Circadian Rhythm -...

How Does a Circadian Clock Work?

Dr. Jay Dunlap, Dartmouth (KITP Bio Networks 3/24/03) 1

Circadian Rhythm

- freerunning, sustainable oscillation with an approximately 24 hour period

- entrainment to daily environmental changes

- universally associated with light and temperature perception

- period length of rhythm is compensated against changes in temperature or metabolic state

- clock operates within defined physiological limits

0 12 0 12 24

Sound recorded from fourCastor canadensis

March 18 - April 22, 1971115°03'W, 51°02'N

.

EUKARYOTAFUNGI

Sponges

ANIMALIA

CiliatesBrown AlgaeDiatoms Protista

Giardia lamblia

Vairimorpha necatrix

Trichomonas foetus

Physarum polycephalumEuglenoids

KinetoplastidsBodonids

Amoebamastigote

Entamoebae invadens

Escherichia coli

CyanobacteriaPlant Chloroplasts Mycoplasma

capricolum

Pseudomonas testosteroniPlant Mitochondria

Agrobacteriumtumefaciens

Dinoflagellates

Dictyostelium discoideumRed Algae

EUBACTERIA

ARCHAEBACTERIA

Mammals-mouse, human

Neurospora

ArabidopsisChlamydomonas

ANIMALIAGonyaulax

Ciliates

TetrahymenaParamecium

Brown Algae

Dinoflagellates

FUNGI

PLANTAE

Insects - Antheraea,Drosophila

Diatoms

Synechococcus

PLANTAE

Circadian Systems in the Universal Tree of Life



WHITE COLLAR-1 & WC-2 in NeurosporaCLK & CYC in Drosophila

CLOCK & BMAL1 in mammals

Negative elements in circadian loops:

FREQUENCY in Neurospora

PERIOD and TIMELESS in Drosophila

PER1, 2, and 3 , CRY1 and CRY2 in mammals

Positive elements in circadian loops:

"Positive Element(s)"

"Negative Element(s)"

output - clock controlled genes

clock gene(s)

rhythmicbiochemistry andphysiology

promoting expression

blocking activity

Common Themes and Elements in Eukaryotic Circadian Feedback Loops

WHITE COLLAR-1 & WC-2 in NeurosporaCLK & CYC in DrosophilaCLOCK & BMAL1 in mammals

Negative elements in eukaryotic circadian loops:

FREQUENCYin NeurosporaPERIOD and TIMELESS in DrosophilaPER1, PER2, PER3 , CRY2, and CRY2in mammals

Positive elements in eukaryotic circadian loops:

output - clock controlled genes rhythmicbiochemistry,physiology,or behavior

Common themes:Transcription/translation based feedback loops, with transcription factors as positive elements

and proteins that block the activity of these factors as negative elements, are central.Rhythmic expression is seen in transcripts and proteins corresponding to the "negative elements.""Positive elements" are heterodimers comprised of two proteins interacting via PAS domains.

Of proteins comprising the heterodimer, one is constitutive and one is rhythmically expressed.Extended sequence similarity links some of the positive elements among all of the systems.At least one of the "negative elements" has a dual role in the cycle, performing both the negative function

in feedback and also a positive function in promoting the expression of one of the heterodimer partners.

clock gene(s)"Negative Element(s)"

blocking activity"Positive Element(s)"

promoting expression



Molecular components of the Neurospora clock

frq

wc-2

wc-1vvd

ccg’s

WC-1

FRQ

FRQ

FRQkinases:CK-1αα, Ck-1ββ,CAMK-1, CKII

turnover

FRQ

FRQ

P

P

P

P

P P

P

-

+

+

+

+

FRQfrq

nightnightday

WC-2

WC-1

WC-2WC-1

WC-2WC-1

nucleus

•WC-1 and WC-2 act positively on the production of frq transcript.

