The BioClock Studio
Winter 2017
presents…
Common Clock Mechanism
Graphics Tool
Produced by Mike Lim, Christopher Tu, Takako Noguchi,
and Susan S Golden
About the Common Clock Mechanism Graphics Tool
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•
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This Common Clock Mechanism Graphics Tool was created to improve student
understanding of transcription-translation feedback loops of circadian clocks across
organisms. Teachers and researchers may freely use and edit graphics in this file with
attribution to the UC San Diego BioClock Studio (CC BY 4.0).
Graphics components were created either in PowerPoint or in Gimp, a free & open source
image editor, and then imported into PowerPoint.
We made our graphics color-blind friendly, using Color Oracle, a free color blindness
simulator. Please see the following links for more tips on generating color-blind friendly
scientific graphics.
Tips for designing scientific figures for color blind readers, Luk
Color Universal Design (CUD)- How to make figures and presentations that are friendly to Colorblind people, Masataka Okabe
Fly Graphic Components
Individual
DBT
CLOCK
PER
CLOCK
Groups
E-Box
JETLAG
TIM
Ub
CYCLE
CRY
CYCLE
CRY
P
Fly Clock Mechanism
Nucleus
©UC San Diego BioClock Studio
tim
tim
per
per
Cytoplasm
Fly Clock Mechanism
Nucleus
©UC San Diego BioClock Studio
tim
tim
per
per
Cytoplasm
Fly Clock Mechanism
Nucleus
©UC San Diego BioClock Studio
tim
tim
per
per
Cytoplasm
Fly Clock Mechanism
Nucleus
©UC San Diego BioClock Studio
tim
tim
per
per
Cytoplasm
F-BOX PROTEIN MECHANISM
Degraded
NEURONAL FIRING
Active Inactive
TTFL
Ca2+
Fly Clock List Legend
Description
Description
DBT
Doubletime, a kinase that phosphorylates PER
E-Box
Enhancer box, a DNA response element in the
promoter region of a gene, to which specific
transcription factors bind
PER
Period, a protein that forms a heterodimer with TIM
and then inhibits its own expression (negative
element)
Black arrow
Movement (into or out of nucleus)
TIM
Timeless, a protein that forms a heterodimer with
PER and then inhibits its own expression (negative
element)
Red Tshape arrow
Inhibition
P
Phosphate group
Sun
Sunlight input during the day
CLOCK
Clock, a transcription factor that forms a heterodimer
with Cycle. With Cycle it binds to E-boxes and
promotes per and tim transcription (positive element)
Moon
No sunlight input during the night
CYCLE
Cycle, a transcription factor that forms a heterodimer
with Clock. With Clock it binds to E-boxes and
promotes per and tim transcription (positive element)
Broken
particles
Degraded protein
CRY
Cryptochrome, a blue-light photoreceptor that initiates
degradation of TIM
JETLAG
a ubiquitin ligase that binds to the CRY-TIM complex
and ubiquitinates TIM to promote TIM’s degradation
Fly Clock Graphics Legend
•
•
CLOCK and CYCLE are transcription factors that form a heterodimer and bind to E-boxes in the nucleus
to promote the expression of the per and tim genes during the day. PER and TIM act in the negative limb
of the Drosophila clock translational transcriptional feedback loop (TTFL). They are transcribed to
produce mRNA in the nucleus, which then travels to the cytoplasm to be translated into proteins. Forming
a heterodimer with one another, PER and TIM return to the nucleus to inhibit their own expression by
binding to and inactivating CLOCK-CYCLE at night. However, if PER does not form a dimer with TIM in
the cytoplasm, DBT, a kinase that phosphorylates PER, will promote the protein’s degradation. DBT
travels into the nucleus with the PER-TIM heterodimer. CRY works as a blue-light sensor in the
Drosophila TTFL. Once it is activated by sunlight, CRY will bind to TIM. The formation of the CRY:TIM
complex promotes the recruitment of JETLAG, a ubiquitin ligase. The complex is then ubiquitinated by
JETLAG to promote TIM and CRY’s light-dependent degradation. In this way, light-activated CRY
promotes TIM’s degradation. Once TIM is removed, PER is degraded due to its instability when it is
phosphorylated and not in complex with TIM.
