090130_Journal_Club

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Published on January 30, 2009

Author: AndrewHires

Source: slideshare.net

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A review of photoactivatable transcription systems.

Photoactivated Transcription 1/29/09 Andrew Hires Svoboda Journal Club

Photoactivated Transcription in Neurospora (Payen 1843) In the warm, moist summer of 1842, bread from army bakeries in Paris was spoiled by massive growth of an orange mold. A commission was set up by the minister of war to investigate the cause of the infestation and to make recommendations. Development of the fungus was a little less abundant than on a piece of the same bread that was exposed to light under conditions that were otherwise identical. Under the first conditions, the fungi remained completely white for more than eight days (see figure b), whereas the illuminated fungi, figure a, a', were covered with red spores. But, remarkably, the white fungi became colored when they were exposed to light for two hours ."

In the warm, moist summer of 1842, bread from army bakeries in Paris was spoiled by massive growth of an orange mold. A commission was set up by the minister of war to investigate the cause of the infestation and to make recommendations.

Development of the fungus was a little less abundant than on a piece of the same bread that was exposed to light under conditions that were otherwise identical. Under the first conditions, the fungi remained completely white for more than eight days (see figure b), whereas the illuminated fungi, figure a, a', were covered with red spores. But, remarkably, the white fungi became colored when they were exposed to light for two hours ."

Species Dependent Strategies Cyanobacteria - bacteriophytochrome Fungi - flavoproteins Plants - cryptochrome & phytochrome Insects - cryptochrome & opsin Mammals - melanopsin (cryptochrome?)

Cyanobacteria - bacteriophytochrome

Fungi - flavoproteins

Plants - cryptochrome & phytochrome

Insects - cryptochrome & opsin

Mammals - melanopsin (cryptochrome?)

Some Examples Red light Bilins (phytochromobilin) Blue light Flavins (FAD, FMN, Deazaflavin) Pterin Kinases Transcription Factors Transcription Factor Interacting Proteins

Red light

Bilins (phytochromobilin)

Blue light

Flavins (FAD, FMN, Deazaflavin)

Pterin

Kinases

Transcription Factors

Transcription Factor Interacting Proteins

Arabidopsis has 5 phytochromes phyA - phyE Covalently attached chromophore 2 states, red (r) and far red absorbing (fr) Absorbtion toggles state Pfr is biologically active Translocates to nucleus Binds PIF3, basic helix-loop-helix protein PIF3 binds CACGTG (G-box) activates preinitiation complex when bound by Pfr

Arabidopsis has 5 phytochromes

phyA - phyE

Covalently attached chromophore

2 states, red (r) and far red absorbing (fr)

Absorbtion toggles state

Pfr is biologically active

Translocates to nucleus

Binds PIF3, basic helix-loop-helix protein

PIF3 binds CACGTG (G-box) activates preinitiation complex when bound by Pfr

phyR : Peak induction within 1 hour

Yeast system Hybrid fusion proteins Phy(pr) + Gal4 DNA Binding Domain PIF3 + Gal4 Activating Domain Binds to Gal1 UAS Photoactivation Activated by 660nm Deactivated by 750nm State changes in milliseconds Indefinitely reversible Needs hydrophobic phycocyanobilin (PCB) in media

Yeast system

Hybrid fusion proteins

Phy(pr) + Gal4 DNA Binding Domain

PIF3 + Gal4 Activating Domain

Binds to Gal1 UAS

Photoactivation

Activated by 660nm

Deactivated by 750nm

State changes in milliseconds

Indefinitely reversible

Needs hydrophobic phycocyanobilin (PCB) in media

phyR : Performance Yeast only grow on selective plate with PCB + light 50:1 LacZ induction, 1/5 expression of wild-type GAL4 system

Yeast only grow on selective plate with PCB + light

phyR : Photon Dosing 1mmol / m 2 ideal photon dose 1 minute pulse best 1mmol 660nm photons = 180 J 180 J / s m 2 = 180W / m 2 for 1s, or 3W / m 2 for 1 minute Filtered fluorescents for long term LEDs for pulsed activation

1mmol / m 2 ideal photon dose

phyR : Temporal precision and reversibility Expression doubles in 1 minute Increased expression stops within 10 minutes of far red pulse

Expression doubles in 1 minute

Increased expression stops within 10 minutes of far red pulse

phyR : Issues GAL4 UAS system Works great in yeast Should work well in flies Gal4 system rarely used in mice No binding to endogenous sites Incorporation of phytocyanobilin Maybe a problem, maybe not (See Tsien’s IFPs with biliverdin) Long wavelength for easy activation, spectral orthagonality, Rapidly reversibile Background from red light / near IR through skull? 1100 amino acids for wt phyA

GAL4 UAS system

Works great in yeast

Should work well in flies

Gal4 system rarely used in mice

No binding to endogenous sites

Incorporation of phytocyanobilin

Maybe a problem, maybe not (See Tsien’s IFPs with biliverdin)

Long wavelength for easy activation, spectral orthagonality,

Rapidly reversibile

Background from red light / near IR through skull?

1100 amino acids for wt phyA

White Collar system

WC : Light activated transcription factor WC-1 & 2 required for circadian rhythm in Neurospora Rapid light induction of frequency expression Replacement of frq with hph works 10:1 light induction Promoter deletions identify 2 light responsive elements KO one, get 50 or 70% drop in expression KO both, get no expression

WC-1 & 2 required for circadian rhythm in Neurospora

Rapid light induction of frequency expression

Replacement of frq with hph works 10:1 light induction

Promoter deletions identify 2 light responsive elements

KO one, get 50 or 70% drop in expression

KO both, get no expression

WC : Light Responsive Element sequence

WC : wc-1 gene structure Light/oxygen/voltage domain Binds FAD noncovalently Per-Arnt-Sim domains for nuclear complex formation Nuclear localization sequence Zinc finger DNA binding domain

Light/oxygen/voltage domain

Binds FAD noncovalently

Per-Arnt-Sim domains for nuclear complex formation

Nuclear localization sequence

Zinc finger DNA binding domain

WC : FAD required for light sensitivity Separate in vitro expression Wc-1 and wc-2 bind heterodimerize without FAD Multimerize with FAD and Light

Separate in vitro expression

Wc-1 and wc-2 bind heterodimerize without FAD

Multimerize with FAD and Light

WC : Photon Dose and Wavelength 2.5mmol photons / m 2 is a full dose Responsive in UV, peak at 465nm Silent above 520nm

2.5mmol photons / m 2 is a full dose

Responsive in UV, peak at 465nm

Silent above 520nm

WC : Two-photon action spectra Much dimmer than FPs Heikal & Webb 2002

Much dimmer than FPs

WC : Issues Direct light modulation of transcription factor FAD omnipresent in mammalian cell mitochondria Probably enough leak to cytosol and nucleus Large size (1147 aa) of wc-1, small wc-2 Short recognition site may have overlap with endogenous sites

Direct light modulation of transcription factor

FAD omnipresent in mammalian cell mitochondria

Probably enough leak to cytosol and nucleus

Large size (1147 aa) of wc-1, small wc-2

Short recognition site may have overlap with endogenous sites

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