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 Chromosome aberrations Gene mutations Random DNA DNA targeting damage repair � 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

Radiation-induced genomic instability in somatic cells Yield of chromatid aberrations in MCF10A cells Yield of chromatid aberrations in MCF10A cells 0.20 0.20 Delayed mutations occur many cell divisions after exposure X 0.15 Everything happens in the directly 0.15 Aberrations/cell Aberrations/cell irradiated cell & mutation induction γ -irradiated occurs at the radiation-damaged sites γ -irradiated 0.10 0.10 control control 0.05 0.05 0.00 0.00 5 10 15 20 25 30 35 40 5 10 15 20 25 30 35 40 Population doublings Population doublings From: Ullrich & Ponnaiya, 1998, Int J Radiat Biol 74, 747

What about the germline? F 0 How to analyse? F 0 F 1 Instability in the non-exposed Mutation offspring of irradiated parents Are they unstable? frequency F 1 F 2 Mutant

Mouse Expanded Simple Tandem Repeat (ESTR) loci � 4-10 bp repeats, 100 bp - 20 kb arrays, non-coding � 4-10 bp repeats, 100 bp - 20 kb arrays, non-coding � Very spontaneous mutation rate (up to 15% per gamete) � Very spontaneous mutation rate (up to 15% per gamete) � Mutations result in the loss/gain of repeats � Mutations result in the loss/gain of repeats Mutant 1 Gain of repeats Progenitor Loss of repeats Mutant 2

ESTR mutation detection in the germline & somatic tissues ♀ ♂ Pedigree approach Single-molecule PCR approach Paternal mutations Mother Mutants Father Maternal mutation

Let’s go transgenerational… 0.5 Gy of fission neutrons F 0 Paternal mutations Mother Father F 1 Maternal mutation F 2 From: Dubrova et al., 2000, Nature 405, 37

Transgenerational germline instability in the F 1 offspring of CBA/H male mice exposed to 0.5 Gy of fission neutrons The non-exposed offspring of The non-exposed offspring of 0.5 0.5 0.5 0.5 irradiated parents are unstable irradiated parents are unstable 0.4 0.4 0.4 0.4 Mutation rate, 95% CI Mutation rate, 95% CI Mutation rate, 95% CI Mutation rate, 95% CI 5.6-fold 5.6-fold 5.6-fold 5.6-fold 5.2-fold 5.2-fold 0.3 0.3 4.5-fold 0.3 0.3 4.5-fold 3.7-fold 3.7-fold 0.2 0.2 0.2 0.2 0.1 0.1 0.1 0.1 0.0 0.0 0.0 0.0 control control F 0 exposed F 0 exposed F 1 males F 1 males F 1 females F 1 females F 1 total F 1 total control control F 0 exposed F 0 exposed F 1 males F 1 males F 1 females F 1 females F 1 total F 1 total Group Group Group Group From: Dubrova et al., 2000, Nature 405, 37

Is transgenerational instability strain-specific?

Fission neutrons, 0.4 Gy: CBA/H; C57BL/6 CBA/H Fission neutrons, 0.4 Gy: CBA/H; C57BL/6 Acute X-rays, 2 Gy: CBA/H Acute X-rays, 2 Gy: CBA/H Acute X-rays, 1 Gy: BALB/c Acute X-rays, 1 Gy: BALB/c BALB/c F 0 ♂ ♀ C57BL/6J F 1 F 1 F 2 F 2 F 3 F 3 From: Barber et al ., 2002, PNAS 99, 6877-82

Transgenerational instability in three inbred mouse strains Control 0.4 0.4 F 1 ESTR mutation rates are elevated in ESTR mutation rates are elevated in F 2 ESTR mutation rate, 95% CI ESTR mutation rate, 95% CI both generations of all inbred strains both generations of all inbred strains 0.3 0.3 0.2 0.2 0.1 0.1 0.0 0.0 C57BL CBA/H BALB/c C57BL CBA/H BALB/c From: Barber et al ., 2002, PNAS 99, 6877-82

Is transgenerational instability tissue-specific?