Spontaneous firing of fly clock neurons increases during day and decreases during night even without
light signals. Maintenance of proper membrane potential is necessary to generate circadian rhythms
along with the TTFL. Membrane potential controls the TTFL through signaling pathways involving
calcium.
Mammalian Graphic Components
Individual
CK1
CLOCK
Groups
E-Box
E-Box
FBXL3
PER
CLOCK
FBXL21
CRY
BMAL1
CRY
BMAL1
Ub
P
FBXLs
Mammalian Clock Mechanism
Nucleus
©UC San Diego BioClock Studio
Cry1,2
Cry1,2
Per1,2,3
Per1,2,3
Cytoplasm
Mammalian Clock Mechanism
Nucleus
©UC San Diego BioClock Studio
Cry1,2
Cry1,2
Per1,2,3
Per1,2,3
Cytoplasm
Mammalian Clock Mechanism
Nucleus
©UC San Diego BioClock Studio
Cry1,2
Cry1,2
Per1,2,3
Per1,2,3
Cytoplasm
Mammalian Clock Mechanism
Nucleus
©UC San Diego BioClock Studio
Cry1,2
Cry1,2
Per1,2,3
Per1,2,3
Cytoplasm
Mammalian Clock Mechanism
Nucleus
©UC San Diego BioClock Studio
Cry1,2
Cry1,2
Per1,2,3
Per1,2,3
Cytoplasm
F-BOX PROTEIN MECHANISM
Cytoplasm
Degraded
slowly
Nucleus
Degraded
slowly
Degraded
Fast
NEURONAL FIRING
Active Inactive
TTFL
CREB
Glu
MAPK, PKA, CAMKII
Ca2+, cAMP
ipRGC
Mammalian Clock List Legend
Description
Description
CK1
Casein Kinase 1, a kinase that phosphorylates PERs.
A homolog of Drosophila DBT
E-Box
Enhancer box, a DNA response element in the
promoter region of a gene, to which specific
transcription factors bind
PERs
PERIOD 1, 2, 3, proteins that form a complex with CRYs
and then inhibit their own expression (negative elements)
FBXL21
F-Box and Leucine Rich Repeat Protein 21, a ubiquitin
ligase that attaches ubiquitin to CRYs, which leads to
their slow degradation in both the cytosol and nucleus.
In the nucleus, it competes with FBXL3.
CRYs
CRYPTOCHROME 1, 2, proteins that form a complex
with PERs and then inhibit their own expression
(negative elements)
FBXL3
F-Box and Leucine Rich Repeat Protein 3, a ubiquitin
ligase that attaches ubiquitin to CRYs, which leads to
their fast degradation in the nucleus.
P
Phosphate group
Black
arrow
Movement (into or out of nucleus)
Ub
Ubiquitin group
Red Tshape
arrow
Inhibition
CLOCK
CLOCK, a transcription factor that forms a heterodimer
with BMAL1. With BMAL1 it binds to E-boxes and
promotes Per and Cry transcription (positive element)
Sun
Sunlight input during the day
Moon
No sunlight input during the night
Brain and Muscle ARNT-Like1, a transcription factor that
forms a heterodimer with CLOCK. With CLOCK it binds
to E-boxes and promotes per and cry transcription. A
homolog of Drosophila CYCLE (positive element)
Broken
particles
Degraded protein
BMAL1
Mammalian Clock Graphics Legend
•
•
CLOCK and BMAL1 are transcription factors that form a heterodimer and bind to E-boxes in the nucleus to promote the
expression of the per and cry genes during the day. PER and CRY act in the negative limb of the mammalian clock
translational transcriptional feedback loop (TTFL). The Per and Cry genes are transcribed to produce mRNA in the nucleus,
which then travels to the cytoplasm to be translated into proteins. Forming a heterodimer with one another, PERs and CRYs
form a complex and return to the nucleus to inhibit their own expression by binding to and inactivating CLOCK-BMAL1.
Stability of PERs is regulated by casein kinases (CK1δ, CK1ε, CK2), enzymes that phosphorylate PERs and promote PER
degradation. Note that CK1 is a homolog of Drosophila DBT. Formation of a large complex of PERs, CRYs, and other
proteins prevents their degradation. Stability of CRYs is regulated by F-box proteins (FBXL3, FBXL21), enzymes that add
ubiquitin chains and promote CRY degradation. FBXL3 is in the nucleus and FBXL21 is in both the nucleus and cytosol.
