Dr. Jaga Giebultowicz Lab 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

Drosophila melanogaster Order: Diptera Commonly called a fruit fly Ideal model organism for studying genetics Ideal for studying genetics because it has a short life cycle, extensive collection of available mutants, low cost, and a sequenced genome

Circadian Rhythms Canton Strain (CS) is the wild type control Negative feedback loop: period (per) and timeless (tim) Positive feedback loop: clock (clk) and cycle (cyc) Per and tim are in the cytoplasm and their transcription is activated by clk and cyc. Clk and cyc bind to the E-box an enhancer element in their promoters. Per and tim transcripts peak during the early evening and their proteins associate with each other and enter the nucleus and suppress clk and cyc Per and tim enter nucleus and inhibit clk and cyc. They are then degraded and clk and cyc can activate transcription again, starting a new cycle

Circadian regulation of response to oxidative stress 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 Example of mutant arrhythmic: wild-type flies become active before lights on in anticipation of lights and per-null flies become active in response to light Levels of hydrogen peroxide peak during the day and decline at night in wild-type flies. They are constant throughout the day in per01 flies. This is previous research that has been completed in Dr. Giebultowicz’s lab

Nrf2 and Keap1 Nrf2 = transcription factor in the leucine zipper family Keap1 = actin binding protein that is Nrf2’s repressor Keap1 restricts Nrf2 to the cytoplasm during unstressed conditions. When an oxidant or electrophile is present Keap1 senses it and releases Nrf2. Nrf2 enters the nucleus and forms a dimer with Maf (Musculo-Aponeurotic Fibrosarcoma) protein. Dimer binds to the ARE (Antioxidant Response Element) causing upregulation of antioxidant enzymes and detoxifying proteins In this picture Oltipraz is sensed by Keap1 and allow Keap1 to release Nrf2 Oltipraz is a drug used in cancer prevention by activating this pathway

Nrf2 in D. melanogaster -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 CncC mRNA is expressed in larvae in the alimentary canal and Nrf2 is expressed in the mammalian digestive tract. CncC is also located in the Malpighian tubules which are detoxifying organs and in the salivary glands gstD1 is an oxidative stress response gene that encodes for a detoxification enzyme. Its transcription unit is preceded by a consensus ARE sequence. gstD1 levels were increased by keap1 knockdown and decreased by CncC knockdown because then CncC was no longer present to bind to ARE and induce transcription of detoxifying enzymes

Circadian genes Transcription Oxidative Repair Mechanisms (gstd1) Pathway Circadian genes Transcription Oxidative Repair Mechanisms (gstd1) ?

Hypothesis -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.

Methods -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 Determine gene expression levels of CncC, Keap1, gstD1 in wild-type and mutant flies

gstd1 mRNA Expression Levels - gstd1 mRNA is cycling with the circadian day. - May not be controlled by period.

cncC mRNA Expression Levels - cncC does not appear to cycle in the circadian day.

keap1 mRNA Expression Levels - - keap1 does not appear to cycle in the circadian day.

gstd1 mRNA Expression Levels - gstd1 appears to cycle in the control flies, but not in the cyc01 flies

cncC mRNA Expression Levels - cncC is expressed at a higher level in the control flies than in the cyc01 flies.

keap1 mRNA Expression Levels - 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

gstd1 mRNA expression levels in differing light conditions -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