Donata Orioli Risposta cellulare al danno Ivana Scovassi RNA Giuseppe Biamonti GENE Alternative Splicing differences are prevalent in cancer pathways ID: 933564
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Slide1
Slide2Slide3Meccanismi
di
riparazione
Donata
Orioli
Risposta
cellulare
al
danno
Ivana
Scovassi
RNA
Giuseppe
Biamonti
Slide4Slide5Slide6GENE
Slide7Slide8Slide9Slide10Slide11Alternative Splicing differences are prevalent in cancer pathways
Slide12EMT is required for invasiveness of epithelial cancer
Most invasive and/or metastatic cancers are characterized by partial or complete EMT
Tumour cell
Fibroblast
Tumour cell under EMT
Immune cell
Epithelial cell
Mesenchymal cell
EMT
EMT
MET
Slide13SRSF1
promotes
the
epithelial-mesenchymal
transition
(EMT)
Vector
SRSF1
Slide14Tumor angiogenesis
Angiogenesis has a crucial role in tumor growth
by allowing oxygen and nutrients to reach proliferating cancer cells
Slide15Alternative Splicing regulation during angiogenesis
The alternative splicing factor Nova2 regulates vascular development and lumen formation
.
Giampietro
C*,
Deflorian
G*, Gallo S* et al. Nature Communications 2015
Vascular lumen
Endothelial cell
Endothelial cell
Slide16Centro d’Analisi Bioinformatiche
per la Genomica (CABGen)
Analisi
microarrays
d’espressione genica
Analisi di dati di Next Generation Sequencing
(RNAseq, ChIP seq, DNAseq, Exome sequencing)
Strumenti per la raccolta dati e lo
study-design
Annotazioni ed analisi
genome-wide
Progettazione e costruzione di
database
relazionali
Sviluppo di nuovi strumenti di
data mining
Analisi statistiche
Accounting per l’uso del cluster computazionale
Resp: Dr. Silvia Bione
Dr. Roberta Carriero
Dr. Paolo Cremaschi
Slide17ANALYSIS OF ALTERNATIVE SPLICING EVENTS
Analysis of the
effects of DNA damage on splicing
profiles
The
CABGen
Pipeline for Alternative Splicing analysis in
NGS
data
MISO
http://genes.mit.edu/burgelab/miso/
DEXSeq
http://www.bioconductor.org/packages/release/bioc/html/DEXSeq.html
RAP Pipeline
Slide18γH2AX in Xeta UV treated cells and 46BR cells
46BR
XP30RO
Chr 11
46BR
XP30RO
Chr 17
Slide19Budding yeast
Main cellular process are conserved from yeast to man
(cell cycle regulation, DNA damage checkpoint, DNA repair mechanisms)
Simple & Cheap model system
Classic and genome-wide Mol/Cell Biol & Genetics techniques can be easily applied
Budding Yeast as model system to study
genome instability and DNA replication stress
Slide20Genome instability in
g
-irradiated human cells
Loucas
& Cornforth
, Radiat Res 2001
Slide21Abdel-Rahman
et al
., PNAS 2001
Genome
instability
in tumor
cells
Truncations
Translocations
Inversions
Duplications
Amplifications
Disease
Human gene
Yeast gene
Cellular function
Ataxia Telangiectasia
ATM
TEL1
Checkpoint
Seckel Syndrome
ATR
MEC1
Checkpoint
AT-like disorder
hMRE11
MRE11
Checkpoint/DNA Recombination
Nijmegen Breakage Syndrome
NBS1
XRS2
Checkpoint/DNA Recombination
Werner Syndrome
WRN
SGS1
DNA Recombination
Bloom Syndrome
BLM
SGS1
DNA Recombination
Rothmund-Thomson Syndromes
RECQ4
HRQ1
DNA Recombination
Roberts syndrome
ESCO2
ECO1
Sister Chromatid Cohesion
Genome instability syndromes
Slide23Genome instability is promoted by a unscheduled use of DNA repair systems (DNA recombination)
DNA lesions can originate during DNA replication
(DNA replication stress)
Slide24Replication fork barriers induce replication stress
RPA
MCM
Replisome
Fork barrier
PCNA
Polε
Polδ
RPA
Replication stress & Genome instability
Slide25Collisions between replication and transcription are a major source of replication stress
Replisome
Head-on
Codirectional
RNA
Pol
RNA
Pol
Replisome
Slide26Normal cells
Senataxin-deficint cells
Alzu
et al
., Cell 2012
Uncoordinated replication-transcription collisions lead to
R-loops accumulation in Senataxin-deficient cells
Slide27Four-way junctions In HU-treated
rad53
cell
Lopes
et al
. Nature 2001
Sogo
et al
. Science 2002
Sister chromatid and/or inter-homologue junctions in MMS-treated
sgs1
cells
Recombination intermediates accumulate in response to replication
stress in checkpoint or BLM helicase mutant cells
Liberi
et al
. Genes Dev 2005
Carotenuto&Liberi DNA repair 2010
IHJ
SCJ
SCJ
Slide28Replication stress
Oncogene
Precancerous cell
DNA damage Respone
Tumor
Genomic instability
Replication stress causes precancerous DNA lesions accumulation
Bartkova
et al
. Nature, 2006
Di Micco
et al
. Nature, 2006
Slide29survivors
Yeast KO collection
lethals
DNA damage treatment
High-throughput Genetic Screenings
Slide30Alessandra Brambati (post-doc)
Luca Zardoni (fellow)
Federica Lopefido (und.Student)
Erika Valeri (und.Student)
Giordano Liberi
Acknowledgements
IGM-CNR
Founding
Slide31IFOM
Foundation
-FIRC Institute of Molecular Oncology, Milan,
Italy
DNA damage response in cancer and ageing
& the role of non coding RNAs
Fabrizio d’Adda di Fagagna
Slide32The DNA Damage Response (DDR)
d’Adda di Fagagna F.
Nature Reviews in Cancer
, 2008
H2AX
53BP1
Slide331 hour
H2AX
pS/TQ
3 days
30 days
60 days
10 days
120 days
X-rays irradiation of non-proliferating human fibroblasts:
Not all DNA damage is repairable!
Slide34Slide35Telomere
Telomere
DDR activation
Persistent DDR
DNA damage repair
Persistent DDR
Slide36Irreparable
telomeric
DNA damage
Oncogene-induced
senescence
Oncogene-induced DNA replication
stress
(Di
Micco
Nature 2006)
Replicative
cellular senescence
Telomere
shortening
i
n proliferating tissues
(d’Adda di Fagagna Nature 2003)
Cellular senescence
initiated by random DD
also in non proliferating
(quiescent/differentiated)
cells
(
Fumagalli
Nature Cell Biology 2012)
Rossiello
F.
et al,
Curr
. Op. in Genetics and Development, 2014
Slide37P
P
P
P
P
P
DNA
d
amage
i
n situ
ligation
followed
by
P
roximity
L
igation
A
ssay
(DI-PLA)
Primary
antibodies
against
biotin
and a DDR marker
Cells
fixation
and
permeabilization
DSB
Induction
DNA
ends
blunting
Linker
ligation
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
Proximity
Ligation
Assay
(PLA):
detects
proximity
between
biotin
and a DDR marker
P
Slide38The DNA Damage Response (DDR)
d’Adda di Fagagna F.
Nature Reviews in Cancer
, 2008
H2AX
53BP1
RNA
(
Francia
Nature 2012)