Radiation genetics, epigenetics and effects on clock genes Yuri E - - PowerPoint PPT Presentation

Radiation genetics, epigenetics and effects on clock genes Yuri E Dubrova

yed2@le.ac.uk Department of Genetics University of Leicester, UK

Radiation genetics = target theory Independently developed in 1949 by NV Timofeev-Ressovsky & DE Lea

Random targeting DNA damage DNA repair Chromosome aberrations Gene mutations Everything happens in the directly irradiated cell & mutation induction occurs at the radiation-damaged sites (targets) The yield of mutations is proportional to the amount of initial DNA damage & efficiency of its repair, i.e. depends on the dose, dose-rate & type of irradiation The risk of exposure to ionising radiation is described by the Linear No-Threshold Model

5 10 15 20 25 30 35 40 Population doublings 0.00 0.05 0.10 0.15 0.20 Aberrations/cell 5 10 15 20 25 30 35 40 Population doublings 0.00 0.05 0.10 0.15 0.20 Aberrations/cell

Yield of chromatid aberrations in MCF10A cells Yield of chromatid aberrations in MCF10A cells γ-irradiated γ-irradiated control control

From: Ullrich & Ponnaiya, 1998, Int J Radiat Biol 74, 747

Radiation-induced genomic instability in somatic cells Delayed mutations occur many cell divisions after exposure Everything happens in the directly irradiated cell & mutation induction

• ccurs at the radiation-damaged sites

X

Mutant

F0 F1 F2 F0 F1

What about the germline?

Are they unstable?

How to analyse?

Mutation frequency Instability in the non-exposed

• ffspring of irradiated parents

Progenitor Mutant 1 Mutant 2 Gain of repeats Loss of repeats

4-10 bp repeats, 100 bp - 20 kb arrays, non-coding Very spontaneous mutation rate (up to 15% per gamete) Mutations result in the loss/gain of repeats 4-10 bp repeats, 100 bp - 20 kb arrays, non-coding Very spontaneous mutation rate (up to 15% per gamete) Mutations result in the loss/gain of repeats Mouse Expanded Simple Tandem Repeat (ESTR) loci

♂ ♀ Mutants

Father Mother Paternal mutations Maternal mutation

Pedigree approach Single-molecule PCR approach

ESTR mutation detection in the germline & somatic tissues

F0 F1 F2 0.5 Gy of fission neutrons

Let’s go transgenerational…

From: Dubrova et al., 2000, Nature 405, 37

Father Mother Paternal mutations Maternal mutation

Transgenerational germline instability in the F1

• ffspring of

CBA/H male mice exposed to 0.5 Gy of fission neutrons

control F0 exposed F1 males F1 females F1 total Group 0.0 0.1 0.2 0.3 0.4 0.5 Mutation rate, 95% CI control F0 exposed F1 males F1 females F1 total Group 0.0 0.1 0.2 0.3 0.4 0.5 Mutation rate, 95% CI

5.6-fold 5.6-fold

control F0 exposed F1 males F1 females F1 total Group 0.0 0.1 0.2 0.3 0.4 0.5 Mutation rate, 95% CI control F0 exposed F1 males F1 females F1 total Group 0.0 0.1 0.2 0.3 0.4 0.5 Mutation rate, 95% CI

5.6-fold 5.6-fold 5.2-fold 5.2-fold 3.7-fold 3.7-fold 4.5-fold 4.5-fold

From: Dubrova et al., 2000, Nature 405, 37

The non-exposed offspring of irradiated parents are unstable The non-exposed offspring of irradiated parents are unstable

Is transgenerational instability strain-specific?

