ss 2009lecture 11 biological sequence analysis 1 v11 circadian clocks in mammals and plants mcclung...

SS 2009 lecture 11Biological Sequence Analysis

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V11 Circadian clocks in mammals and plants

McClung Plant Cell 18, 792 (2006)

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

Biological Sequence Analysis2

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 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 property of a general mechanism that buffers the clock

against changes in cellular metabolism.

What effect does temperature usually have on chemical reactions?


SS 2009 lecture 11


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)

SS 2009 lecture 11

SS 2009 lecture 11Biological Sequence Analysis

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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.


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.


SS 2009 lecture 11


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.

Eckel-Mahan & Sassone-Corsi,Nat. Struct. Mol. Biol. 16, 462 (2009)

SS 2009 lecture 11

Paolo Sassone-Corsi,UC Irvine


Basic molecular elements of the mammalian clock

This is the minimal scheme for the

mammalian clock.

It requires several interconnecting

transcriptional, translational and post-

translational loops to achieve gene

expression with circadian periodicitySancar,Nat. Struct. Mol. Biol. 15, 23 (2008)

SS 2009 lecture 11

(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.

(c) The PER and CRY proteins

dimerize, enter the nucleus

and inhibit CLOCK-BMAL1–

activated transcription.


Full (?) circuit of circadian rhythms in mammals

Ko & Takahashi Hum Mol Genet 15, R271 (2006)

SS 2009 lecture 11

CK1: casein kinase

Rev-erb, ROR: retinoic acid-related orphan nuclear receptors

Cdg: clock-controlled gene(s)

PER: period

CRY: cryptochrome


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 2009 lecture 11


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.


SS 2009 lecture 11


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.


SS 2009 lecture 11



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.


SS 2009 lecture 11



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)

SS 2009 lecture 11


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 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 2009 lecture 11


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.

Harmer et al. Science 290, 2110 (2000)SS 2009 lecture 11

3. Lecture WS 2006/07 Bioinformatics III 16

Energy conversion: Photosynthetic Unit

Structure suggested by

force field based

molecular docking.

http://www.ks.uiuc.edu/Research/vmd/gallery


Synchronized production of photoprotective pigments


„Phenolic sunscreen“

Substances absorb light inthe visible and UV range.

SS 2009 lecture 11


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 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 2009 lecture 11


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 ...


SS 2009 lecture 11


Genes implicated in cell elongation are circadian-regulated

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

SS 2009 lecture 11

(B) Proposed mode

of action of the

products of these

clock-controlled

genes in cell wall

remodeling.


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.

SS 2009 lecture 11


Review: 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 2009 lecture 11


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 protein-

1 (PDP1) activates the transcription of Clk.Gallego et al. Nat.Rev.Mol.Cell.Biol. 8, 140 (2007)

SS 2009 lecture 11


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 2009 lecture 11


Multiple roles of CK1 in the mammalian circadian clock

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 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 (here

PER1). In some cell types, CKI

activity promotes the cytoplasmic

accumulation of PER1, whereas in

others it mediates the nuclear

translocation of PER1.

SS 2009 lecture 11


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)

SS 2009 lecture 11


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)

SS 2009 lecture 11


additional slides (not used)

SS 2009 lecture 11


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 2009 lecture 11


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.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.

SS 2009 lecture 11

Homologous genes regulate the

Drosophila melanogaster and vertebrate

(Neurospora crassa ) clocks, although

some details might differ.

ss 2009lecture 11 biological sequence analysis 1 v11 circadian clocks in mammals and plants mcclung...

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