[email protected] Department of Genetics University of ... · 56 transcripts @ FDR 0.05 FDR < .05 The...
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Transgenerational effects of parental exposure Yuri E Dubrova [email protected] Department of Genetics University of Leicester, UK
Transcript of [email protected] Department of Genetics University of ... · 56 transcripts @ FDR 0.05 FDR < .05 The...
Transgenerational effects of parental exposure
Yuri E Dubrova
Department of Genetics
University of Leicester, UK
5 10 15 20 25 30 35 40
Population doublings
0.00
0.05
0.10
0.15
0.20
Ab
erra
tion
s/ce
llRadiation-induced genomic instability
γ-irradiated
control F0
F1 Are they unstable?
F0
F1
F2
0.5 Gy of
fission neutrons
Fat
her
Mo
ther
Paternal
mutations
Maternal
mutation
Expanded simple tandem
repeat (ESTR) loci
= microsatellite loci
ESTR mutation rates were evaluated in the germline of:
irradiated F0 males = mutation induction
non-exposed F1 offspring = transgenerational instability
From: Dubrova et al., 2000, Nature 405, 37
Let’s go transgenerational…
Transgenerational germline instability in the F1 offspring of
CBA/H male mice exposed to 0.5 Gy of fission neutrons
5.6-fold
4.5-fold
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
From: Barber et al., 2002, PNAS 99, 6877-82
Transgenerational instability in three inbred mouse strains
C57BL CBA/H BALB/c0.0
0.1
0.2
0.3
0.4
ES
TR
mu
tatio
n r
ate
, 95
% C
I
Control
F1
F2 ESTR mutation rates are elevated in
both generations of all inbred strains
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
From: Barber et al., 2006, Oncogene 25, 7336-42; 2009, Mutat Res 664, 6-12
ESTR mutation rates are equally elevated
in the germline & somatic tissues
Early developmental onset of instability
Is transgenerational instability
specific for tandem repeat loci?
CBA/Ca BALB/c0
5
10
15
20
Hp
rt m
uta
tion
freq
uen
cy
x 1
0-6
, 95
% C
I
24 30 33 36 39 42 48
Hours after partial hepatectomy
0.3
0.4
0.5
0.6
Freq
uen
cy o
f chrm
oso
me a
berratio
ns
Transgenerational instability
at the mouse hprt locus
Chromosome aberrations in the
F1 offspring of irradiated rats
From: Barber et al., 2006, Oncogene 25, 7336-42 From: Vorobtsova, 2000, Mutagenesis 15, 33-38
hprt is X-linked gene
♂ ♀
♂ ♂ ♀ ♀
XY XX
XY XY
A genome-wide destabilisation
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
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
ESTR instability in the F1 offspring of mutagen-treated male mice
sperm
bone marrow
Instability signal is initiated by
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
Mechanisms
♂
Transmission of an epigenetic
instability signal to the
offspring & its manifestation
F1
♂
F0
Initiation of an epigenetic
instability signal in the directly
exposed male germ cells
Endogenous DNA damage in controls
& the F1 offspring of irradiated males
Control
F1
Control
F1
CBA/Ca BALB/c3
4
5
6
7
8
9
10
11
Mea
n C
om
et ta
il, 95
% C
I
1.9-fold 2-fold
CBA/Ca BALB/c0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.1
Mea
n n
um
ber
γ-H2AX
foci, 9
5%
CI
2.3-fold
1.7-fold
Single-strand DNA breaks
Comet assay, bone marrow
Double-strand DNA breaks
γ-H2AX assay, spleen
From: Barber et al., 2006, Oncogene 25, 7336-42
DNA repair in the F1 offspring of irradiated males
0 20 40 60 0 20 40 60
Time post-treatment, min
0
10
20
30
40
Ta
il DN
A
CBA/Ca BALB/c
Ex vivo exposure of bone
marrow to X-rays, 10 Gy
Alkaline Comet
The efficiency of DNA repair in
the offspring is not compromised
From: Barber et al., 2006, Oncogene 25, 7336-42
Oxidative stress
DNA damage:
modified bases
single-strand breaks
double-strand breaks
Hallmark:
Accumulation of
oxidatively damaged
nucleotides in DNA
The efficiency of DNA in the F1 offspring is OK
No sign of oxidative stress in the F1 offspring
What else?