F0 F1 F1 F2 F2 F3 F3

♂ ♀

CBA/H BALB/c C57BL/6J

Fission neutrons, 0.4 Gy: CBA/H; C57BL/6 Acute X-rays, 2 Gy: CBA/H Acute X-rays, 1 Gy: BALB/c Fission neutrons, 0.4 Gy: CBA/H; C57BL/6 Acute X-rays, 2 Gy: CBA/H Acute X-rays, 1 Gy: BALB/c

From: Barber et al., 2002, PNAS 99, 6877-82

C57BL CBA/H BALB/c

0.0 0.1 0.2 0.3 0.4 ESTR mutation rate, 95% CI

C57BL CBA/H BALB/c

0.0 0.1 0.2 0.3 0.4 ESTR mutation rate, 95% CI

From: Barber et al., 2002, PNAS 99, 6877-82

Control F1 F2

ESTR mutation rates are elevated in both generations of all inbred strains ESTR mutation rates are elevated in both generations of all inbred strains Transgenerational instability in three inbred mouse strains

Is transgenerational instability tissue-specific?

From: Barber et al., 2006, Oncogene 25, 7336-42; 2009, Mutat Res 664, 6-12

Transgenerational instability in the germline & somatic tissues

BALB/c CBA/Ca

ESTR mutation rates are equally elevated in the germline & somatic tissues ESTR mutation rates are equally elevated in the germline & somatic tissues

Is transgenerational instability specific for tandem repeat loci?

CBA/Ca BALB/c 5 10 15 20 Hprt mutation frequency x 10-6, 95% CI 24 30 33 36 39 42 48

Hours after partial hepatectomy 0.3 0.4 0.5 0.6 Frequency of chrmosome aberrations

Chromosome aberrations in the F1

• ffspring of irradiated rats

From: Barber et al., 2006, Oncogene 25, 7336-42 From: Vorobtsova, 2000, Mutagenesis 15, 33-38

3.3-fold

Control F1

3.7-fold

F1

• f irradiated males

F1

• f irradiated males

Control Control

Transgenerational instability at the mouse hprt locus A genome-wide destabilisation A genome-wide destabilisation

hprt is X-linked gene ♂ ♀ ♂ ♂ ♀ ♀ XY XX XY XY

For how long can a transgenerational signal survive in the irradiated males?

<1 week Sperm ♂

♀

Instability? Adult

3 weeks Spermatids

♀

♂ Instability? Adult

6 weeks Spermatogonia

♀

♂ Instability? Adult

Primordial stem cells

♀

♂ Instability? in utero

• tids
• gonia

sperm

• gonia in utero

sperm

• gonia in utero

Stage of paternal irradiation 1 2 3 4 5

Ratio to control, s.e.

CBA/H germline BALB/c germline BALB/c bone marrow

From: Barber et al., 2002, PNAS 99, 6877-82; 2006, Oncogene 25, 7336-42; 2009, Mutat Res 664, 6-12; Hatch et al., 2007, Oncogene, 26, 4720-4

Transgenerational effects manifest in the offspring regardless the stage of paternal irradiation

3 weeks 6 weeks 1 week 1 week 6 weeks 6 weeks in utero in utero

Can paternal exposure to chemical mutagens destabilise the F1 genomes?

Anticancer drug cyclophosphamide, CPP alkylated monoadducts & crosslinks results in base substitutions crosslinks can result in DSBs after replication/repair Alkylating agent ethynitoesurea, ENU mostly base damage results in base substitutions ~ no ENU-induced DSBs Anticancer drug mitomycin C, MMC alkylated monoadducts & crosslinks base substitutions crosslinks can result in DSBs Anticancer drug procarbazine, PCH alkylated monoadducts free radical species base substitutions & SSBs

sperm bone marrow

From: Barber et al., 2002, PNAS 99, 6877-82 Dubrova et al., 2008, Environ Mol Mutagen 49, 308-11 Glen, Dubrova 2012, PNAS 109, 2984

ESTR instability in the F1

• ffspring of mutagen-treated male mice

Instability signal is initiated by a generalised DNA damage Instability signal is initiated by a generalised DNA damage

Is transgenerational instability sex-specific?

