DNA recombination

Meccanismi di riparazione
Donata Orioli
Risposta cellulare al danno
Ivana Scovassi
RNA
Giuseppe Biamonti
GENE
Alternative Splicing differences are prevalent in cancer
pathways
EMT is required for invasiveness of epithelial cancer
EMT
EMT
Epithelial cell
Mesenchymal cell
Tumour cell
Tumour cell under EMT
Fibroblast
Immune cell
Most invasive and/or metastatic cancers are characterized by partial or
complete EMT
SRSF1 promotes the epithelial-mesenchymal
transition (EMT)
Vector
SRSF1
Tumor angiogenesis
Angiogenesis has a crucial role in tumor growth
by allowing oxygen and nutrients to reach proliferating cancer cells
Alternative Splicing regulation during angiogenesis
Endothelial cell
Endothelial cell
Vascular lumen
The alternative splicing factor Nova2 regulates vascular
development and lumen formation.
Giampietro C*, Deflorian G*, Gallo S* et al. Nature Communications 2015
Centro d’Analisi Bioinformatiche
per la Genomica (CABGen)
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Analisi microarrays d’espressione genica
Analisi di dati di Next Generation Sequencing
(RNAseq, ChIP seq, DNAseq, Exome sequencing)
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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
ANALYSIS 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/
RAP Pipeline
DEXSeq
http://www.bioconductor.org/packages/release/bioc/html/DEXSeq.ht
ml
γH2AX in Xeta UV treated cells and 46BR cells
15
20
15
number_of_peaks
number_of_peaks
10
10
5
5
0
46BR
0
0
50
100
Location
46BR
0
20
40
60
80
Location
10.0
15
number_of_peaks
number_of_peaks
7.5
10
5
5.0
2.5
0
0.0
0
XP30RO
50
100
Location
Chr 11
XP30RO
0
20
40
Location
Chr 17
60
80
Budding Yeast as model system to study
genome instability and DNA replication stress
Budding 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
Genome instability in g-irradiated human cells
Loucas & Cornforth, Radiat Res 2001
Genome
instability
in tumor
cells
Truncations
Translocations
Inversions
Duplications
Amplifications
Abdel-Rahman et al., PNAS 2001
Genome instability syndromes
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 is promoted by a unscheduled use
of DNA repair systems (DNA recombination)
DNA lesions can originate during DNA replication
(DNA replication stress)
Replication fork barriers induce replication stress
PCNA
Polδ
Replisome
Polε
R
P
A
RPA
MCM
Fork barrier
Replication stress & Genome instability
Collisions between replication and transcription
are a major source of replication stress
Replisome
RNA Pol
Replisome
RNA Pol
Uncoordinated replication-transcription collisions lead to
R-loops accumulation in Senataxin-deficient cells
Normal cells
Senataxin-deficint cells
Alzu et al., Cell 2012
Recombination intermediates accumulate in response to replication
stress in checkpoint or BLM helicase mutant cells
Four-way junctions In HU-treated rad53 cell
Sister chromatid and/or inter-homologue
junctions in MMS-treated sgs1 cells
IHJ
SCJ
Lopes et al. Nature 2001
Sogo et al. Science 2002
SCJ
Liberi et al. Genes Dev 2005
Carotenuto&Liberi DNA repair 2010
Replication stress causes precancerous DNA
lesions accumulation
Oncogene
Replication stress
DNA damage Respone
Genomic instability
Precancerous cell
Tumor
Bartkova et al. Nature, 2006
Di Micco et al. Nature, 2006
High-throughput Genetic Screenings
Yeast KO collection
DNA damage treatment
survivors
lethals
Acknowledgements
Alessandra Brambati (post-doc)
Luca Zardoni (fellow)
Federica Lopefido (und.Student)
Erika Valeri (und.Student)
Giordano Liberi
IGM-CNR
Founding
IFOM 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
The DNA Damage Response (DDR)
H2AX
53BP1
d’Adda di Fagagna F. Nature Reviews in Cancer, 2008
Not all DNA damage is repairable!
X-rays irradiation of non-proliferating human fibroblasts:
gH2AX
pS/TQ
1 hour
3 days
10 days
30 days
60 days
120 days
Telomere
Telomere
DDR activation
DNA damage repair
Persistent DDR
Persistent DDR
Replicative
cellular senescence
Cellular senescence
initiated by random DD
also in non proliferating
(quiescent/differentiated) cells
Telomere shortening
in proliferating tissues
(Fumagalli Nature Cell Biology 2012)
(d’Adda di Fagagna Nature 2003)
Irreparable telomeric DNA damage
Oncogene-induced
senescence
Oncogene-induced DNA replication stress
(Di Micco Nature 2006)
Rossiello F. et al, Curr. Op. in Genetics and Development, 2014
DNA damage in situ ligation
followed by Proximity Ligation Assay
(DI-PLA)
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P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
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Cells fixation and
permeabilization
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DNA ends blunting
Linker ligation
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Primary antibodies against biotin
and a DDR marker
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Proximity Ligation Assay (PLA):
detects proximity between biotin
and a DDR marker
P
P
P
P
P
P
P
P
P
P
P
P
P
DSB Induction
P
P
The DNA Damage Response (DDR)
H2AX
53BP1
RNA
(Francia
Nature 2012)
d’Adda di Fagagna F. Nature Reviews in Cancer, 2008