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Temporal regulation of the cell cycle in the Drosophila
wing during metamorphosis
Bu#$aLab,UniversityofMichigan
Time
Distinct states of cell cycle exit:
Kerry
Flegel
YiqinMa
ShyamaNandakumar
RosalineSun
G0
Reversible
Quiescence
Senescence
Terminal differentiation
Drosophila metamorphosis – remarkably cool, and complicated
Cellshape/idenGty
HairdifferenGaGon
Cellcyclechanges
Morphologicalchanges
The transition to G0 in the fly wing
DNA
0h APF
(After Puparium
Formation )
2h APF
Mitosis
Proliferation
S-phase
time
6h APF
24h APF
36h APF
Adult
Differentiation
18h APF
S-pase /neurons
mitoses / neurons
TemporaleventsareregulatedbythehormoneEcdysone
Ashburner1989
Howisecdysonesignalingconnectedtothecellcycle?
Regulators
M
ratelimiGngforentryto
mitosis
Stg
G2
Stg(cdc25c)
CycB/Cdk1
G1
CycE
ratelimiGngforS
phaseentry
S
Wee/Myt1
Y.Guo,K.Flegel
Howisecdysonesignalingconnectedtothecellcycle?
Time
-10h
2h
6h
18h
24h
36h
Wing
Abdominal
histoblasts
20HE
20HE
EcR/USP
EcR/USP
Broad
proliferaGng
20-HE
string
G2arrest
Broad
G0arrest
string
20-HE
miR-965
Stg
Stg
G2arrest
Mentry
Verma&Cohen
Y.Guo,K.Flegel
temporal
pa$erning
Hormone Vein/intervein
signaling
S-phase
Delta
CycA
Schubiger and Palka
The transition to G0 in the fly wing
DNA
0h APF
(After Puparium
Formation )
2h APF
Mitosis
Proliferation
S-phase
time
6h APF
24h APF
36h APF
Adult
Differentiation
18h APF
S-pase /neurons
mitoses / neurons
Two stages of G0 imply multiple
levels of cell cycle shut down
Normal cell cycle exit
E2F
0h
24h
28h
36h
Mitosis
E2F
How do we manipulate
cell cycle exit?
ReGnoblastoma
E2FtranscripGonfactor
Mitosis
M
E2F
Stg
G2
G1
CycE
S
Two stages of G0 imply multiple
levels of cell cycle shut down
Robust cell cycle exit
Normal cell cycle exit
E2F
0h
24h
How is robust G0
actively maintained
despite high level of cell
cycle regulators?
Robust G0
Flexible G0
28h
36h
Mitosis
E2F
42-46h
Mitosis
E2F
YiqinMa
Common “lockdown” models
Key cell cycle genes recruited to
periphery/constitutive heterochromatin
VanSteenselLab
Key cell cycle genes silenced by PRC
or HP1 heterochromatin formation
Dynlacht,MacLellanLabs
Origins become inaccessible
CrescenziLab
Regulatory element accessibility + RNAseq during metamorphosis
FAIRE-seq (Formaldehyde-Assisted Isolation of Regulatory Elements)
Terrence S. Furey Nature Reviews
Genetics 13, 840-852 (December 2012)
Dan McKay
UNC Chapel Hill
ChromaGnaccessibilitychangesatthenablocusduringlarval->pupalstages
L3wing
24hAPF
Becomesaccessible
36hAPF
44hAPF
72hAPF
Becomesinaccessible
nab
UNCHANGED
%Peaks
TSS/promoter
DistancetoTSS
DYNAMICAT24H
1stintron+1-5kb
upstream
DYNAMICAT44H
Chromatin accessibility changes at cell cycle genes after cell
cycle exit
Stggenelocus
CycEgenelocus
L3
Early
G0
mid
G0
Late
G0
[023
8]
[017
]
[014
]
[021
]
[0299
]
[021]
[015]
[012]
Enhancer
E2.2
E2.3
E5.3
E6.7
ProliferaGng PostmitoGc
…but few chromatin accessibility changes at most
other cell cycle genes
CyclinB
PCNA
CycA
Chromatin accessibility changes at the few
“Generals”, not the many “Soldiers”
M
ratelimiGngforentryto
mitosis
Stg
G2
G1
CycE
S
ratelimiGngforS
phaseentry
AddingexogenousCycE+StgtoE2FbypassesG0
Bypassing G0 in the fly wing using E2F+CycE+Stg
> 44 hr APF
Wing
Actin
mitoses
wingepithelium
55hrAPF
Actin
Actin
What drives the changes in accessibility at cell cycle genes in G0?
ManyquesGonsremain:
•  WhathappenstoaccessibilitywhenwebypassG0?(causeorconsequence?)
•  HowdoesMi-2impactchromaGnaccessibilityatcellcyclegenes?
•  DoesprevenGngchangesinaccessibilityleadtoamoreflexibleG0state?
•  CanwereverseanestablishedG0stateandre-opentheinaccessiblespots?
Acknowledgements
Bu#$aLab
KerryFlegel
RosalineSun
YiqinMa
OlgaGrushko
ShyamaNandakumar
Reagents:
BloomingtonStockCenter
BobDuronio
Collaborators:
DanMcKay(FAIRE-seq)
Funding:
AmericanCancerSociety
NIHNIA
U.MichiganBSSPAward