The offspring of irradiated females are stable

Irradiated in utero Adult irradiation

From: Barber et al., 2009, Mutat Res 664, 6-12; Abouzeid Ali et al., 2012, Mutat Res 732, 21-5

Mechanisms

~ 1,000 genes are involved in maintaining genome stability in mammals (DNA repair, apoptosis, cell cycle arrest etc) max spontaneous mutation rate 10-6 per locus exposure to 1 Gy of X-rays results in ~ a 3-fold increase in mutation rate if ANY radiation-induced mutation at ANY of 1,000 genes is DOMINANT and can substantially compromise the genome stability, then 1000 x 3 x 10-6 = 0.3% of the F1

• ffspring should be unstable

according to our data ~100% of the F1 offspring of

• f irradiated males are unstable

Some back of the envelope exercises…

The mechanisms must be epigenetic

♂

Initiation of an epigenetic instability signal in the directly exposed male germ cells Transmission of an epigenetic instability signal to the

• ffspring & its manifestation

F1

Measuring DNA damage in vivo

The alkaline Comet assay The γH2AX assay Mostly single-strand DNA breaks + some DNA adducts Double-strand DNA breaks only

CBA/Ca BALB/c 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 Mean number γ-H2AX foci, 95% CI

From: Barber et al., 2006, Oncogene 25, 7336-42

Endogenous DNA damage in controls & the F1

• ffspring of irradiated males

CBA/Ca BALB/c 3 4 5 6 7 8 9 10 11 Mean Comet tail, 95% CI

Control F1 Control F1

1.9-fold 1.9-fold 2-fold 2-fold 2.3-fold 2.3-fold 1.7-fold 1.7-fold

Single-strand DNA breaks Comet assay, bone marrow Double-strand DNA breaks γ-H2AX assay, spleen

20 40 60 20 40 60 Time post-treatment, min 10 20 30 40 Tail DNA 20 40 60 20 40 60 Time post-treatment, min 10 20 30 40 Tail DNA

CBA/Ca CBA/Ca BALB/c BALB/c

Control F1 Ex vivo exposure

• f bone marrow

to X-rays, 10 Gy Alkaline Comet Ex vivo exposure

• f bone marrow

to X-rays, 10 Gy Alkaline Comet

From: Barber et al., 2006, Oncogene 25, 7336-42

The efficiency of DNA in the F1

• ffspring is not compromised

The efficiency of DNA in the F1

• ffspring is not compromised

DNA repair in the F1

• ffspring of irradiated males

Oxidative stress

DNA damage: modified bases single-strand breaks double-strand breaks Hallmark: Accumulation of

• xidatively damaged

nucleotides in DNA

Oxidative stress

DNA damage: modified bases single-strand breaks double-strand breaks Hallmark: Accumulation of

• xidatively damaged

nucleotides in DNA

From: Barber et al., 2006, Oncogene 25, 7336-42

CBA/Ca BALB/c 1.2 1.4 1.6 1.8 2.0 2.2 2.4 Mean Comet tail, 95% CI CBA/Ca BALB/c 1.2 1.4 1.6 1.8 2.0 2.2 2.4 Mean Comet tail, 95% CI

Control F1

Oxidative DNA damage in the F1

• ffspring (FPG Comet)

The efficiency of DNA in the F1 offspring is OK No sign of oxidative stress in the F1 offspring What else? The efficiency of DNA in the F1 offspring is OK No sign of oxidative stress in the F1 offspring What else?