Addition of ubiquitin chains by FBXL3 results in fast degradation and that by FBXL21 results in slow degradation of CRYs. In
the nucleus, FBXL21 competes with FBXL3 for ubiquitination of the CRYs, opposing the fast degradation initiated by FBXL3.
A light signal is delivered from the retina to the suprachiasmatic nucleus (SCN), the master circadian clock tissue of
mammals, by intrinsically photosensitive retinal ganglion cells (ipRGC). Glutamate (Glu) release from ipRGCs excites SCN
neurons. Neuronal excitation modulates the TTFL through a signaling pathway involving calcium and cyclic adenosine
monophosphate (cAMP). In the downstream pathway, mitogen-activated protein kinases (MAPKs), protein kinase A (PKA),
and calcium/calmodulin-dependent protein kinase II (PKII) are activated. Finally, cAMP response element-binding protein
(CREB) activation modulates expression of several clock genes. Spontaneous firing of SCN neurons increases during the
day and decreases during the night even without light signaling. Maintenance of proper membrane potential is necessary to
generate circadian rhythms along with the TTFL.
Plant Graphic Components
Individual
TOC1
EE
CCA1
ZTL
LHY
PHYB
Ub
Groups
Plant Clock Mechanism
Nucleus
©UC San Diego BioClock Studio
TOC1
TOC1
LHY
LHY
CCA1
CCA1
Cytoplasm
Plant Clock Mechanism
Nucleus
©UC San Diego BioClock Studio
TOC1
TOC1
LHY
LHY
CCA1
CCA1
Cytoplasm
Plant Clock Mechanism
Nucleus
©UC San Diego BioClock Studio
TOC1
TOC1
LHY
LHY
CCA1
CCA1
Cytoplasm
Plant Clock List Legend
Description
Description
LHY
Late Elongated Hypocotyl, a protein that forms a
heterodimer with CCA1 and then inhibits the expression
of TOC1 during the day
EE
Evening Element, Enhancer box, a DNA response
element in the promoter region of a gene, to which
specific transcription factors bind
CCA1
Circadian Clock Associated 1, a protein that forms a
heterodimer with LHY and then inhibits the expression of
TOC1 during the day
Blue
arrow
light activation of CCA1 through PHYB
TOC1
Timing of CAB Expression 1, a protein that inhibits the
expression of LHY and CCA1 at night
Black
arrow
Movement (into or out of nucleus)
Ub
Ubiquitin group
Red Tshape
arrow
Inhibition
ZTL
ZEITLUPE, an F-box protein ubiquitin ligase that controls
the degradation of TOC1 in a light-dependent manner
Sun
Sunlight input during the day
PHYB
Phytochrome B, a red/far-red light photoreceptor
Moon
No sunlight input during the night
Broken
particles
Degraded protein
Plant Clock Explanation Legend
•
LHY and CCA1 are transcription factors that are translated during the day. They bind to the EE
(evening element) of the promoter region of the TOC1 gene and other evening-phased genes in
the nucleus, inhibiting their expression during the day. At night, TOC1 is transcribed and
translated, and TOC1 inactivates LHY and CCA1 gene expression. In the late night, the quantity
of TOC1 protein decreases gradually due to ubiquitination by ZTL, which leads to TOC1
degradation. As a result, LHY and CCA1 are no longer repressed by the end of the night and they
turn on by the beginning of the day. PHYB connects red-light signaling with the circadian clock.
Extra Components
References
• Mendoza-Viveros et al. Cell Mol Life Sci. 2017 Mar;74(6):1035-1059.
• Hirano et al. Nat Struct Mol Biol. 2016 Dec 6;23(12):1053-1060.
• Shim et al. Plant Physiol. 2017 Jan;173(1):5-15.
Acknowledgments
Dr Todd H Holmes (UC Irvine), Dr Jerome S Menet (Texas A&M University)
Dr Jose Pruneda-Paz (UC San Diego), Dr Paul E Hardin (Texas A&M University)
Dr Erik D Herzog (Washington University in St. Louis)
The BioClock Studio is an undergraduate class supported in part by a grant to Dr
Susan S Golden at UC San Diego from the Howard Hughes Medical Institute through
the Science Education Program.