The effect of chromatin structure
on DNA damage signaling
Dr. Rebecca Burgess
Misteli Lab
Cell Biology of Genomes Group
National Cancer Institute
Bethesda, MD
DNA is compacted into chromatin structures using
histone proteins
11 nm
30 nm
Chromatin condenses into chromosomes during mitosis
Histone Octamer Crystal Structure
H3 tail
H4 tail
Luger et al., Nature 1997
H2B tail
H2A tail
Histone tails are heavily modified
Phosphorylation
Methylation
Acetylation
Ubiquitylation
Sumoylation
Ribosylation
H3 K14 Acetyl
bromo
BRG1SWI/SNF
H3 K9 methyl
chromo
HP1
ATP-driven nucleosome remodeling
euchromatin
heterochromatin
SUV3-9
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
Can chromatin control the cellular response to DNA damage?
Ionizing radiation
UV light
e.g. X-rays, gamma rays
Replication errors
Chemical
carcinogens
&
Cellular metabolites
e.g. reactive oxygen species
Chemotherapeutics
Cell system for visualizing a specific genomic location
LacO array
x256
mCherry-lacI
LacI
LacI
LacI
mCherry
mCherry
mCherry
U2OS cells with 2 LacO integrations into the genome
Interphase nucleus
mCherry-lacI
DAPI-stained DNA
Mitotic Chromosomes
Dig-labelled LacO probe
DAPI-stained DNA
Cell system for creating localized chromatin domains
LacO array
Normal
x256
mCherry-lacI
LacI
LacI
LacI
mCherry
mCherry
mCherry
Lac repressor (LacI) fusions to
chromatin proteins
mCherry-lacI
LacI
LacI
LacI
mCherry
mCherry
mCherry
Condensed
Heterochromatin factor
Expanded
mCherry-lacI
Euchromatin
factor
LacI
LacI
LacI
mCherry
mCherry
mCherry
The cellular response to DNA double-strand breaks (DDR)
Double-strand
break ends
Mre11-Rad50-Nbs1 M
R
R M
N
N
ATM
P
Damage recognition and
ATM activation
Ku70/80
DNA-PK
P
M
P
ATM
P
P
P
P
R
R MN
M
P
N
R MDC1
P
N
Cell cycle checkpoint
activation
CDC25
ATR
P
CHK1
SMC1
Single-strand DNA
53BP1
P
Effector kinase
activation
CHK2
Apoptosis
p53
P
P
Signal amplification and
transduction by mediators
P
PML
DNA repair
(NHEJ, HR)
BRCA1
BRCA2
Downstream effectors
Cells undergo many changes during 3D migration
•Cytoskeleton reorganization
•Adhesion complexes
•Signaling molecules
•Endocytic pathways
•Nuclear changes
Friedl et al., COCB 2011
Closely-spaced “bed of nails”
More widely-spaced “forest”
20 m high,
10 m diameter with
8 m pillar spacing
5 m high
1 m diameter pillars with
1 m pillar spacing
(du Roure et al., PNAS 2005)
8 m
12 m
The nucleus is 5-10 times stiffer than the
surrounding cytoskeleton
Effects of cell migration on chromatin structure
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
From: Gerlitz and Bustin, Trends Cell Biol. 2011
U2OS cells
DAPI
8 m pillar spacing
53BP1
HeLa cells