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DNA damage and p53: target gene selection and signaling dynamics
Jacob Stewart-OrnsteinDept. Computational and Systems Biology
University of Pittsburgh & Hillman Cancer Center
Stress is Universal
Stress Response
Molecular Pathways that respond to cellular damage and:
(1) Adjust the cell to its new environment
(2) Repair Any Damage
=Environmental FluctuationsPoisonsInfectionRadiation
Stress responses are ubiquitous and conserved
Vihervaara… Lis, Nat. Rev. Gen., 2018
Conserved from yeast, Human
Nutrient (m)TOR, RAS, GCN2/4Heat HSF1Oxidative NRF2Hypoxia HIF1ER IRE1/XBP1, PERK, ATF6DNA damage ATM/ATR, tp53
Gro
wth
Rat
eRe
spon
se
Time (~hrs)
Stress responses act to restore homeostasis
1mM DTT2mM DTT
Pincus et al., 2010, PLOS Bio
UPR response in Budding Yeast
Resp
onse
In multicellular systems stress responses specify cell fate
Damage
Cell deathResumption of growth
Resp
onse
Cell death
Dose of DNA damage determines cell fate
ApoptosisOr
Senescence
Cell cycle arrest+ Repair
damage
γH2AXDapi
How can the cell determine its DNA damage status?
ApoptosisOr
Senescence
Cell cycle arrest+ Repair
damage
# breaksAppropriate Transcriptional response
γH2AXDapi
Tissue specificity of DNA damage signaling
DAPI
,Tun
el(5
Gy IR
) Small Intestine Spleen
# breaks Cell fate
Outline
How universal is the transcriptional response to DNA damage?
(1)
(2)
How does the cell respond proportionately to DNA damage?
IR dose
When cells are treated with radiation
# Breaks (~dose2)
Viability
γH2AX
DAPI
IR dose
What is the transcriptional response to IR?
# Breaks (~dose2)
γH2AX
Transcriptional Response?
Linear Saturating
dose
Resp
onse
doseRe
spon
se
What signaling Pathways are activated by DNA damage?
Z-Score ΔGene Expression
Num
ber o
f Gen
es
Human Cell Lines
x12
RNAseq + Ionizing RadiationWhat pathways are enriched?
The transcriptional response to radiation is dominated by p53
Z-Score ΔGene Expression
Num
ber o
f Gen
es
Human Cell Lines
p53 (e-21)
x12
Highly conservedDNA damage responsive factor
p53
The transcriptional response to radiation is dominated by p53
Z-Score ΔGene Expression
Num
ber o
f Gen
es
Human Cell Lines
p53 (e-21)
x12
Z-Score ΔGene Expression
Mouse Tissues
p53 (e-35)
x5
1 2 3 4 5 6
Log2 Fold Change WT
-2
-1
0
1
2
3
4
5
6
Log2
Fol
d C
hang
e p5
3sh
Knockdown of p53 largely eliminates the short term transcriptional response to radiation in epithelial cells
Z-Score ΔGene Expression
Num
ber o
f Gen
es
p53 (e-21)
Significantly induced transcripts
90% (43/48)p53 dependent
p53 Determines DNA damage Sensitivity
Lowe… Jacks, 1993, nature
thymocytes
p53-/-
p53+/-
p53+/+
p53 Determines DNA damage Sensitivity
thymocytes
Lowe… Jacks, 1993, nature
p53-/-
p53+/-
p53+/+
Untreated Fitness
-2 -1 0 1 2
Cis
plat
in T
reat
ed F
itnes
s
-2
-1.5
-1
-0.5
0
0.5
1
1.5
2 p53
CAS9 Screen A549
p53 is the key DNA damage responsive transcription factor
ATM
p53
DNA
Cell Cycle Arrest
Apoptosis
DNA Repair
DeLeo et al., 1979 Crawford et al., 1979Finlay et al., 1989Farmer et al., 1992El-Deiry et al., 1993Harper et al., 1993…
Metabolism
Hypoxia
Oxidative Stress
TranslationalStress
IR dose
How does the cell respond transcriptionally to DNA damage?
# Breaks (~dose2)
p53?