•FRQ acts negatively on the production of its own transcript. (negative feedback loop)

•FRQ acts positively on the production of wc-2 transcript and WC-1. (positive feedback loops)

•FRQ becomes phosphorylated which leads to its turnover

-

•The frq/FRQ oscillations are essential for the generation of all circadian rhythms in Neurospora.

•Changes in frq/FRQ levels are directly translated into changes in the circadian rhythms in Neurospora.

Cry1

Cry2

PER1

LIGHT

Per1Per2

ccg's

Clock-controlled processes

SCN neuronal activity rhythmsneuropeptide release

overt rhythms in the organism

Per3

PER3PER2

phosphorylation &

turnover ?

complexformation

Nucleus

PERyPERx::(andothers)

BCCBMAL1::CLOCK

Cell

mammalian neuron

Cry1

Cry2

Cry1ccg's

Clock-controlled processes:stress responsesdevelopmental regulationintermediary metabolism

LIGHT

frqkinase

turnover

Nucleus

FRQPP

processing

Cell

Neurospora

wc-1

vvd

FRQWCC

WCC

translation

WCC

wc-2

PP

FRQproteins

Cry2

bmal

two eukaryotic circadian oscillators



Circadian rhythm

•Biological rhythm with a period of ~24 hours•Endogenous and self-sustaining under constant environmental conditions•Temperature compensated within the physiological range of the organism •Environmental signals (light and temperature changes) can entrain or reset the rhythm•Organized at the cellular level

Van D. GoochUniversity of Minnesota

A Circadian Rhythm in Development on Racetubes

PRC movie

ENTRAINMENTLight or temperature treatment results in changes in the timing of conidial banding.

Van D. GoochUniversity of Minnesota

QuickTime™ and aSorenson Video decompressorare needed to see this picture.



WC-1

Time

FRQproteins

ccg's


caseinkinase II

turnover

NucleusPP

FRQPP

processing

Cell

wc-1

vvd

FRQWCC

WCC

translation

WCC

wc-2

Light

frq

Molecular Components of theNeurospora Clock

FRQFRQfrqfrq

frq +

mRNA

203

118FRQ

11 18 25 47DD 33 4015 22 29 36 43 51 1815 hoursCT 0 8 16 16 4 84 12 20 0 4 12 208

frq + frq10

0

5

10

15

20

0

5

10

15

20

11 15 18 22 25 29 33 36 40 43 47 51

Hours in Constant Darkness

FRQfrq+

mRNArRNA

Rhythms in frq mRNA and FRQ protein



205

116

0 3 7 12 15 19 0 3 7 12 15 19CTIP + λλPPaseExtract

FRQ is Phosphorylated in a Time-of-Day-Specific Manner

High temp

Low temp

.

frq

513I

513D

64200.2

0.4

0.6

0.8

1.0

1.2

bd513I513D

Hrs after addition of CHX

Rel

ativ

e F

RQ

leve

ls

Phosphorylation site at aa 513 is important for FRQ degradation

+

PERIOD LENGTH

ARR

35 hr

22 hrYL 5/99

0 1 3 5 0 1 3 5 0 1 3 5control 513I 513D

Hrs:

FRQ

SER->ILE SER->ASP



.

T ot al L ys at e Nu cl ear

p C L 1 1 p C L 1 1

AUG#1AUG#3

N-terminal NLS frq(pCL11)

p C L 1 1 p C L 1 1

A.

Re-insertion of the NLS restores nuclearlocalization to polypeptide bearing it.

NLSfrq +

1st AUG3rd AUG

frq ORF

frq ORF1st AUG

3rd AUG

frq

+

frq

∆∆ NLS

frq

(nu

ll)10

frq

+

frq

∆∆ NLS

Re-insertion of the NLS restores FRQ function.C.

NLS

Nu cl ear

∆∆ AU

G#!

