EPIGENETIC CHANGES IN RADIATION- INDUCED GENOME INSTABILITY AND CARCINOGENESIS: POWER, PROMISE AND OPPORTUNITIES

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1 EPIGENETIC CHANGES IN RADIATION- INDUCED GENOME INSTABILITY AND CARCINOGENESIS: POWER, PROMISE AND OPPORTUNITIES Olga Kovalchuk, MD/PhD University of Lethbridge, AB, Canada

2 Sources of radiation exposure: Environmental and occupational exposure Diagnostic and therapeutic exposure One third of people are likely to get cancer More than half will receive radiotherapy

3 RADIATION EFFECTS Direct or targeted Effects in the directly exposed cells Indirect or non-targeted effects Effects in the neighboring unexposed cells Persisting effects in the progeny of exposed cells

4 Indirect/Non-targeted Effects of Exposure to Ionizing Radiation Effects in unexposed cells and their progeny - in cells not directly hit. Genomic instability Bystander Effects Transgenerational effects

5 Genome instability molecular mechanisms "Genetic" - a heritable change in the DNA sequence GENETIC EPIGENETIC Epigenetic - information contained in chromatin and mediated via short RNAs, other than the actual DNA sequence. Epigenetic information defines a heritable specific gene expression pattern

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7 EPIGENETIC CHANGES Epigenetic alterations changes induced in cells that alter expression of the information on transcriptional, translational, or post-translational levels without change in DNA sequence Methylation of DNA Modifications of histones RNA-mediated modifications DNMT1 DNMT3a DNMT3b Me P U sirna, mirna, pirna Control Treated SAM SAH A A - acetylation Me - methylation P - phosphorylation U - ubiquitination

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9 SMALL INTERFERING RNAS Small non-coding RNAs refers nt regulatory RNAs acting as a major epigenetic regulators of cellular processes. A few classes of small RNAs that differ with respect to their size, nucleotide composition, biogenesis and mode of action were discovered in mammals. 1. microrna: nt, down-regulate protein translation, transcript cleavage; 2. pirna: nt, transposon silencing, establishment of epigenetic patterns in the germ line of developing embryo; 3. sirna (21-22 nt): regulation of transposable elements, pseudogenes and endogenous genes. Other classes of small regulatory RNAs with unknown function may be derived from active trna, rrna and snorna. Non-coding RNAs dominate transcriptional output in mammals, they are developmentally and stress regulated. Large number of mirnas are involved in cancer as either tumor-suppressors or oncogenes.

10 micrornas (mirs) play an integral role in gene regulation - 21nt, single stranded, RNA molecules result from the processing of primary, double-stranded transcripts Drosha Pre-miR Dicer Pasha Pri-miR NUCLEUS AAAAAAAAAA AAAAAAAAA RISC Translational Repression CYTOPLASM

11 pirnas - a distinct class of 24- to 30-nucleotidelong RNAs are generated by Dicer-independent mechanism. pirnas associate with Piwi-class Argonaute proteins. Function: germline development, silencing of selfish DNA elements, and in maintaining germline DNA integrity. Klattenhoff & Theurkauf, Development, 2006

12 LOW DOSE RADIATION-INDUCED EPIGENETIC CHANGES IN AN ANIMAL MODEL EPIGENETICS OF RADIATION-INDUCED IN VIVO BYSTANDER EFFECT EPIGENETIC MECHANISMS INVOLVED IN THE TRANSGENERTIONAL GENOME INSTABILITY THERAPEUTIC AND DIAGNOSTIC EXPOSURE CHALLENGES

13 LOW DOSE RADIATION-INDUCED EPIGENETIC CHANGES IN AN ANIMAL MODEL EPIGENETICS OF RADIATION-INDUCED IN VIVO BYSTANDER EFFECT EPIGENETIC MECHANISMS INVOLVED IN THE TRANSGENERTIONAL GENOME INSTABILITY THERAPEUTIC AND DIAGNOSTIC EXPOSURE CHALLENGES

