Chromatin remodeling may enforce cell senescence

1
Chromatin remodeling may enforce cell
senescence (ING family PHD proteins help set the
histone code) Karl Riabowol Southern Alberta
Cancer Research Institute University of Calgary
Alberta, Canada Second SENS meeting Cambridge
UK September 7-11, 2005
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The Hayflick Limit in fibroblasts
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Telomere length is linked to mortality due
to infectious diseases and heart disease (Cawthon
et al. The Lancet 361, 363-365 2003)
Mortality rate ratio of persons with short
telomeres (bottom 25) compared to those with
long telomeres (top 75) for Heart Disease
3.18 1 (p0.008) Infectious
diseases 8.54 1 (p0.015) Cancer
1.43 1 (p0.625)
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Human Diploid Fibroblast in vitro replicative
aging
26 MPD
78 MPD
85 MPD
9 kbp Telomere length
4 kbp
The Hayflick Limit
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Correlates of cell aging gene expression
SA-??Galactosidase
Hs68-34
Hs68-84
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Cell aging OIPS/SIPS as barriers against
cancer?
Proliferation Competent (Controlled Cell Growth
and Proliferation)
Viral Oncoproteins Tag, E1A, E6/E7
p53 Rb
M1
M2
SENESCENCE (Genetically programmed growth arrest)
Telomerase Activated (or Alt)
CRISIS (Critically short telomeres)
IMMORTALIZATION
Additional Mutations
Oncogene/Stress-induced premature
senescence (OIPS/SIPS)
ONCOGENIC TRANSFORMATION
(Uncontrolled Cell Growth and Proliferation)
PNAS 92 8348-8352 (1995).
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Isolation of the ING1 growth suppressor
cDNAs
LTR
neo
LTR
(-)
subtractive hybridization screen Senescent cell
library select 200
packaging
random-fragment library cloned
cells
into retroviral vector LNCX
viral
infection
PCR-isolation of putative
transforming fragments from
tumours and subcloning into
retroviral vector LNCX
normal mouse mammary
tumour formation
epithelial cells
in nude mice
focus forming assay
cloning
full-length
cDNA
Soft agar assay
identify splicing isoforms, other ING members
Nature Genetics, 1996
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4 of 5 INhibitor of Growth (ING) genes are
telomeric
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Evolutionary relatedness of ING proteins from all
species examined
Five vertebrate INGs were derived from three
ancestral sequences
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(No Transcript)
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Modification of histone tails regulates chromatin
configuration through opening and closing
nucleosomes
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ING proteins are found in HAT HDAC
complexes that regulate nucleosome
stability through acetylation of histones
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Increased ING1 in senescent cells correlates with
formation of SAHF
Hs68-36
Merge
Phalloidin (Actin)
?-ING1
DNA
Hs68-86
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ING proteins bind chromatin more avidly in
senescent cells (as estimated by Chromatin
Immunoprecipitation (ChIP) assays)
?-ING1 ?-AcH4 ?-PKC Beads
ChIP
Y S Y S Y S Y S
8.0 kb
0.5 kb
(equal number of cells harvested)
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Blocking ING1 or ING2 expression extends
proliferative lifespan (now also being tested in
nematode mouse models)
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Retroviral Vector Control
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a-sense ING1 Retrovirus
Number of colonies
20
10
0
40-99 100-159 160-219 220-280
Number of Hs68 (MRC5) cells per colony
MCB 17 2014-2019 (1997)/MCB 25 6639-6648 (2005)
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Heterochromatization Heterochromatinization in
replicative senescence
Metaphase Chromosome
120-nm fiber
HATs HDACs (methylases-demethylases kinases-phos
phatases)
30-nm fiber
H3
H1
HDAC
HDAC
Ac
H2A
Nucleosome
H4
10-nm fiber
2-nm DNA fiber
H2B
HDAC
Histone Octamer with 146 bp of DNA
Heterochromatinization Senescence
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Collaborators Alan Casson Mike Cole Andreas von
Deimling Curt Harris Caren Helbing Daniel
Larocque Steve McMahon Yutaka Miura Stephane
Richard Gesche Tallen Matthias Truss
Collaborators-C David Bazett-Jones Tara
Beattie Michel Biosvert Chris Brown Doug
Demetrick Lisa DeFeancesco Peter Forsyth Randy
Johnston Robbie Loewith Paul Mains Dan Muruve
Igor Garkavtsev Wei Gong Yasuo Hara Hiromi
Kataoka Jingjing Luo Lana Pastyryeva Jason
Quarrie Mike Russell Michelle Scott
Keiko Suzuki Tatsuya Toyama Diego
Vieyra Support NCIC MRC/CIHR AHFMR ACB Big Rock
Derrick Rancourt Donna Senger Christoph
Sensen Dallan Young The Lab Team Pinaki Bose Phil
Berardi Donna Boland Xiaolan Feng
The lab crew