Meccanismi di riparazione - PowerPoint Presentation

Slide1

Slide2

Slide3

Meccanismi

di

riparazione

Donata

Orioli

Risposta

cellulare

al

danno

Ivana

Scovassi

RNA

Giuseppe

Biamonti

Slide4

Slide5

Slide6

GENE

Slide7

Slide8

Slide9

Slide10

Slide11

Alternative Splicing differences are prevalent in cancer pathways

Slide12

EMT 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

Slide13

SRSF1

promotes

the

epithelial-mesenchymal

transition

(EMT)

Vector

SRSF1

Slide14

Tumor angiogenesis

Angiogenesis has a crucial role in tumor growth

by allowing oxygen and nutrients to reach proliferating cancer cells

Slide15

Alternative 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

Slide16

Centro 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

Slide17

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/

DEXSeq

http://www.bioconductor.org/packages/release/bioc/html/DEXSeq.html

RAP Pipeline

Slide18

γH2AX in Xeta UV treated cells and 46BR cells

46BR

XP30RO

Chr 11

46BR

XP30RO

Chr 17

Slide19

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

Budding Yeast as model system to study

genome instability and DNA replication stress

Slide20

Genome instability in

g

-irradiated human cells

Loucas

& Cornforth

, Radiat Res 2001

Slide21

Abdel-Rahman

et al

., PNAS 2001

Genome

instability

in tumor

cells

Truncations

Translocations

Inversions

Duplications

Amplifications

Slide22

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

Slide23

Genome instability is promoted by a unscheduled use of DNA repair systems (DNA recombination)

DNA lesions can originate during DNA replication

(DNA replication stress)

Slide24

Replication fork barriers induce replication stress

RPA

MCM

Replisome

Fork barrier

PCNA

Polε

Polδ

RPA

Replication stress & Genome instability

Slide25

Collisions between replication and transcription are a major source of replication stress

Replisome

Head-on

Codirectional

RNA

Pol

RNA

Pol

Replisome

Slide26

Normal cells

Senataxin-deficint cells

Alzu

et al

., Cell 2012

Uncoordinated replication-transcription collisions lead to

R-loops accumulation in Senataxin-deficient cells

Slide27

Four-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

Slide28

Replication 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

Slide29

survivors

Yeast KO collection

lethals

DNA damage treatment

High-throughput Genetic Screenings

Slide30

Alessandra Brambati (post-doc)

Luca Zardoni (fellow)

Federica Lopefido (und.Student)

Erika Valeri (und.Student)

Giordano Liberi

Acknowledgements

IGM-CNR

Founding

Slide31

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

Slide32

The DNA Damage Response (DDR)

d’Adda di Fagagna F.

Nature Reviews in Cancer

, 2008

H2AX

53BP1

Slide33

1 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!

Slide34

Slide35

Telomere

Telomere

DDR activation

Persistent DDR

DNA damage repair

Persistent DDR

Slide36

Irreparable

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

Slide37

P

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

Slide38

The DNA Damage Response (DDR)

d’Adda di Fagagna F.

Nature Reviews in Cancer

, 2008

H2AX

53BP1

RNA

(

Francia

Nature 2012)