∆∆ AU

G#3

frq

+

frq

+

phosphatase t reated

T ot al L ys at e

B.

frq +/25°C

pCL11/25°C

pCL11/27°C

pCL11/22°C

frq ∆∆NLS/ 25°C

FRQ acts in the

nucleus

Molecular Components within the Neurospora Circadian Oscillator

FRQproteins ccg's


LIGHT

frqCK 2

turnover

Nucleus

FRQPP

processing

Cell

wc-1

vvdWCC

translation

WCC

wc-2

FRQPP

WCC



WC-1 and WC-2 are par t of theNeurospora clock

5 15304560

-light +light

2 ' li ght pulse

5 153045 60 5 15304560

5 15304560

frq

frq WTwc-1

DD LP DD LP DD LP

ER33 ∆∆wc-2 WTfrq

frq0 2 6 1014 18 2226

FRQ

2 6 10 14 18 2226 30 34 38 42 46 50 54 58

LL, 4°C -> DD, 25°C

WT4°

frq

0 2 6 101418 2226

FRQ

2 6 10 14 18 22 26 30 34 38 42 46 50 54 58

wc-1frq

0 2 6 1014 18 2226

FRQ

2 6 10 14 18 22 26 30 34 3842 46 50 54 58

wc-2[ER33]

4°

4°dark

loss of function

wc-2

White Collar-1 (WC-1) and White Collar-2 (WC-2)

•WC-1 and WC-2 are required for the light induction of frq transcript.

•WC-1 and WC-2 heterodimerize via their PAS domains.

•WC-1 and WC-2 contain zinc-finger (Zn) DNA binding domains and activation domains (AD).

WC-2PAS ZnAD 530 aa

WC-1LOV PAS PAS ZnAD AD 1167aa



.

Anti-WC2 co-immunoprecipitatesFRQ and WC1

FRQ

WC2

WC1

I PIt o t a l e x t r a c t

DD26

I PI

DD14

FRQ

WC2

WC1

A Partially Functional Allele of wc-2 Displays Altered Period Length and

Temperature Compensation

WTER24

Temperature (�

C)

Per

iod

len

gth

(h

ou

rs)

20

22

24

26

28

30

20 22 24 26 28 30

wild type

wc-2ER24



WC-1

FRQ

WC1 vs. FRQ in frq+

0

1

2

3

4

5

6

7

8

9

0

1

2

3

4

5

6

4 12 20 28 36 44 52 60 68

WC1 vs. FRQ in frq 7

0

5

10

15

20

25

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

4 12 20 28 36 44 52 60 68

W C1

FRQ

Hours in constant darkness Hours in constant darkness

Protein cycling in frq+ Protein cycling in frq7WC-1

FRQ

Levels of WC-1 and FRQ cycle in Antiphase to One Another

..

ATGqa-2promoter

FRQ ORF

+

=

quinic

acid

glucose( )

( )

VII R

qa-1S qa-1F(activator)(repressor)

Indu cible FRQ Protein Production

qa-1Fqa-1S

FRQ

protein

3’ UTR



FRQ promotes the Synthesis of WC-1 through a Post-transcriptional

Mechanism

+frq10::qaFRQ

+frq+

ααWC-1QA

ααFRQ

--

1 2 3 1 2 3 1 2 3 1 2 3

QAfrq+

wc-1EtBr stainedmembrane

- -+ +frq10::qaFRQ

wc-1 RNArRNA

- + +

frq+

-

50

100

frq10::qaFRQ

- + +frq+-

frq10::qaFRQ

relativeamountofprotein

0 4 8 12 16 20 0 4 8 12 16 20

QA pulse cont r ol

ααWC-1

ααFRQ

hr oftreatment

16

8

00 8 16

Time (hr)

relativeamount

ofprotein

ααFRQ(longer exp.)