14 Low dose radiation-induced epigenetic changes in an animal model : Objective: to dissect the epigenetic basis of induction of the low dose radiationinduced genome instability and adaptive response and the specific fundamental roles of epigenetic changes (i.e. DNA methylation, histone modifications and mirnas) in their generation. Approach: we utilize an in vivo murine model to study epigenetic alterations in the radiation-target organs thymus and spleen in context of low dose radiation effects and adaptive responses. We also archive and analyze other tissues gonades, brain and liver. Results: Exposure Time points Organs Endpoints 0 Gy (sham) 0.01 Gy 0.1 Gy 1 Gy 10 x 0.01 Gy 0.01Gy prime followed by 1 Gy challenge 6 hours 96 hours 4 weeks thymus spleen Global and locus-specific DNA methylation analysis Global histone modification analysis Analysis of micrornaome DNA damage analysis by H2AX foci Genome stability analysis Gene expression analysis In this study, we for the first time found that low dose radiation (LDR) exposure causes profound and tissue-specific epigenetic changes in the exposed tissues We established that LDR exposure affects methylation of repetitive elements in the genome, causes changes in histone methylation, acethylation and phosphorylation Importantly, LDR causes profound and persistent effects on small RNAs profiles. MicroRNAs are excellent biomarkers of LDR exposure. LDR exposure causes tissue-specific changes in gene expression. We identified several novel biomarkers of LDR exposure.

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18 LOW DOSE RADIATION-INDUCED EPIGENETIC CHANGES IN AN ANIMAL MODEL EPIGENETICS OF RADIATION-INDUCED IN VIVO BYSTANDER EFFECT EPIGENETIC MECHANISMS INVOLVED IN THE TRANSGENERTIONAL GENOME INSTABILITY THERAPEUTIC AND DIAGNOSTIC EXPOSURE CHALLENGES

19 Radiation-induced bystander effects in spleen CT control animal CT control animal 0 Gy 0 Gy B - completely exposed animal B - completely exposed animal 1 Gy 1 Gy H - head exposed animal H - head exposed animal lead shielding 1 Gy lead shielding 1 Gy BALBc C57BL/6

20 The main findings are: Cranial irradiation leads to: elevated levels of DNA damage increased p53 expression altered levels of cellular proliferation and apoptosis altered DNA methylation levels The observed bystander changes were not caused by radiation scattering, and were observed in two different mouse strains, C57BL/6 and BALB/c, although certain strain specificity was noticed.

21 Whole body and cranial radiation exposure induces the significant and sex-specific loss of DNA methylation in the murine spleen BS IR CT male * * * * 96 H 6 H BS female IR CT * * 96 H 6 H % of unmethylated CCGG sites in genome as compared to control CT control animals; IR body-exposed animals; BS bystander/head-exposed animals

22 The other sex-specific responses to whole-body and cranial exposure in the spleen tissue included: Changes in apoptosis Proliferation changes MicroRNAome alterations Changes in the gene expression Sex hormones are involved in the sex-specificity of the radiation responses

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24 Sequencing: Illumina GAIIx; Single-End; multiplexed; 36 cycles; 6 samples per lane, each sample was run in duplicate across two lanes. Reference: Rat, rn4 UCSC, downloaded from Illumina igenome site. Amongst various changes, we identified a predicted gene, which was strikingly upregulated in all of the male brain regions studied, but completely unaffected in females. Sequence within these coordinates did not contain any long open reading frames (ORFs), so the gene is most probably non-coding. There were no CpG islands found in the vicinity (2000 bp up- and downstream) of the gene. High confidence transcription binding sites as predicted by TFSEARCH software include CdxA, GATA-1, SRY, HFH-2, p300 and a couple of others. Interestingly, SRY is the sex determination factor which is expressed only in males, that may explain why this gene is upregulated in sex-specific manner. On the other hand, p300 is the histone acetyltransferase, which points at why this gene can stay activated long time after exposure?

