Circadian Regulation of Oxidative Stress Response Genes, CncC, and Keap1 in Drosophila melanogaster

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Oxidative Stress Response Genes, cncc, keap1, and
gstd1 A Circadian Connection?
Marisa McAllister Dr. Jaga Giebultowicz Lab
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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

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Drosophila melanogaster

• Order Diptera
• Commonly called a fruit fly
• Ideal model organism for studying genetics

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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)

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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

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Nrf2 and Keap1

• Nrf2 transcription factor in the leucine zipper
  family
• Keap1 actin binding protein that is Nrf2s
  repressor

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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

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Pathway

• Circadian genes
• Transcription
• Oxidative Repair Mechanisms (gstd1)

?
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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.

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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

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gstd1 mRNA Expression Levels
- gstd1 mRNA is cycling with the circadian day. -
May not be controlled by period.
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cncC mRNA Expression Levels
- cncC does not appear to cycle in the circadian
day.
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keap1 mRNA Expression Levels
-
- keap1 does not appear to cycle in the circadian
day.
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gstd1 mRNA Expression Levels
- gstd1 appears to cycle in the control flies,
but not in the cyc01 flies
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cncC mRNA Expression Levels
- cncC is expressed at a higher level in the
control flies than in the cyc01 flies.
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keap1 mRNA Expression Levels
- keap1 is expressed at a higher level in the
control flies than in the cyc01 flies.
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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

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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
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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.
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Acknowledgements

• Jaga Giebultowicz
• Laura Beaver
• Giebultowicz Lab Members
• Kevin Ahern
• University Honors College
• HHMI
• URISC