The effect of chromatin structure on DNA damage signaling
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Transcript of The effect of chromatin structure on DNA damage signaling
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The effect of chromatin structure on DNA damage signaling
Dr. Rebecca BurgessMisteli Lab
Cell Biology of Genomes GroupNational Cancer Institute
Bethesda, MD
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30 nm
11 nm
DNA is compacted into chromatin structures using histone proteins
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Chromatin condenses into chromosomes during mitosis
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Histone Octamer Crystal Structure
Luger et al., Nature 1997
H3 tail
H4 tail
H2A tailH2B tail
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Histone tails are heavily modified
PhosphorylationMethylationAcetylation UbiquitylationSumoylationRibosylation
H3 K9 methyl SUV3-9
heterochromatin
H3 K14 Acetyl
euchromatin
BRG1 SWI/SNF
ATP-driven nucleosome remodeling
bromo
HP1HP1chromo
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Chromatin marks can control DNA-level processes such as gene expression/transcription
A “Histone Code?”
Extension of the information contained in the DNA
Histone marks dictate genome dynamics in a combinatorial manner: - Who can interact - When and where the genomic information is accessed
Tight regulation protects against the dangers of uncontrolled access to the genome
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UV lightIonizing radiatione.g. X-rays, gamma rays
Chemical carcinogens
&
ChemotherapeuticsCellular metabolitese.g. reactive oxygen species
Replication errors
Can chromatin control the cellular response to DNA damage?
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U2OS cells with 2 LacO integrations into the genome
LacI
mCherry
LacO arrayx256
LacI
mCherry
LacI
mCherrymCherry-lacI
Interphase nucleus Mitotic Chromosomes
Cell system for visualizing a specific genomic location
mCherry-lacIDAPI-stained DNA
Dig-labelled LacO probeDAPI-stained DNA
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LacI
mCherry
NormalLacO arrayx256
LacI
mCherry
LacI
mCherrymCherry-lacI
Heterochromatin factor
Expanded
CondensedLacI
mCherry
LacI
mCherry
LacI
mCherry
Euchromatin factor
LacI
mCherry
LacI
mCherry
LacI
mCherry
Lac repressor (LacI) fusions to chromatin proteins
mCherry-lacI
mCherry-lacI
Cell system for creating localized chromatin domains
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g-H2AX domain
Double-strand break ends
MRN
ATM
PPP M R
N
MRN
MRN P
P
ATM
CHK2
MDC1
53BP1
P P
MRN
P
Downstream effectors
PP
P
P
Damage recognition and ATM activation
Signal amplification and transduction by mediators
Cell cycle checkpoint activation
DNA repair(NHEJ, HR)
Apoptosis
CHK1
p53
Effector kinase activation
CDC25
P
BRCA1
ATR
DNA-PK
Ku70/80
Single-strand DNA
P
SMC1 PML BRCA2
Mre11-Rad50-Nbs1
The cellular response to DNA double-strand breaks (DDR)
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•Cytoskeleton reorganization
•Adhesion complexes
•Signaling molecules
•Endocytic pathways
•Nuclear changes
Cells undergo many changes during 3D migration
Friedl et al., COCB 2011
Do the nuclear changes of migrating cells affect their capacity to repair DNA damage?
Can this be harnessed to alter the effects of DNA damaging cancer drugs on metastasizing cells?
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The nucleus is 5-10 times stiffer than the surrounding cytoskeleton
8 m
12 m
5 m high1 m diameter pillars with 1 m pillar spacing
(du Roure et al., PNAS 2005)
Closely-spaced “bed of nails”
20 m high,10 m diameter with8 m pillar spacing
More widely-spaced “forest”
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From: Gerlitz and Bustin, Trends Cell Biol. 2011
Condensation of chromatin occurs upon cell migration in a restrictive matrix (altered H1 motility, increased H3K9me3)
Gerlitz, et al., Traffic 2007
Chromatin condensation is required for migration of melanocytes
Gerlitz and Bustin, JCS 2010
Effects of cell migration on chromatin structure
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U2OS cells 8 m pillar spacing HeLa cells
DAPI 53BP1
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Cell migration is associated with pathologies such as chronic inflammatory diseases, and formation of metastases.
…but is also an essential part of embryonic development, the immune response and wound healing.