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 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 Groups DBT CLOCK E-Box JETLAG CLOCK PER TIM Ub P CYCLE CRY CYCLE CRY TN: Glow effects tends shrink when copy and pasted as a picture. I made the glow bigger.

Fly Clock Mechanism Nucleus tim tim per per Cytoplasm ©UC San Diego BioClock Studio

Fly Clock Mechanism Nucleus tim tim per per Cytoplasm ©UC San Diego BioClock Studio

Fly Clock Mechanism Nucleus tim tim per per Cytoplasm ©UC San Diego BioClock Studio

Fly Clock Mechanism Nucleus tim tim per per Cytoplasm ©UC San Diego BioClock Studio

F-box Protein Mechanism Degraded

Neuronal firing Active Inactive TTFL Ca2+

Fly Clock List Legend 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 T-shape 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 I spelled out Clock and Cycle, but I am not sure if they should be upper case (?)

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. Minor editing to the new text

Mammalian Graphic Components Individual Groups CK1 E-Box E-Box CLOCK FBXL3 CLOCK PER FBXL21 CRY BMAL1 Ub P CRY BMAL1 FBXLs

Mammalian Clock Mechanism Nucleus Cry1,2 Cry1,2 Per1,2,3 Per1,2,3 Cytoplasm ©UC San Diego BioClock Studio

Mammalian Clock Mechanism Nucleus Cry1,2 Cry1,2 Per1,2,3 Per1,2,3 Cytoplasm ©UC San Diego BioClock Studio

Mammalian Clock Mechanism Nucleus Cry1,2 Cry1,2 Per1,2,3 Per1,2,3 Cytoplasm ©UC San Diego BioClock Studio

Mammalian Clock Mechanism Nucleus Cry1,2 Cry1,2 Per1,2,3 Per1,2,3 Cytoplasm ©UC San Diego BioClock Studio

Mammalian Clock Mechanism Nucleus Cry1,2 Cry1,2 Per1,2,3 Per1,2,3 Cytoplasm ©UC San Diego BioClock Studio

F-BOX PROTEIN Mechanism Cytoplasm Nucleus Degraded slowly Degraded slowly Degraded Fast TN: FBXL21 & 3 degradation process was updated.

Neuronal firing Active Inactive TTFL CREB MAPK, PKA, CAMKII Glu Ca2+, cAMP ipRGC

Mammalian Clock List Legend 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 T-shape 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 BMAL1 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 I spelled out clock in the legend since we don’t use CLK on the graphics. Still not sure about upper case letters. I can’t find the feedback email that mentioned the nomenclatures!

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 Groups TOC1 EE CCA1 ZTL LHY Ub PHYB

Plant Clock Mechanism Nucleus Cytoplasm TOC1 TOC1 LHY LHY CCA1 CCA1 ©UC San Diego BioClock Studio

Plant Clock Mechanism Nucleus Cytoplasm TOC1 TOC1 LHY LHY CCA1 CCA1 ©UC San Diego BioClock Studio

Plant Clock Mechanism Nucleus Cytoplasm TOC1 TOC1 LHY LHY CCA1 CCA1 ©UC San Diego BioClock Studio

Plant Clock List Legend 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 T-shape 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.