Transcriptome analysis of transgenerational effects

F0 F1 CBA/Ca F0 F1 1 Gy of acute X-rays

♂ ♀

♂ ♀

1 Gy of acute X-rays

♂ ♀ ♂ ♀

BALB/c RNA extraction Kidney Liver Spleen Brain NimbleGen 12x135K expression arrays:

• 135,000 probes per array;

45-60mer long

• Complete coverage of the mouse

transcriptome (42,576 transcripts)

• 3-4 probes per transcript
• 12 arrays per slide

• 5
• 4
• 3
• 2
• 1
• log10(rank of P)

2 4 6 8 10 12 14

• log10(P)

FDR <.05; 56 transcripts

Probabilities for the effects of paternal irradiation on F1 gene expression

GO categories: GO:0048511 Rhythmic process P=1.25 x 10-9 GO:0007623 Circadian rhythm P=1.52 x 10-7 GO:0006355 Regulation of transcription, P=1.62 x 10-6 DNA-dependent

• 5
• 4
• 3
• 2
• 1
• log10(rank of P)

2 4 6 8 10 12 14

• log10(P)

FDR <.05; 56 transcripts GO categories: GO:0048511 Rhythmic process, 6 genes P=1.25 x 10-9 GO:0007623 Circadian rhythm, 5 genes P=1.52 x 10-7 GO:0006355 Regulation of transcription, P=1.62 x 10-6 DNA-dependent, 11 genes

Dbp Per2 Npas2 Npas2 Arntl Nr1d2 Arntl Per3 Tef Mtf1 Nfil3 Ppard Lxh2

Compromised gene expression in the F1

• ffspring

Dbp Per2 Per3 Tef Nr1d2 Mtf1 Lhx2 Nfil3 Ppard Arntl Npas2

• 6
• 5
• 4
• 3
• 2
• 1

1 2 3

Ratio F1/control, log2

means

down-regulated up-regulated

Circadian transcripts in mouse liver

Circadian trascriptome & circadian metabolism in mice

From: Maywood et al., 2007, Cold Spring Harb Symp Quant Biol 72, 85 Akhtar et al., 2002, Curr Biol 12, 540

And so what?

Control X-rays, F1 X-rays, F2 Group 20 40 60 80 Tumor induction, % (95% CI)

Sham-treated Sham-treated Sham-treated

Control X-rays, F1 X-rays, F2 Group 20 40 60 80 Tumor induction, % (95% CI) Control X-rays, F1 X-rays, F2 Group 20 40 60 80 Tumor induction, % (95% CI)

Sham-treated Sham-treated Sham-treated TPA-treated TPA-treated TPA-treated

From: Vorobtsova et al., 1993, Mutat. Res. 287, 207-216

Incidence of skin tumour in the offspring of irradiated male mice

Transgenerational effects in the children of irradiated parents

From: Tawn et al., 2005, Mutat Res 523, 198-206; Aghajanyan & Suskov, 2009, Mutat Res 523, 52-7

control families irradiated families

Chernobyl clean-up workers

Unstable?

Childhood cancer survivors

survivors partners children

Stable? Stable?

Experiment one: Male mice exposed to 10 – 100 cGy acute γ-rays

• r 100 cGy chronic γ-rays

Experiment two: Male mice exposed to clinically-relevant doses

• f 3 anticancer drugs:

Cyclophosphamide Mitomycin C Procarbazine

♂

♀ Sperm, brain, bone marrow From mice to humans....

10 25 50 100 100 Paternal dose, cGy 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 Ratio to control, s.e.

Paternal exposure to acute & chronic γ-rays

sperm brain acute chonic

Dose per single radiotherapy procedure

Cyclophosphamide Mitomycin C Procacbazine 1.0 1.5 2.0 2.5 Ratio to control, s.e.

150 mg/kg 5 mg/kg 50 mg/kg

sperm bone marrow

Paternal exposure to anticancer drugs

From: Glen, Dubrova 2012, PNAS 109, 2984

Doses per single chemotherapy procedure Instability signal is triggered by a stress-like response in irradiated cells