CBA/Ca BALB/c1.2
1.4
1.6
1.8
2.0
2.2
2.4
Mea
n C
om
et tail, 9
5%
CI
Control
F1
Oxidative DNA damage in the F1 offspring (FPG Comet)
From: Barber et al., 2006, Oncogene 25, 7336-42
-1.2 -0.8 -0.4 0.0 0.4 0.8 1.2
Mean fold-change, log2
0
2
4
6
8
10
12
14
Pro
ba
bility
, -log
10
56 transcripts @ FDR 0.05
FDR < .05
The effects of paternal irradiation on F1 gene expression
Dbp
Dbp
Per2
Npas2Arntl
Arntl Npas2
TefPer3ArntlMtf1
PpardNr1d2 Nfil3Lhx2
-1.2 -0.8 -0.4 0.0 0.4 0.8 1.2
Mean fold-change, log2
0
2
4
6
8
10
12
14
Pro
ba
bility
, -log
10
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 From: Gomes et al., Mutat Res 787, 33-37
Circadian trascriptome & circadian metabolism in mice
Circadian transcriptional regulators in liver Circadian transcripts
in mouse liver
Among them those involved in:
DNA replication, recombination & repair
cell division & cell death
From: Koike et al., 2012, Science 338, 349; Akhtar et al., 2002, Curr Biol 12, 540;
Maywood et al., 2007, Cold Spring Harb Symp Quant Biol 72, 85
And what is transmitted?
From: Taudt et al., 2016, Nat Rev Genet 17, 319-332
What can we expect to find?
Spermatogenesis in mice & humans
Diploid spermatogonia
Meiotic primary spermatocytes
Meiotic secondary spermatocytes
Post-meiotic spermatids
Sperm cells
Transcription
X
<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
Ra
tio to
co
ntr
ol, s.e
.
CBA/H
germline
BALB/c
germline
BALB/c
bone marrow
3 weeks
6 weeks
1 week
1 week
6 weeks
6 weeks in utero
in utero
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
Potential transgenerational mechanisms involving sperm RNAs
Extacellular vesicles, Evs may be regarded as
the transgenerational messenger
From: Chen et al., 2016, Nat Rev Genet 17, 733-743
And so what?
Transgenerational effects in the children of irradiated parents
Childhood cancer survivors
survivors
partners
children
Stable?
Stable?
control families
irradiated families
Chernobyl clean-up workers
Unstable?
From: Tawn et al., 2005, Mutat Res 523, 198-206; Aghajanyan & Suskov, 2009, Mutat Res 523, 52-7
♂ ♀
Sperm, brain, bone marrow
Experiment one:
Male mice exposed to
10 – 100 cGy acute γ-rays
or 100 cGy chronic γ-rays
Experiment two:
Male mice exposed to
clinically-relevant doses
of 3 anticancer drugs:
Cyclophosphamide
Mitomycin C
Procarbazine
From mice to humans....
From: Mughal et al., 2012, PLoS ONE 7, e41300
Paternal exposure to acute & chronic γ-rays
10 25 50 100 100
Paternal dose, cGy
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
Ra
tio to
co
ntr
ol, s.e
.
sperm
brain
acute chronic
Cyclophosphamide Mitomycin C Procacbazine
1.0
1.5
2.0
2.5
Ra
tio to
co
ntr
ol, 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
Dose per single
radiotherapy
procedure
Doses per single chemotherapy procedure
Instability may not exist among the
children of irradiated cancer patients
The children of cancer patients
treated by these drugs may be unstable
Conclusions
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!
Acknowledgements
Dubrova’s lab
Ruth Barber Colin Glen Safeer Mughal Andre Gomes Hamdy Ali Abouzeid
Dominic Kelly Robert Hardwick Mariel Voutounou Carole Yauk Tim Hatch
Peter Hickenbotham Morag Shanks Carles Vilarino-Guell Bruno Gutierrez Isabelle Roux
Karen Burr Karen Monger Julia Brown Natalya Topchiy Peter Black
Demetria Pavlou Adeolu Adewoye
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