γH2AX
dose dose
p53?
Acute response to DNA damage is not dose sensitiveLo
g2 D
ose
dose
p53
+DNA damage
p53
Longer Term response to DNA damage is dose dependent Lo
g2 D
ose
p53
dose
Across 12 human cell lines the acute p53 response is dose invariant, but the longer term response depends on DNA damage
Models of p53 signalingDuration of signaling is dose sensitive
Resp
onse
Resp
onse
Need real time reporters of p53 signaling!
A live cell reporter of p53 activity reveals complex dynamics
p53-YFPMCF7 (breast cancer)
In response to DNA damage a negative feedback loop drives oscillations of p53
ATM
p53
MDM2
DNA
p53-YFP
In response to DNA damage a negative feedback loop drives oscillations of p53
ATM
p53
MDM2
DNA
0 5 10 15 20 25 30 3530
40
50
60
0 5 10 15 20 25 30 35100
150
200
250
p53
MDM2
P53-
YFP(
AU)
p53-YFP
In response to DNA damage a negative feedback loop drives oscillations of p53
ATM
p53
MDM2
DNA
0 5 10 15 20 25 30 3530
40
50
60
0 5 10 15 20 25 30 35100
150
200
250
Time (hrs)
0 2 4 6 8
Auto
corre
latio
n
-0.5
0
0.5
1
Time (hrs)
0 1 2 3 4
Cro
ssco
rrela
tion
-0.2
0
0.2
0.4
0.6
p53
MDM2
P53 period ~5hrP53->MDM2
P53-
YFP(
AU)
Recapitulates results from Lahav, 2004
Given oscillations, how is dose encoded?
ATM
p53
MDM2
DNA
vs
ATM
p53
MDM2
DNA
0 5 10 15 20 25-0.5
0
0.5
1
1.5
2
2.5
3
3.5
4
P53
(AU
)P5
3 (A
U)
0 5 10 15 20 25-0.5
0
0.5
1
1.5
2
2.5
3
3.5
4
Low IR (1Gy)
High IR (8Gy)
In MCF7 number of ‘pulses’ of p53 is controlled by doseSignaling is duration encoded
Dose proportional # PulsesDose is not proportional to Amplitude
Time after IR (hrs)
p53-
YFP
(AU
)
Dose (Gy)
Analysis of p53 dynamics across 12 cell lines
Time Time
Low IR (1Gy) High IR (8Gy)
p53-
YFP
(AU
)
Dose (Gy)
Other cell lines show both duration and amplitude control
Low IR (1Gy) High IR (8Gy)
Time Time
0 5 10 15 20 25
0
1
2
3
4
5
6
0 5 10 15 20 25
0
2
4
6
8
10
In A549s amplitude of p53 is regulated by doseP5
3 (A
U)
P53
(AU
)
Low IR (1Gy)
High IR (8Gy)
Time after IR (hrs)
Low IR (1Gy)
High IR (8Gy)
Why do some cell lines show amplitude vs duration response to DNA damage dose?
Time
Low IR (1Gy) High IR (8Gy)
Ampl
itude
Duration
MCF7
A549
High IR (8Gy)
Time
P53
(AU
)P5
3 (A
U)
Drug screen
Chemical Screen for p53 dose response behavior
Kinase inhibitor screen for dynamics
High IR (8Gy)
Time
P53
(AU
)P5
3 (A
U)
Drug screen
In maximum A549 amplitude of p53 is regulated by dose
ATM
p53
MDM2
DNA
pATM DNA damage (AU)
DNA damage (AU)AT
M a
ctAT
M a
ct
MCF7
A549
Cell types with low ATM levels show reduced dynamic rangeIn p53 signaling
p53
p53
-4 -3 -2 -1 0 1 20
1
2
3
4
5
6
freq
uenc
y
Log ATMact (AU)
A549UO31UACC62
MCF7HCT116
Cell types with low ATM levels show reduced dynamic rangeIn p53 signaling
Time
8Gy IR
Stewart-Ornstein and Lahav., Science Signaling, 2017Stewart-Ornstein and Lahav., Current Opinion is Systems Biology, 2017
A general model reconciling p53 response and ATM activity
ATM
p53
MDM2
DNA*DNA
Outline
Do all cells respond to DNA damage in the same way?Is the p53 transcriptional response uniform?