50

100

50

100

WC-1

Time

FRQproteins

ccg's


caseinkinase II

turnover

NucleusPP

FRQPP

processing

Cell

wc-1

vvd

FRQWCC

WCC

translation

WCC

wc-2

Light

frq

Molecular Components of theNeurospora Clock

FRQFRQfrqfrq



42342618100

2

4

6

8

10

1221°C FRQ28°C FRQ

Time in DD (hr)

Leve

l of F

RQ

42342618100

2

4

6

8

10

12

Time in DD(hr)

Leve

l of f

rq

FRQ

frq

1318 24 29 3440

21 Co

1318 24 29 3440

28 Co

rRNA

amidoblack

Hrs:

FRQ Oscillates around a Higher Levelat Higher Temperatures

21°C frq28°C frq

CT 0 12dawn dusk

CTSTEP UP: All points on the low tempcurve are low compared to the high tempcurve, so the clock is reset to the timecorresponding to the low point in FRQ - dawn.

CT 0 12dawn dusk

CTStep Down: All points on the high tempcurve are high compared to the low temp curve,so the clock is reset to the time correspondingto the high point in FRQ - late day.

time of day

amo

un

t o

f cl

ock

co

mp

on

ent

Clock Resetting by Temperature Steps



.

ribosomal RNA

10'

20'

30'

60'

120'

frq RNA

seconds of light ( 25 lux )

0'

Light-Induced Clock Resetting Increases in Proportion to Light Induction of frq

1

5

1 0

level of frq RNA vs. light

seconds of light (25 lux)

0 10 20 30 60 120

frq RNA

ribosomal RNA

0

2

4

PhaseAdvance

(hrs)

0 4 8 12 16 2018

AdvanceDelay

0

-6

6

12

frq

mR

NA

leve

l

Subjective DAY NIGHTNIGHT ligh

t-in

duce

d cl

ock

rese

ttin

g

hrs in dark- light

frq

+ light28 32 36 40 44 48 28 32 36 40 44 48

frqRNA

Advance

Delay

Resetting the Neurospora Clock with Light



Digoxygenin staining of SCN +/- light

Shigeyoshi et al,Cell, 1997

frq

frq

How are light signals perceived and transduced to the clock?

•frq transcript is rapidly and highly induced in response to light.

nightnightday

?

?

?

+WC-2WC-1

•WC-1 and WC-2 are required for the light induction of frq transcript.

nightnightday



The frq promoter contains two Light Response Elements (LRE) necessary and sufficient for light induction

� distal

& �

proxwt

� distal

� prox

frq RNA*

*

* = light

= dark

proximalLRE

distalLRE

ClaI MscI SnaBI BglII MscI +1

~1200 bp ~200 bp

ααW

C-1

ααW

C-2

ααF

RQ

wc-

1ER

45

wc-

2KO

frq

KO

self non-self mut.self

1 4 532 6 7 8 10 119 12 13 14 1615 17

The LREs are bound by a WC-1/WC-2 containing complex

WC-2WC-1



The distal and proximal LREs are bound by distinct complexes in the light and dark

Light

Dark

Light

Dark

proximal LRE distal LRE

growth conditions

What causes the change in mobility of the WCC/LRE complex in the light?

•phosphorylation of WC-1/WC-2

•conformational change of WC-1/WC-2

•binding of additional factor(s)

•combination of the above

•other?

?

PPP

P

P PP



VVD

PYP

W C-2

W C-1

PAS domain

DNA binding domain

activation domain

His kinasedomain

chromophore

100 aa

Real and Potential Neurospora Photoreceptors

Response regulator-receiver

GAF+ chromophore

PHOT1

PHYA

PHY-2

PHY-1

CRY

CRY

known photoreceptors from other systems

LOV domain

NOP-1

Nuclear Protein Extracts are Photoreactive

growth conditions D DL L

in vitrolight treatment

L L

Protein extractions, binding reactions and gel running performed in the dark (red lighting).



Is the in vitro light-induced change in mobili ty of the WCC/LRE complex occurr ing at biologically relevant…

•amounts of light (ill uminance)?

•quali ties of light (wavelength)?