Transgenerational instability in the germline & somatic tissues BALB/c CBA/Ca ESTR mutation rates are equally elevated ESTR mutation rates are equally elevated in the germline & somatic tissues in the germline & somatic tissues From: Barber et al ., 2006, Oncogene 25, 7336-42; 2009, Mutat Res 664 , 6-12

Is transgenerational instability specific for tandem repeat loci?

Transgenerational instability Chromosome aberrations in the at the mouse hprt locus F 1 offspring of irradiated rats F 1 of irradiated males Frequency of chrmosome aberrations F 1 of irradiated males Hprt mutation frequency x 10 -6 , 95% CI Control 0.6 20 A genome-wide destabilisation A genome-wide destabilisation F 1 0.5 15 3.3-fold 10 0.4 hprt is X-linked gene Control Control 5 3.7-fold 0.3 24 30 33 36 39 42 48 0 XX XY CBA/Ca BALB/c Hours after partial hepatectomy ♀ ♂ From: Barber et al ., 2006, Oncogene 25, 7336-42 From: Vorobtsova, 2000, Mutagenesis 15 , 33-38 ♂ ♀ ♂ ♀ XY XY

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

Adult <1 week ♂ ♀ Sperm Instability?

Adult 3 weeks ♂ ♀ Spermatids Instability?

Adult 6 weeks ♂ ♀ Spermatogonia Instability?

in utero ♂ ♀ Primordial stem cells Instability?

CBA/H BALB/c BALB/c 5 germline germline bone marrow Transgenerational effects manifest 6 weeks 4 in the offspring regardless the stage of paternal irradiation 3 weeks Ratio to control, s.e. 3 6 weeks in utero 1 week 1 week 2 6 weeks in utero 1 -tids -gonia sperm -gonia in utero sperm -gonia in utero Stage of paternal irradiation 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

Can paternal exposure to chemical mutagens destabilise the F 1 genomes?

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

ESTR instability in the F 1 offspring of mutagen-treated male mice sperm bone marrow Instability signal is initiated by Instability signal is initiated by a generalised DNA damage a generalised DNA damage 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

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

Some back of the envelope exercises… � ~ 1,000 genes are involved in maintaining genome stability in mammals (DNA repair, apoptosis, cell cycle arrest etc) The mechanisms must be epigenetic � 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 F 1 offspring should be unstable � according to our data ~100% of the F 1 offspring of of irradiated males are unstable

♂ Initiation of an epigenetic instability signal in the directly exposed male germ cells F 1 Transmission of an epigenetic instability signal to the offspring & its manifestation

Measuring DNA damage in vivo The alkaline Comet assay The γ H2AX assay Mostly single-strand DNA breaks Double-strand DNA breaks only + some DNA adducts

Endogenous DNA damage in controls & the F 1 offspring of irradiated males Single-strand DNA breaks Double-strand DNA breaks Comet assay, bone marrow γ -H2AX assay, spleen 11 1.1 1.0 10 Control Control Mean number γ -H2AX foci, 95% CI 0.9 Mean Comet tail, 95% CI 9 F 1 F 1 0.8 8 1.9-fold 1.9-fold 0.7 1.7-fold 7 1.7-fold 2-fold 2-fold 0.6 6 0.5 2.3-fold 2.3-fold 5 0.4 4 0.3 3 0.2 CBA/Ca BALB/c CBA/Ca BALB/c From: Barber et al ., 2006, Oncogene 25, 7336-42

DNA repair in the F 1 offspring of irradiated males CBA/Ca BALB/c CBA/Ca BALB/c 40 40 Control F 1 � Ex vivo exposure � Ex vivo exposure 30 30 of bone marrow of bone marrow to X-rays, 10 Gy to X-rays, 10 Gy Tail DNA Tail DNA � Alkaline Comet � Alkaline Comet 20 20 10 10 The efficiency of DNA in the The efficiency of DNA in the F 1 offspring is not compromised F 1 offspring is not compromised 0 0 0 20 40 60 0 20 40 60 0 20 40 60 0 20 40 60 Time post-treatment, min Time post-treatment, min From: Barber et al ., 2006, Oncogene 25, 7336-42