DNA Damage and P53: Target Gene Selection and Signaling Dynamics
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DNA damage and p53: target gene selection and signaling dynamics Jacob Stewart-Ornstein Dept. Computational and Systems Biology University of Pittsburgh & Hillman Cancer Center Stress is Universal Environmental Fluctuations Poisons = Infection Radiation Stress Response Molecular Pathways that respond to cellular damage and: (1) Adjust the cell to its new environment (2) Repair Any Damage Stress responses are ubiquitous and conserved Conserved from yeast, Human Nutrient (m)TOR, RAS, GCN2/4 Heat HSF1 Oxidative NRF2 Hypoxia HIF1 ER IRE1/XBP1, PERK, ATF6 DNA damage ATM/ATR, tp53 Vihervaara… Lis, Nat. Rev. Gen., 2018 Stress responses act to restore homeostasis UPR response in Budding Yeast Response 1mM DTT 2mM DTT Growth Rate Time (~hrs) Pincus et al., 2010, PLOS Bio In multicellular systems stress responses specify cell fate Response Damage Cell death Response Resumption Cell death of growth Dose of DNA damage determines cell fate damage γH2AX Dapi Cell cycle arrest Apoptosis + Repair Or Senescence How can the cell determine its DNA damage status? damage γH2AX Dapi Apoptosis Cell cycle arrest Or + Repair Senescence Appropriate # breaks Transcriptional response Tissue specificity of DNA damage signaling # breaks Cell fate Small Intestine Spleen (5Gy IR) (5Gy Tunel , DAPI Outline (1) How does the cell respond proportionately to DNA damage? (2) How universal is the transcriptional response to DNA damage? When cells are treated with radiation IR dose # Breaks (~dose2) γH2AX Viability DAPI What is the transcriptional response to IR? IR dose # Breaks (~dose2) γH2AX Transcriptional Response? Linear Saturating Response Response dose dose What signaling Pathways are activated by DNA damage? Human Cell Lines x12 RNAseq + Ionizing Radiation What pathways are enriched? Number of of Number Genes Z-Score ΔGene Expression The transcriptional response to radiation is dominated by p53 Human Cell Lines x12 p53 Number of of Number Genes p53 (e-21) Highly conserved DNA damage responsive factor Z-Score ΔGene Expression The transcriptional response to radiation is dominated by p53 Human Cell Lines Mouse Tissues x12 x5 Number of of Number Genes p53 (e-21) p53 (e-35) Z-Score ΔGene Expression Z-Score ΔGene Expression Knockdown of p53 largely eliminates the short term transcriptional response to radiation in epithelial cells Significantly induced transcripts 6 5 4 3 2 1 Number of of Number Genes 0 p53 (e-21) Log2 Fold Change p53sh 90% (43/48) -1 p53 dependent -2 1 2 3 4 5 6 Log2 Fold Change WT Z-Score ΔGene Expression p53 Determines DNA damage Sensitivity thymocytes p53-/- p53+/- p53+/+ Lowe… Jacks, 1993, nature p53 Determines DNA damage Sensitivity thymocytes 2 p53 1.5 -/- p53 1 0.5 0 -0.5 p53+/- -1 Cisplatin Treated Fitness -1.5 +/+ p53 -2 -2 -1 0 1 2 Untreated Fitness CAS9 Screen A549 Lowe… Jacks, 1993, nature p53 is the key DNA damage responsive transcription factor DNA ATM Cell Cycle Arrest Oxidative Stress Apoptosis p53 Hypoxia DNA Repair Translational Stress DeLeo et al., 1979 Crawford et al., 1979 Metabolism Finlay et al., 1989 Farmer et al., 1992 El-Deiry et al., 1993 Harper et al., 1993 … How does the cell respond transcriptionally to DNA damage? IR dose # Breaks (~dose2) γH2AX p53? p53? dose dose Acute response to DNA damage is not dose sensitive +DNA damage p53 Log2 Dose p53 dose Longer Term response to DNA damage is dose dependent p53 Log2 Dose 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 signaling Duration of signaling is dose sensitive Response Response Need real time reporters of p53 signaling! A live cell reporter of p53 activity reveals complex dynamics MCF7 (breast cancer) p53-YFP In response to DNA damage a negative feedback loop drives oscillations of p53 DNA ATM p53 p53-YFP MDM2 In response to DNA damage a negative feedback loop drives oscillations of p53 60 p53 50 DNA YFP(AU) - 40 30 P53 0 5 10 15 20 25 30 35 250 MDM2 ATM 200 150 100 p53 0 5 10 