High-dose acute paternal exposure to a number of mutagens can significantly destabilise the genomes of their offspring Transgenerational instability is a genome-wide phenomenon which affects the frequency of chromosome aberrations and gene mutations Transgenerational instability is triggered in the directly exposed germ cells by a stress-like response to a generalised DNA damage Transgenerational instability is attributed to the presence of a persistent subset of endogenous DNA lesions Transgenerational instability is attributed to the epigenetic changes affecting the expression of a subset of genes, involved in rhythmic process & regulation of transcription Transgenerational instability may represent an important component of the long-term genetic risk of human exposure to mutagens, but we need HUMAN data to prove it! High-dose acute paternal exposure to a number of mutagens can significantly destabilise the genomes of their offspring Transgenerational instability is a genome-wide phenomenon which affects the frequency of chromosome aberrations and gene mutations Transgenerational instability is triggered in the directly exposed germ cells by a stress-like response to a generalised DNA damage Transgenerational instability is attributed to the presence of a persistent subset of endogenous DNA lesions Transgenerational instability is attributed to the epigenetic changes affecting the expression of a subset of genes, involved in rhythmic process & regulation of transcription Transgenerational instability may represent an important component of the long-term genetic risk of human exposure to mutagens, but we need HUMAN data to prove it! Conclusions

Dubrova’s lab

Ruth Barber Colin Glen Safeer Mughal Andre Gomes Robert Hardwick Carole Yauk Mariel Voutounou Tim Hatch Dominic Kelly Peter Hickenbotham Morag Shanks Carles Vilarino-Guell Karen Monger Bruno Gutierrez Karen Burr Julia Brown Natalya Topchiy Isabelle Roux Peter Black Demetria Pavlou Hamdy Ali Abouzeid MRC Radiation and Genome Stability Unit, Harwell, UK Mark Plumb Emma Boulton Jan Fennelly Dudley Goodhead Department of Cancer Studies and Molecular Medicine, University of Leicester, UK George “Don” Jones Gabriela Almeida Comet Assay NI Vavilov Institute of General Genetics, Moscow, Russia Chronic irradiation Alexander Rubanovich Andrey Myazin Medical Radiological Research Centre, Obninsk, Russia Chronic irradiation Leonid Zhavoronkov Yuri Semin Albert Brovin Valentina Glushakova Valentina Posadskaya Olga Izmet’eva MRC Toxicology Unit, Leicester, UK Anticancer drugs Andy Smith Centre for Molecular Genetics and Toxicology, University of Wales, Swansea, UK George Johnson James Parry Hprt assay Catholic University of Nijmegen, The Netherlands Peter de Boer Alwin Derijck Godfried van der Heijden Sperm irradiation Gray Cancer Institute, Northwood, UK γH2AX assay Kai Rothkamm

Dubrova’s lab

Ruth Barber Colin Glen Safeer Mughal Andre Gomes Robert Hardwick Carole Yauk Mariel Voutounou Tim Hatch Dominic Kelly Peter Hickenbotham Morag Shanks Carles Vilarino-Guell Karen Monger Bruno Gutierrez Karen Burr Julia Brown Natalya Topchiy Isabelle Roux Peter Black Demetria Pavlou Hamdy Ali Abouzeid MRC Radiation and Genome Stability Unit, Harwell, UK Mark Plumb Emma Boulton Jan Fennelly Dudley Goodhead Department of Cancer Studies and Molecular Medicine, University of Leicester, UK George “Don” Jones Gabriela Almeida Comet Assay NI Vavilov Institute of General Genetics, Moscow, Russia Chronic irradiation Alexander Rubanovich Andrey Myazin Medical Radiological Research Centre, Obninsk, Russia Chronic irradiation Leonid Zhavoronkov Yuri Semin Albert Brovin Valentina Glushakova Valentina Posadskaya Olga Izmet’eva MRC Toxicology Unit, Leicester, UK Anticancer drugs Andy Smith Centre for Molecular Genetics and Toxicology, University of Wales, Swansea, UK George Johnson James Parry Hprt assay Catholic University of Nijmegen, The Netherlands Peter de Boer Alwin Derijck Godfried van der Heijden Sperm irradiation Gray Cancer Institute, Northwood, UK γH2AX assay Kai Rothkamm

Acknowledgements

The EMF Biological Research Trust