Summer Undergraduate Research Program - Fred Hutchinson ...

2009 Best Poster Presentation Award
34 A genetic screen for regulators of cell cycle exit
Charlotte Besson ,Jennifer L. Bandura, Huaqi Jiang, Derek Nickerson, and Bruce A. Edgar
Fred Hutchinson Cancer Research Center, Division of Basic Sciences, 1100 Fairview Ave. N., Seattle, WA 98109
1. Abstract:
3. Results: 2L mutant lines #28 and #30 4. Conclusion / Discussion
Uncontrolled proliferation is one of the main characteristics of a
cancer cell, usually triggered by mutations in cell cycle regulator genes. Some #30 mutant cells are cycling at 24h APF
Cdc2 might be involved in cell cycle exit
Most cells permanently exit the cell cycle during terminal differentiation,
but mechanisms involved in cell cycle exit and in its conservation after
differentiation are not totally understood. A feedback loop exists between
E2F and Cyclin/Cdk complexes involved in G1/S transition (A). In
Drosophila, it had been shown that upon terminal differentiation, this
feedback loop disappears (B). Furthermore, it has been shown that RBF
and Dap alone cannot explain cell cycle exit, indicating that unknown
inhibitors of E2F and/or CyclinE/Cdk2 are involved in that process(C). To
identify what those inhibitors are, we performed a genetic screen, set up
to identify new genes involved in silencing cell cycle gene transcription
upon differentiation in the eye.
Cycling cells Upon differenciation Double insurance
mecanism
2. Background:
pcna-miniW + and genetic screen
(A) The pcna-miniW + (A) The pcna-miniW reporter : A single E2F binding site within the PCNA
+ reporter : A single E2F binding site within the PCNA
enhancer/promoter is necessary for G1/S-directed gene expression.
The white gene results in red eye color in flies if expressed at 1-2 days
after puparium formation (APF), a time when cell divisions have
normally ceased, so as E2F activity. Flies with a W- background
carrying this reporter had been mutagenised with EMS.
(B) When cell cycle exit isn’t delayed, flies have a white eye phenotype.
(C) When a mutation induces a cell cycle exit delay or bypass, E2F is still
activated during the white gene critical period. We then screened for
flies with a red eye phenotype.
A
A’
DNA GFP PH3
(A,A’) - Mutant clones of #30 in pupal eyes 24H APF presenting a PH3
staining, showing that those cell D’ are still undergoing mitosis at
this time point.
(B,B’) - Mutant clones of #30 in pupal eyes 24H APF presenting a BrdU
incorporation staining, showing that those cells are still
undergoing S-phase phase at this time point.
Mutant clones are marked by the absence of GFP
Some #30 mutant cells are undergoing mitosis
at 28h APF
A’
163
GFP
PH3
#30 mutant clones in pupal eyes 28H APF presenting a PH3 staining,
showing that those cell are still undergoing mitosis.
Mutant clones are marked by the absence of GFP
#28 and #30 are mutated in cdc2 ED L1 L2 L3 Pupa Adult
A.
B
cdc2E1-9 B
cdc2 and # 28 mutant clones:
B
- Delay of cell cycle exit from 24h APF to ~ 28h APF
- Not obvious when clones were induced by hsFLP at 60H AED
High levels of cdc2 are required in asymmetric divisions. Low levels of
cdc2 do not affect mitosis entry, but disturb asymmetrically localized
proteins during mitosis. (Tio et al, 2001)
miniW +
E2F
A.
PCNA
ATG
~230 bp PCNA enhancer /
promoter
miniW
W
B.
- promoter
Background
10 Candidat genes:Mutant Lines Available for 3
CG5091 Glucosyltransferase activity (1 line)
Cdc2 Cyclin-dependent protein kinase activity (5 lines)
Da Transcription factor
Screening
EMS mutagenesis C. #28 (687): C=>T
#30 (274): G=>A
Arginine
Proline
B. WT
24h APF
W
(A) Recombination mapping predicted
10821061 +/- ~334kb. We
complementation crosses with
ED L1 L2 L3 Pupa Adult
Cell cycle exit upon
reduce the region of interest
region).
+
Cell cycle exit upon
differentiation
C. Delayed cell cycle exit 24h APF
W
ED L1 L2 L3 Pupa Adult
region).
(B) The 2L: 10381211; 10406409 region
and #30 mutant lines failed to complement
for cdc2, indicating that those lines
partner.
(C) After sequencing cdc2 in both line
#28 and #30 are mutant for cdc2
+
C. Delayed cell cycle exit
#28 and #30 are mutant for cdc2
as an amorph allele: cdc2E1-9 10 Candidat genes:Mutant Lines Available for 3 Complementation
Glucosyltransferase activity (1 line) Yes
dependent protein kinase activity (5 lines) NO
(2 lines) Yes
Mapping confirmation: rescue experiments (for lethality and cell
cycle exit phenotype : system Gal-4, UAS-cdc2)
Does a KO of cdc2 present the same phenotype? (RNAi line
experiment)
Are mutant clones developmentally delayed? (Staining for
differentiation markers)
Arginine => Glutamine (123)
Proline => Serine(242)
Do levels of cdc2 affect the cell cycle exit phenotype? (Heat shock
at different time points, temperature sensitive allele of cdc2)
predicted that mutations should be at 2L
tested regions in orange by
deficiency lines. This allowed us to 5. References
ED L1 L2 L3 Pupa Adult
Cell cycle exit upon
differentiation
to 2L: 10381211; 10406409. (red
region had 10 candidates genes. #28
complement with 5 deficiency lines
lines are mutated in cdc2 or in a cdc2
lines, we have been able to show that
2. #30 had previously been described
Buttitta, L. A., Katzaroff, A. J., Perez, C. L., de la Cruz, A., and Edgar, B. A.
(2007) A double-assurance mechanism controls cell cycle exit upon
terminal differentiation in Drosophila. Developmental Cell 12: 631-643.
Thacker, S. A., Bonnette, P. C., and Duronio, R. J. (2003) The contribution
of E2F-regulated transcription to Drosophila PCNA gene function.
Current Biology 13: 53-58.
Tio, M., Udolph, G., Yang, X. et al (2001). Cdc2 links the Drosophila cell
cycle and asymmetric division machineries. Nature, 409: 1063-1067
B’
B’
10821061
GFP BrdU
The Cell cycle exit phenotype is not evident when clones
are induced later (with an hsFLP system, instead of eyFLP):
an low levels of cdc2 affect cell cycle exit?
Cdc2 and Cell cycle exit phenotype:
Hypothesis
Cdc2 level EyFLP hsFLP
24 h
APF
Threshold
level of cdc2:
For cell cycle
exit phenotype
For G2/M
transition
Cdc 2 needs to be present in high level during differentiation to
induce cell cycle exit.
Work in progress