15 20 25 30 35 p53-YFP MDM2 In response to DNA damage a negative feedback loop drives oscillations of p53 60 p53 50 DNA YFP(AU) - 40 30 P53 0 5 10 15 20 25 30 35 250 MDM2 ATM 200 150 100 p53 0 5 10 15 20 25 30 35 P53->MDM2 P53 period ~5hr MDM2 1 0.6 0.4 0.5 0.2 0 0 Autocorrelation Crosscorrelation -0.5 -0.2 0 2 4 6 8 0 1 2 3 4 Time (hrs) Time (hrs) Recapitulates results from Lahav, 2004 Given oscillations, how is dose encoded? DNA DNA ATM ATM p53 vs p53 MDM2 MDM2 In MCF7 number of ‘pulses’ of p53 is controlled by dose Signaling is duration encoded 4 3.5 Low IR (1Gy) 3 2.5 2 1.5 P53 (AU) 1 0.5 0 -0.5 0 5 10 15 20 25 Dose proportional # Pulses 4 Dose is not proportional to Amplitude 3.5 High IR (8Gy) 3 2.5 2 1.5 1 P53 (AU) 0.5 0 -0.5 0 5 10 15 20 25 Time after IR (hrs) Analysis of p53 dynamics across 12 cell lines Dose (Gy) Low IR (1Gy) High IR (8Gy) YFP (AU) - p53 Time Time Other cell lines show both duration and amplitude control Dose (Gy) Low IR (1Gy) High IR (8Gy) YFP (AU) - p53 Time Time In A549s amplitude of p53 is regulated by dose 6 Low IR (1Gy) Low IR (1Gy) 5 4 3 P53 (AU) 2 1 0 0 5 10 15 20 25 10 High IR (8Gy) 8 High IR (8Gy) 6 4 P53 (AU) 2 0 0 5 10 15 20 25 Time after IR (hrs) Why do some cell lines show amplitude vs duration response to DNA damage dose? Low IR (1Gy) High IR (8Gy) Duration MCF7 Time A549 Amplitude Chemical Screen for p53 dose response behavior High IR (8Gy) Kinase inhibitor screen for dynamics P53 (AU) Drug screen P53 (AU) Time In maximum A549 amplitude of p53 is regulated by dose High IR (8Gy) P53 (AU) Drug screen P53 (AU) Time Cell types with low ATM levels show reduced dynamic range In p53 signaling DNA MCF7 ATM act ATM p53 ATM pATM DNA damage (AU) p53 A549 ATM act ATM p53 MDM2 DNA damage (AU) Cell types with low ATM levels show reduced dynamic range In p53 signaling 8Gy IR A549 Time UO31 UACC62 6 5 4 MCF7 3 HCT116 2 frequency 1 0 -4 -3 -2 -1 0 1 2 Log ATMact (AU) A general model reconciling p53 response and ATM activity DNA DNA* ATM p53 MDM2 Stewart-Ornstein and Lahav., Science Signaling, 2017 Stewart-Ornstein and Lahav., Current Opinion is Systems Biology, 2017 Outline (1) 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 (2) Do all cells respond to DNA damage in the same way? Is the p53 transcriptional response uniform? A diverse set of cell lines to model variation in DNA damage response 12 Human Cell Lines x12 All express WT p53 Range of Radiosensitivites EC50: 1-3Gy Before and After DNA damage p53 levels are comparable Across 12 cell lines Fold change after damage 1.5-3.5 2 1.5 1 0.5 p53 Fold Change p53 Fold Change log2 p53 Change Fold 0 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 IL1A GREB1 ~5% of p53 binding sites show variability across cell lines IL1A GREB1 Why would p53 bind different locations in different cell lines? Cofactors or Chromatin 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 MCF7 LOXIMVI GREB1 IL1A variation in p53 binding can be explained by chromatin accessibility More accessible in LOXIMVI Differential atac seq signaling explains 22% of the variance in p53 binding More accessible in MCF7 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 p21 PUMA MDM2 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 Tight Regulation by Pioneering activity at chromatin ‘poised’ sites Cell type specific ‘Universal’ Binding sites DNA binding sites Are there physiological cell states that alter p53 binding? Mesenchymal and Estrogen+ Cell States are associated with novel p53 binding IL1A GREB1 EMT A549 cells when treated with TGFB undergo EMT Does p53 binding change? +TGFB Axl dapi A549 cells forced to undergo EMT show p53 binding at the IL1 locus +TGFB Axl dapi rep1 ChIPseq p53 +TGFB rep2 +TGFB IL1 expression in +TGFB EMTed dapi Axl A549 cells depends on p53 p53 ChIPseq Log2 siTP53/siCTR p53 IL1A IL1B 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? Master regulator Linear Transducer / Signal integrator Damage Cell Fate 1 Cell Fate 2 Modified from Bieging and Attardi - Trends in Cell Bio, 2015.