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