The effect of chromatin structure on DNA damage signaling

Dr. Rebecca BurgessMisteli Lab

Cell Biology of Genomes GroupNational Cancer Institute

Bethesda, MD

30 nm

11 nm

DNA is compacted into chromatin structures using histone proteins

Chromatin condenses into chromosomes during mitosis

Histone Octamer Crystal Structure

Luger et al., Nature 1997

H3 tail

H4 tail

H2A tailH2B tail

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

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

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?

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

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

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)

•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?

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”

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

U2OS cells 8 m pillar spacing HeLa cells

DAPI 53BP1

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.