Transcript Circadian Regulation of Oxidative Stress Response Genes, CncC

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