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TitleMolecular Clocks and Light Signalling
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Total Pages308
Table of Contents
	Chair’s introduction
	Non-rod, non-cone photoreception in rodents and teleost fish
	Cryptochromes and inner retinal non-visual irradiance detection
	General discussion I
	Light signalling in Cryptochrome-deficient mice
	Circadian light input in plants, flies, and mammals
	Orphan nuclear receptors, molecular clockwork, and the entrainment of peripheral oscillators
	General discussion
	SCN: ringmaster of the circadian circus or conductor of the circadian orchestra?
	On the communication pathways between the central pacemaker and peripheral oscillators
	Central and peripheral circadian oscillators in Drosophila
	Integration of molecular rhythms in mammalian circadian system
	Circadian transcriptional output in the SCN and liver of the mouse
	The molecular workings of the Neurospora biological clock
	Expression of clock gene products in the suprachiasmatic nucleus in relation to circadian behaviour
	Circadian rhythms in Drosophila
	The role of phosphorylation and degradation of hPer proteins oscillation in normal human fibroblasts
	Regulation of daily locomotor activity and sleep by hypothalamic EGF receptor signalling
	CK1 and GSK-3 in the Drosophila and mammalian circadian clock
	Final general discussion
	Closing remarks
	Index of contributors
	Subject index
Document Text Contents
Page 1



Novartis Foundation Symposium 253


Page 154

tim transcription can begin. In the Clk loop, Clk transcription is repressed by
CLK^CYC during the early/mid day (*ZT 04), and derepressed by rising levels
of PER^TIM during the mid-evening (*ZT 16), which bind CLK^CYC to
promote Clk transcription. A critical issue is how CLK^CYC repress Clk. The
lack of canonical E-box binding sites for CLK^CYC in and around Clk
suggests that repression occurs indirectly, probably through the activation of a
transcriptional repressor. A prime candidate for such a repressor is the basic-
zipper (bZIP) transcription factor VRILLE (VRI) because (1) vri is activated by
CLK^CYC, (2) over-expression of VRI reduces or eliminates expression of two
CLK^CYC dependent transcripts (per and tim), and (3) VRI acts as a repressor
genetically since over-expression leads to long period rhythms and reducing vri
gene dosage results in a short period rhythm (Blau & Young 1999).
If VRI directly repressesClk expression, this would predict that VRImust cycle

in antiphase to Clk mRNA, that VRI binding sites will be present in the circadian
regulatory region of Clk, and that VRI over-expression will repress Clk mRNA
levels in vivo. These predictions were tested in the following ways. First, an
antibody generated against VRI shows that it cycles in phase with vri mRNA and
antiphase toClkmRNA (Fig. 1). Second, the VRIDNAbinding domain is almost
identical to that of themammalian transcription factor E4BP4 (George&Terracol
1997), suggesting that it binds the same target sequence. Several perfect or near-
perfect E4BP4 target sequences are found within an 8.0 kb Clk genomic fragment
that mediatesClkmRNA cycling, and VRI binds strongly to several of these sites.
Third, when VRI over-expression is induced in a cyc01 mutant background, in
which Clk is constitutively expressed at peak levels (Glossop et al 1999), Clk
mRNA levels fall to about half of their peak value. These results ful¢l the
predictions above, thus identifying VRI as an integral component of the
interlocked feedback loop whose role is to repress Clk transcription (Fig. 2).
The mammalian circadian oscillator is also comprised of two interacting

feedback loops: a Per/Cry loop and a Bmal1 loop (reviewed in Reppert &Weaver
2001, Glossop &Hardin 2002). The Per/Cry loop is analogous to the per/tim loop
in Drosophila. Initially, mammalian CLOCK forms heterodimers with BMAL1
(the mammalian CYC homologue) and drives rhythmic transcription of three per
homologues (Per1, Per2 and Per3) and two cry homologues (Cry1 andCry2). PER
proteins (at least PER1 and PER2) then form complexes with CRYproteins, move
into the nucleus and repress CLOCK^BMAL1-dependent expression. The Bmal1
loop is analogous to the Clk loop inDrosophila. Bmal1 is ¢rst activated in a PER2-
dependent manner, and later repressed in a CLOCK^BMAL1-dependent manner.
As in Drosophila, this repression occurs indirectly, but in this case CLOCK^
BMAL1 activates Rev-Erba, which encodes an orphan nuclear receptor that
represses Bmal1 transcription (Preitner et al 2002). Rising levels of PER^CRY in
the nucleus then repress Rev-Erba, thus relieving the repression of Bmal1 and


Page 155

initiating the next cycle (Preitner et al 2002). Although the interlocked feedback
loops of £ies and mammals are mechanistically similar, they di¡er in three ways.
First, mammalian CRY appears to have taken the place of TIM in that it binds to
PER and promotes PERnuclear localization (Reppert&Weaver 2001,Glossop&
Hardin 2002). Second, the regulation of Bmal1 and Clock in mammals is switched
compared to their £y homologues: Bmal1 and Drosophila Clk are rhythmically
expressed whereas cyc and mammalian Clock are constitutively expressed
(Reppert & Weaver 2001, Glossop & Hardin 2002). Third, the bZIP
transcription factor VRI represses Clk in Drosophila, but the orphan nuclear
receptor REV-ERBa represses Bmal1 in mammals (Preitner et al 2002). Each of
these di¡erences concerns the identity of factors that carry out conserved
regulatory steps within the feedback mechanism, indicating that each of these
steps is important for circadian oscillator function regardless of the factor that
carries out that step.
The same interlocked feedback loop mechanism is thought to operate in

circadian oscillator cells throughout the Drosophila circadian system. However,
studies of the blue light photoreceptor CRYPTOCHROME (CRY) suggest that
central and peripheral oscillator mechanisms in Drosophila are not the same.
Drosophila CRY was initially identi¢ed as a photoreceptor that mediates light


