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

2.
Photoactivated Transcription in Neurospora (Payen 1843) <ul><li>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. </li></ul><ul><li>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 .&quot; </li></ul>

3.
Species Dependent Strategies <ul><li>Cyanobacteria - bacteriophytochrome </li></ul><ul><li>Fungi - flavoproteins </li></ul><ul><li>Plants - cryptochrome & phytochrome </li></ul><ul><li>Insects - cryptochrome & opsin </li></ul><ul><li>Mammals - melanopsin (cryptochrome?) </li></ul>

4.
Some Examples <ul><li>Red light </li></ul><ul><ul><li>Bilins (phytochromobilin) </li></ul></ul><ul><li>Blue light </li></ul><ul><ul><li>Flavins (FAD, FMN, Deazaflavin) </li></ul></ul><ul><ul><li>Pterin </li></ul></ul><ul><li>Kinases </li></ul><ul><li>Transcription Factors </li></ul><ul><li>Transcription Factor Interacting Proteins </li></ul>

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

6.
phyR : Peak induction within 1 hour

7.
<ul><li>Yeast system </li></ul><ul><li>Hybrid fusion proteins </li></ul><ul><ul><li>Phy(pr) + Gal4 DNA Binding Domain </li></ul></ul><ul><ul><li>PIF3 + Gal4 Activating Domain </li></ul></ul><ul><ul><li>Binds to Gal1 UAS </li></ul></ul><ul><li>Photoactivation </li></ul><ul><ul><li>Activated by 660nm </li></ul></ul><ul><ul><li>Deactivated by 750nm </li></ul></ul><ul><ul><li>State changes in milliseconds </li></ul></ul><ul><ul><li>Indefinitely reversible </li></ul></ul><ul><li>Needs hydrophobic phycocyanobilin (PCB) in media </li></ul>

8.
phyR : Performance <ul><li>Yeast only grow on selective plate with PCB + light </li></ul>50:1 LacZ induction, 1/5 expression of wild-type GAL4 system

9.
phyR : Photon Dosing <ul><li>1mmol / m 2 ideal photon dose </li></ul>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

10.
phyR : Temporal precision and reversibility <ul><li>Expression doubles in 1 minute </li></ul><ul><li>Increased expression stops within 10 minutes of far red pulse </li></ul>

11.
phyR : Issues <ul><li>GAL4 UAS system </li></ul><ul><ul><li>Works great in yeast </li></ul></ul><ul><ul><li>Should work well in flies </li></ul></ul><ul><ul><li>Gal4 system rarely used in mice </li></ul></ul><ul><ul><li>No binding to endogenous sites </li></ul></ul><ul><li>Incorporation of phytocyanobilin </li></ul><ul><ul><li>Maybe a problem, maybe not (See Tsien’s IFPs with biliverdin) </li></ul></ul><ul><li>Long wavelength for easy activation, spectral orthagonality, </li></ul><ul><li>Rapidly reversibile </li></ul><ul><li>Background from red light / near IR through skull? </li></ul><ul><li>1100 amino acids for wt phyA </li></ul>

12.
White Collar system

13.
WC : Light activated transcription factor <ul><li>WC-1 & 2 required for circadian rhythm in Neurospora </li></ul><ul><li>Rapid light induction of frequency expression </li></ul><ul><li>Replacement of frq with hph works 10:1 light induction </li></ul><ul><li>Promoter deletions identify 2 light responsive elements </li></ul><ul><li>KO one, get 50 or 70% drop in expression </li></ul><ul><li>KO both, get no expression </li></ul>

14.
WC : Light Responsive Element sequence

15.
WC : wc-1 gene structure <ul><li>Light/oxygen/voltage domain </li></ul><ul><ul><li>Binds FAD noncovalently </li></ul></ul><ul><li>Per-Arnt-Sim domains for nuclear complex formation </li></ul><ul><li>Nuclear localization sequence </li></ul><ul><li>Zinc finger DNA binding domain </li></ul>

16.
WC : FAD required for light sensitivity <ul><li>Separate in vitro expression </li></ul><ul><li>Wc-1 and wc-2 bind heterodimerize without FAD </li></ul><ul><li>Multimerize with FAD and Light </li></ul>

17.
WC : Photon Dose and Wavelength <ul><li>2.5mmol photons / m 2 is a full dose </li></ul><ul><li>Responsive in UV, peak at 465nm </li></ul><ul><li>Silent above 520nm </li></ul>

18.
WC : Two-photon action spectra <ul><li>Much dimmer than FPs </li></ul>Heikal & Webb 2002

19.
WC : Issues <ul><li>Direct light modulation of transcription factor </li></ul><ul><li>FAD omnipresent in mammalian cell mitochondria </li></ul><ul><ul><li>Probably enough leak to cytosol and nucleus </li></ul></ul><ul><li>Large size (1147 aa) of wc-1, small wc-2 </li></ul><ul><li>Short recognition site may have overlap with endogenous sites </li></ul>