Light (umol photons/m2)

0.0

0.2

0.4

0.6

0.8

1.0

0 10 20 30 40 60 180

300

900

600

1800

1200

2700

1800

0

9000

3600

Rel

ativ

e B

and

Inte

nsi

ty

The in vitro light-induced WCC/LRE mobil ity change occurs at biologically relevant light intensities.

in vivo threshhold

1

5

10

In vivo light responses

0 8 16 24 48 96

frqmRNA

0

2

4 PhaseAdvance

(hrs)

Light (umol photons/m2)

Crosthwaite et al, Cell, 1995



Wavelength (nm)0 410 455 540430 475

0.0

0.2

0.4

0.6

0.8

1.0

400 450 500 550

Rel

ativ

e B

and

Inte

nsi

ty

1012

1013

Ban

din

g In

hib

ition

(1/erg s

-1cm-2)

Sargent and Briggs Plant Phys. 1967

The in vitro light-induced WCC/LRE mobility change occurs at biologically relevant wavelengths.

Slower Complex

dark light

Faster Complex

D L

WC-1 and WC-2 generated in vitro can bind to the LREs as both the faster and slower migrating complexes



Slower Complex

dark dark lightlight

- FAD +FAD

Faster Complex

WC-1 +FADWC-1

WC-2

D L

D LD LD L

FAD imparts light regulation to the WC-1/WC-2 complex

FAD


• frq transcript is rapidly and highly induced in response to light.

• The frq promoter contains two cis-acting Light Response Element (LREs).

• The LREs are bound by a WC-1/WC-2 heterodimeric complex in the dark.

• Absorption of light by a FAD chromophore bound by WC-1 triggers the multimerization of the WCs presumptively allowing the multiple WC activation domains to act.

proximalLRE

distalLRE

+1

frq promoter

FAD FAD

Therefore, WC-1 is a circadian blue light photoreceptor.



frq

wc-2

wc-1vvd

ccg’s

WC-1

FRQ

FRQ

FRQkinases:CK-1αα, Ck-1ββ,CAMK-1, CKII

turnover

FRQ

FRQ

P

P

P

P

P P

P

-

+

+

+

+

FRQfrq

nightnightday

WC-2

WC-1

WC-2WC-1

WC-2WC-1

nucleus

•WC-1 and WC-2 act positively on the production of frq transcript in the dark.

-

0 2412 36

The distal LRE is necessary for overt and molecular rhythmicity

Light Darkwt

� distal

wt

4 25oC

� distal

0 2412 36

frq RNA FRQ protein

� distal

wthours in dark hours in dark



The distal LRE is sufficient to drive rhythmic transcriptionThe distal LRE is also a circadian element.

6 12 18 24

endogenousfrq

vectorhph

reporter

distal LRE

6 12 18 24hours in dark

*

hours in dark

proximal LRE

hp

hTATA

hph{LRE}

The distal LRE is rhythmically bound by the WCC

12 3630 422418DDCT 23 2216 3115

12 18 24 30 36 42DD

FRQBinding

CT 23 2216 3115

WC-1

WC-2

FRQ

12 3630 422418 DDCT23 2216 3115



•FRQ negatively regulates its own transcript.

•FRQ physically interacts with WC-1 and WC-2.

•Oscillations in FRQ levels are phased appropriately and suggest an attractive means of generating oscillations in WCC binding.

Does FRQ regulate WCC binding to the distal LRE?

