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Download Circadian Regulation of Oxidative Stress Response Genes, CncC
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Oxidative Stress Response Genes, cncc, keap1, and gstd1: A Circadian Connection? Marisa McAllister Dr. Jaga Giebultowicz Lab Circadian Rhythms in Daily Life Controls rhythms in daily life functions Rhythms controlled by genes Internal clock disrupted during travel = jetlag evening or rotating work shifts (15%) Associated with insomnia, diabetes, depression, heart attacks, and cancer Working hypothesis: controls the repair of oxidative stress Oxidative stress has been implicated as a cause of aging -Order: Diptera -Commonly called a fruit fly -Ideal model organism for studying genetics -Canton Strain (CS) is the wild type control -Negative feedback loop: period (per) and timeless (tim) -Positive feedback loop: clock (clk) and cycle (cyc) -per01 is the mutant loss-of-function gene -The time of day at which flies were exposed to H2O2 affected how well they were able to repair damage - per01 mutants are more susceptible to H2O2 than CSp flies at 9am and 5am -H2O2 levels in wild-type flies were higher during the day than at night -Nrf2 = transcription factor in the leucine zipper family -Keap1 = actin binding protein that is Nrf2’s repressor -A homolog of Nrf2 has been found in D. melanogaster -The gene cncC has a similar location, function, and N terminus to Nrf2 -gstD1 is an oxidative stress response gene that cncC activates Circadian genes Transcription ? Oxidative Repair Mechanisms (gstd1) -Clock genes regulate the response to oxidative stress by stimulating expression of cncC/keap1. -Prediction: expression levels of keap1, cncC and gstd1 will cycle in a 24 hour period. -Prediction: expression of keap1 and cncC, will be reduced in the per01 or cyc01 mutants. -Prediction: gstD1 should be down-regulated in the mutants. -Collect 5-day old male CSp, per01, CSc and cyc01 flies every 4 hours for 24 hours -Separate heads and bodies -Run quantitative real time polymerase chain reaction Relative Expression (RQ) gstd1 mRNA Expression Levels 2.0 Control per01 1.5 1.0 0.5 0.0 0 4 8 12 16 20 24 Hours after lights on - gstd1 mRNA is cycling with the circadian day. - May not be controlled by period. Relative Expression (RQ) cncC mRNA Expression Levels 2.0 Control per01 1.5 1.0 0.5 0.0 0 4 8 12 16 20 24 Hours after lights on - cncC does not appear to cycle in the circadian day. Relative Expression (RQ) keap1 mRNA Expression Levels 3 Control per01 2 1 0 0 4 8 12 16 20 24 Hours after lights on - keap1 does not appear to cycle in the circadian day. Relative Expression (RQ) gstd1 mRNA Expression Levels 2.5 Control Cyc 01 2.0 1.5 1.0 0.5 0.0 0 4 8 12 16 20 24 Hours after lights on - gstd1 appears to cycle in the control flies, but not in the cyc01 flies Relative Expression (RQ) cncC mRNA Expression Levels 3 Control Cyc 01 2 1 0 0 4 8 12 16 20 24 Hours after lights on - cncC is expressed at a higher level in the control flies than in the cyc01 flies. Relative Expression (RQ) keap1 mRNA Expression Levels 4 Control Cyc 01 3 2 1 0 0 4 8 12 16 20 24 Hours after lights on - keap1 is expressed at a higher level in the control flies than in the cyc01 flies. Circadian Rhythms and Light If a gene is controlled by the circadian clock it should be: Rhythmic in light-dark conditions Rhythmic in constant darkness Arrhythmic in constant light Relative Expression (RQ) gstd1 mRNA expression levels in differing light conditions 1.5 Light-Dark Constant Darkness Constant Light 1.0 0.5 0.0 8 20 8 20 8 20 Hours after lights on -gstd1 appears to be a rhythmic gene because it is cycling in LD and DD, but not cycling in LL Summary/Conclusions Specific findings: keap1 and cncC do not appear to cycle in a 24 hour period. Expression of keap1 and cncC are reduced in cyc01 mutants compared to controls. Expression of keap1 and cncC are similar in per01 mutants compared to controls. gstd1 appears to cycle in a circadian day with a peak at 5pm and a trough at 5am. Conclusion: -Unclear whether clock genes regulate the response to oxidative stress through the cncC/keap1 pathway. -gstd1 may be regulated by the circadian clock independently of cncC/keap1. Acknowledgements Jaga Giebultowicz Laura Beaver Giebultowicz Lab Members Kevin Ahern University Honors College HHMI URISC