(1)
(2)
How does the cell respond proportionately to DNA damage?
-duration or amplitude of p53 signaling are proportional to DNA damage-balance between amplitude and duration signaling is cell lines specific-ATM is a regulator of the dynamic range of p53
A diverse set of cell lines to model variation in DNA damage response
x12
12 Human Cell Lines
All express WT p53
Range of Radiosensitivites
EC50: 1-3Gy
Before and After DNA damage p53 levels are comparableAcross 12 cell lines
p53
Fold
Cha
nge
0
0.5
1
1.5
2
p53
Fold
Cha
nge
log2
Fold change after damage
1.5-3.5
What is p53 doing in each cell line?
What is p53 binding to?
ChIP-seq to measure p53 binding in the genome
p53 binding in the genome is quite stereotyped
2hrs after IR treatment (4Gy)
Different cell lines show quantitatively similar binding
Pearson R in dataset ranged from 0.5-0.75
p53 binding is stereotyped, with some exceptions
Some universal, some cell type specific p53 targets
GREB1IL1A
~5% of p53 binding sites show variability across cell linesGREB1IL1A
Why would p53 bind different locations in different cell lines?
Cofactors orChromatin Access?
Variation in p53 binding can be explained by chromatin accessibility (measured with Mu-Transposase ATACseq)
MCF7
LOXIMVI
variation in p53 binding can be explained by chromatin accessibility
GREB1 IL1A
MCF7
LOXIMVI
variation in p53 binding can be explained by chromatin accessibility
Differential atac seqsignaling explains 22% of the variance in p53 binding
More accessible in MCF7
More accessible in LOXIMVI
Does chromatin regulate p53 binding or does p53 regulate chromatin?
Regulated by Chromatin “pioneer factor”
No. For core targets of p53 chromatin does not need to be open
PUMA MDM2p21
Consistent with Genome Res. 2015 Sammons... Berger.
Treatment with DNA methylase inhibitor to test effect of increased chromatin accessibility
Decitabine
Treatment with DNA methylase inhibitor alters chromatin state in MCF7 cells and opens up new p53 binding sites
Decitabine
p53 is both a pioneer and a follower
Pioneering activity at ‘poised’ sites
Tight Regulation by chromatin
‘Universal’ Binding sitesCell type specificDNA binding sites
Are there physiological cell states that alter p53 binding?
Mesenchymal and Estrogen+ Cell States are associated with novel p53 binding
GREB1IL1A
EMT
A549 cells when treated with TGFB undergo EMTDoes p53 binding change?
+TGFB
Axldapi
A549 cells forced to undergo EMT show p53 binding at the IL1 locus+T
GFB
+TGFB
Axldapi
p53
ChIP
seq
rep1
rep2
IL1 expression in EMTed A549 cells depends on p53+T
GFB
+TGFB
Axldapi
p53
ChIP
seq
p53
IL1A
IL1B
Log2
siTP
53/s
iCTR
What happens to p53 binding when a cell becomes cancerous?
57% of cancers have WT p53
Conclusions
Dose of DNA damage alters the duration and amplitude of p53 signaling-balance is controlled by ATM levels
Chromatin tunes p53 binding imparting cell type specificity to the DNA damage response-- Some binding sites are ‘poised’ for p53 binding-- Other binding sites require exogenous stimuli
How to think about p53?
Linear TransducerMaster regulator
/Signal integrator
Modified from Bieging and Attardi - Trends in Cell Bio, 2015.
Damage
Cell Fate 1 Cell Fate 2
LahavAdrian GranadaCaroline MockTonia HafnerGalit Lahav
NovartisBill Forrester
Acknowledgements
Current FundingNCI R00 (JSO)Hillman Cancer Center
Past FundingLudwig Cancer CenterNIGMS Novartis
Stewart-Ornstein Lab
WeisslederRalph WeisslederYoshi Iwamoto
Jacob Stewart-OrnsteinPinakin Pandya Lyubov KubloShayla Goller
LAB