FIG. 2. Model of the interlocked feedback loop mechanism in Drosophila. The per/tim loop
(left) and dClk loop (right) are shown. Transcriptional activator genes, black italics;
transcriptional activator proteins, black capitals; transcriptional repressor genes, grey italics;
transcriptional repressor proteins, grey capitals. Filled arrows, transcriptional activation; open
arrows, translation; bars, repression.

Page 307

rods 9, 12, 80
ROR (Retinoic-acid receptor related Orphan

Receptor) 90, 93, 100, 176
RORa 90, 91, 92
RORb 90, 92
ROREs (ROR elements) 90, 91
RORg 90, 92
RT-PCR 229, 235^236, 241, 242


salmon (Salmo salar) 5
secretogranin III 177
secretory granule neuroendocrine protein 1

Ser513, FRQ phosphorylation 187
serotonergic-positive a¡erents 208
serum shock 129, 167, 241^242
Shaggy (SGG) 225, 268, 270, 275
shaggy 230, 239
sleep syndromes 239, 241, 246^247, 272, 274
sleep^wake cycle, TGFa 254, 256^257
Slob 282
Slowpoke 177
small ventral lateral neurons 141
smelt ¢sh 8
somatic cell melanopsin 28
somatostatin 176
spermatogenesis 132
Spermophilus 208
STAT3 45
subparaventricular zone (SPZ) 251, 266
suprachiasmatic nucleus (SCN) 32, 78,

110^121, 161, 171^173, 203^217
aromatic L-amino acid decarboxylase 165
casein kinase 1d 220
clock proteins 204^206
cryptochrome 59, 80, 204, 206
cycling genes 174^176
diurnal versus nocturnal species 208, 210
electrical rhythms 57, 59^61, 66^67
food entrainment 102^103
light entrainment 206^208
locomotor activity 251, 252
masking 220^222
metallothionein 1 activator 177^178
mRNA cycling 174
neuronal signalling 176^177
neuropeptide release 176^177
NIH 3T3 co-culture 129
non-photic cues 208

peptidergic signalling 176, 210, 212
PER 204, 206, 208
Per1/Per2 165^167
peripheral clocks 64, 69^71, 113^115,

126^136, 214
phosphorylated MAP kinase 206
photic signalling to 33^34
photoresponse in vitamin A-depleted

mutants 53
retinal role 115
rod and cone loss 12
somatostatin 176
TGFa 254
transplants 66^67, 69^71, 136, 251
vasopressin 176

Synecococcus 283


tau 270, 272
TEF 162
teleost ¢sh 4, 5^8, 17

peripheral clock entrainment 96^98
peripheral tissue compensation 181
regulation, Neurospora 187^188

testis 132^133
TIM 162

circadian function in mammals 273
Drosophila 225, 267^268
nucleus 278
phosphorylation 225, 239, 268, 270,

tim 140^141, 225, 230
Timeless 32, 36 see also TIM
tmt-opsin 28
TOC1 76, 85
transcriptional pro¢ling 171^180
transducin 5
transforming growth factor a (TGFa) 173,

252, 254^257, 259^260
transparency 159
twilight 45
Tyr216 phosphorylation 275


ubiquitin-proteasome pathway 244, 246
ubiquitination 164^165
ultraradian rhythm 257


Page 308


VA (vertebrate ancient) opsin 8

valproate 273
vascular smooth muscle cells 167
vasopressin (AVP) 176
vertebrate ancient (VA) opsin 5^8, 17
VIP 210, 219
vitamin A-based chromophore 3, 4^5
vitamin A binding proteins 30
VIVID 186^187, 199
voles 101
VPAC2 64, 210, 212, 219
Vpac2 knock-out mice 218
VRILLE (VRI) 142, 146, 148, 150, 154^155,

226, 268
VVD 186^187, 199
vvd 187


waved-2 257, 259
WC-1 (White Collar 1)
Bmal similarity 87
C-Box binding 192
FRQ interaction 185^186
LOV domain 190
LRE-bound complexes 188, 190
photoreceptor role 190, 198^199, 201^202

wc-1 (white collar 1) 185

WC-2 (White Collar 2)
C-Box binding 192
FRQ interaction 185^186
LRE-bound complexes 188, 190
period length mutants 183

wc-2 (white collar 2) 185
white collar complex (WCC) 185
C-Box binding 192


xenobiotic metabolism 177
Xenopus laevis
melanophores 4, 14, 17
melanopsin 15, 17, 27


yeast two-hybrid screen 36, 50^51


Z3 127^129
melanopsin 15, 27
peripheral clocks 127
vertebrate ancient opsin 5
Z3 127^129

zeitgeber, feeding times 96
zeitgedachtnis, bees 123^124
Zif268 80


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