


Conservation of Circadian clock mechanisms

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.


Conservation of mechanism in the Drosophila melanogaster andNeurospora crassa circadian clocks. Homologous genes regulate the Drosophila melanogaster and vertebrate clocks, although some details might differ.

SS 2008 lecture 6


The clock mechanism in Neurospora crassa

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.


The white collar complex (WCC) activates the transcription of the frequency (frq) gene. The FRQ protein positively and negatively regulates the WCC.

SS 2008 lecture 6


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.


SS 2008 lecture 6


Multiple roles of CK1 in the mammalian circadian clock

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 ubiquitin-mediated 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.


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.

SS 2008 lecture 6


Circadian rhythm disorders

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.


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.

SS 2008 lecture 6


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)

SS 2008 lecture 6


But: is this all?

Kay & Schroeder Science 318, 1730 (2007)

SS 2008 lecture 6


[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)

SS 2008 lecture 6


(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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V6 Circadian clocks in Arabidopsis thaliana

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