25 LOW DOSE RADIATION-INDUCED EPIGENETIC CHANGES IN AN ANIMAL MODEL EPIGENETICS OF RADIATION-INDUCED IN VIVO BYSTANDER EFFECT EPIGENETIC MECHANISMS INVOLVED IN THE TRANSGENERTIONAL GENOME INSTABILITY THERAPEUTIC AND DIAGNOSTIC EXPOSURE CHALLENGES

26 Epigenetic mechanisms involved in the transgenertional genome instability?????

27 Paternal radiation exposure affects DNA methylaton of LINE 1 and SINE B2 retrotransposable elements in the thymus tissue of the progeny Relative qpcr amplification LINE1 * SINE B2 4 * 2 0 CT EX CT EX

28 Change from control, % Paternal radiation exposure decreases LSH protein levels in the thymus of progeny in vivo LSH1 levels CT * EX LSH1 loading

29 Paternal radiation exposure results in micrornaome deregulation in the thymic tissue of the unexposed progeny DICER positive cells per field of view progeny of control progeny of exposed 3 DICER levels 2 p progeny of control progeny of exposed

30 Radiation exposure alters micrornaome of mouse testes

31 Relative qpcr amplification Change from control, % Radiation exposure alters mirna expression and methylation of LINE 1 and SINE B2 retrotransposons in male germline A B mirna Fold induction Target protein mir-29a 2.33 DNMT3a, DNMT3b mir-29b 1.23 DNMT3a, DNMT3b DNMT3a actin DNMT3a levels CT * EX C CT LINE1 SINE B2 * EX CT 1 2 EX

32 ANALYSIS OF RASIRNA (PIRNA) MOLECULES BY DEEP SEQUENCING all LTR LINE SINE low complexity DNA simple repeat CT EX

33 Parental germline Progeny DNA damage? Aberrant setting of methylation marks Altered DNA methylation Altered mirnaome Loss of pirnaome/ rasirnaome? Aberrant gene (including small RNAs) gene expression?? Downstream snowball effects?

34 LOW DOSE RADIATION-INDUCED EPIGENETIC CHANGES IN AN ANIMAL MODEL EPIGENETICS OF RADIATION-INDUCED IN VIVO BYSTANDER EFFECT EPIGENETIC MECHANISMS INVOLVED IN THE TRANSGENERTIONAL GENOME INSTABILITY THERAPEUTIC AND DIAGNOSTIC EXPOSURE CHALLENGES

35 Analysis of DNA methylation, gene expression and apoptosis in brain cancer cells lines reveals a potential anti-tumor effect of low dose radiation in neuroblastoma and an opposite tumor-promoting effect in malignant glioma

36 Gene-specific DNA methylation and gene expression changes induced by low dose radiation in human neuroblastoma (A-172 and IMR-32) and glioma cells (SK-N-BE) DNA methylation A-172 IMR-32 SK-B-NE 24 hours hours Gene expression A-172 IMR-32 SK-B-NE 24 hours hours Correlation between the levels of gene expression, methylation and apoptosis in the studied neuroblastoma and glioma cells DNA methylation A-172 IMR-32 SK-B-NE 24 hours hours Gene expression A-172 IMR-32 SK-B-NE 24 hours hours Apoptosis A-172 IMR-32 SK-B-NE 24 hours hours

37 Epigenetics a new frontier in radiation research

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39 Advances are made by answering questions. Discoveries are made by questioning answers. Bernard Haisch

40 Kovalchuk group Slava Ilnytsky Jody Filkowski Matt Merrifield Bo Wang Dongping Li Rocio Rodriguez-Juarez Anna Kovalchuk Lidia Luzhna Corinne Sidler Alumni Jason Novak Jan Tamminga Kristy Kutanzi Igor Koturbash Mike Lowings Jonathan Loree James Meservy Natasha Singh Joel Stimson Munima Alam Paul Walz Acknowledgements Collaborators: Bryan Kolb, CCBN Eugene Berezikov, Hubrecht Institute Igor Pogribny, NCTR William Bonner, NCI/NIH Olga Martin, PeterMaccallum Cancer Center, Ausralia Funding: CIHR Institute of Gender, Sex and Health Chair Program

41 When used right, technology becomes an accelerator of momentum, not a creator of it. Contacts: Dr. Igor Kovalchuk igor.kovalchuk@uleth.ca