WC-2

WC-1

FRQ

Gel Shift

Hrs Induction

Strain

0 04 421

wtQA::FRQ

0 1 2 4

wtQA::FRQ

0 4 Hrs Induction

Strain

Bin

din

g

FR

Q

0

1

2

3

4

5

6

7

0

4

8

12

16

20

24

Increases in FRQ cause a decrease in WCC binding



Dawn ~CT0/24•FRQ increasing•WCC binding decreasing•frq transcription high

Midnight ~CT18-22•FRQ precipitously turns over and is low•WCC binding increasing or maximal•frq low, but increasing

Midday ~CT6-10•FRQ increasing to maximum•WCC binding decreasing to minimum•frq decreasing

Dusk ~CT12•FRQ decreasing, becoming phosphorylated•WCC binding low•frq low

FRQfrq

nightday

binding

Model of the Neurospora Clock (in constant darkness)

AcknowledgementsClock Genes and Clock Mechanism

Hildur ColotSue Crosthwaite

Mi ShiDeana Denault

Allan Froehlich

Kwangwon Lee

Antonio Pregueiro

Jill Wahleithner

Hitoshi Okamura

Giles Duffield

Bill SchwartzKobe Univ.

Mouse

U. Mass. Worcester

Neurospora

Han Cho

Light RegulationChristian Heintzen

Yi Liu

Allan Froehlich

Clock-Controlled Genes

Bruce Roe

Univ. OklahomaBernd Meurers

RW Johnson PRI

Mari ShinoharaMinou Nowrousian

Jennifer LorosJay Dunlap

Carol Ringelberg

Carsten Schwerdtfeger

Arun Mehra



23 22 21 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1Fraction #

Rel

ativ

e P

rote

in

WC-1/lightWC-2/light

WC-2/darkWC-1/dark

WC-1

WC-2

dark

** *

22 21 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1TLFraction #

+light WC-1

WC-2

ΒΒ-amylase200kDa

*

BSA66kDa

* *

thyroglobin669kDa

23 22 21 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1Fraction #

TL

Light-Induced Shift of WC-1/WC-2 into a Higher Molecular Weight Complex

?



Dawn•FRQ increasing•WCC binding high•frq transcription high

Midnight•FRQ decreasing•WCC binding increasing•frq low, but increasing

Midday•FRQ high•WCC binding high•frq transcription high

Dusk•FRQ high, but decreasing•WCC binding decreasing•frq decreasing

FRQfrq

nightday

binding

Model of the Neurospora Clock (under light/ dark cycles)

Deletion of the proximal LRE results in a light-specific phase delay

wt

4 25oC

� prox

wt

Light Dark

� prox

~12 hr delay

� prox

wthours in dark

frq RNA

0 12 24 36

FRQ protein

0 12 24 36hours in dark



WC-2WC-1

Consensus sequence for WC-1/WC-2 binding

distal LRE

CGTCCTGATGCCGCTGCAAGACCGATGACGCTGCAAAATTGAGATCTA**** ****

CGCAGAGGACCCTGAACTTTTCGATCCGCTCGATCCCCTGGAA

proximal LRE

**** ****

proximal

distal

CGATCCGATCTGATGCGATGCGATACCGATAATACGCTal-3

CGCT

CCGCTACGCTCCGCA

CCCT

CGAT_____CGCTconsensus

The WC-1 LOV domain shares homology with LOV domains found in blue light photoreceptors

LOV WC-1PAS PAS ZnAD AD

chromophore

Crosson and Moffat, 2001



= flavin

Flavin adenine dinucleotideFAD

Flavin mononucleotideFMN

1 4 532 6 7 8 10 119 12 13 14 1615 17 18 19 2021

WC-1 +FADWC-1

WC-2 WC-2 +FAD

D LD L

D LD L

D L

D L

D L

D LD LD L

D L

D L

D L

L D

L

D

WC-1, bound to FAD, is the Neurospora circadian photoreceptor and does not require WC-2 for photoreception

FAD

FAD is required -compare 11 thru 16

WC-1 is the receptor -compare 20 vs 19

Both WC-1 and WC-2 are needed - lanes 1-10




•frq transcript is rapidly and highly induced in response to light.

•The frq promoter contains two cis-acting Light Response Element (LREs).

• The LREs are bound by a WC-1/WC-2 (WCC) containing complex.

• Light causes decreased mobility of the WCC/LRE.

proximalLRE

distalLRE

+1

frq promoter

? ?

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