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The effect of chromatin structure on DNA damage signaling

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.

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The effect of chromatin structure on DNA damage signaling

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  1. The effect of chromatin structure on DNA damage signaling Dr. Rebecca Burgess Misteli Lab Cell Biology of Genomes Group National Cancer Institute Bethesda, MD

  2. DNA is compacted into chromatin structures using histone proteins 11 nm 30 nm

  3. Chromatin condenses into chromosomes during mitosis

  4. Histone Octamer Crystal Structure H3 tail H4 tail H2A tail H2B tail Luger et al., Nature 1997

  5. Histone tails are heavily modified Phosphorylation Methylation Acetylation Ubiquitylation Sumoylation Ribosylation H3 K9 methyl SUV3-9 HP1 HP1 H3 K14 Acetyl chromo bromo BRG1 SWI/SNF ATP-driven nucleosome remodeling heterochromatin euchromatin

  6. Chromatin marks can control DNA-level processes such as gene expression/transcription A “Histone Code?”Extension of the information contained in the DNAHistone marks dictate genome dynamics in a combinatorial manner: - Who can interact - When and where the genomic information is accessedTight regulation protects against the dangers of uncontrolled access to the genome

  7. Can chromatin control the cellular response to DNA damage? Ionizing radiation e.g. X-rays, gamma rays UV light Replication errors Chemical carcinogens & Chemotherapeutics Cellular metabolites e.g. reactive oxygen species

  8. 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 MitoticChromosomes mCherry-lacI DAPI-stained DNA Dig-labelledLacO probe DAPI-stained DNA

  9. Cell system for creating localized chromatin domains LacO array x256 Normal mCherry-lacI LacI LacI LacI mCherry mCherry mCherry Lac repressor (LacI) fusions to chromatin proteins Condensed LacI LacI LacI mCherry mCherry mCherry mCherry-lacI Heterochromatin factor Expanded LacI LacI LacI mCherry-lacI mCherry mCherry mCherry Euchromatin factor

  10. The cellular response to DNA double-strand breaks (DDR) Double-strand break ends Damage recognition and ATM activation Mre11-Rad50-Nbs1 Ku70/80 ATM DNA-PK P P P R R R R M M M M N N N N Signal amplification and transduction by mediators g-H2AX domain P P P P P P P P P P MDC1 Single-strand DNA ATM ATR 53BP1 R M N Effectorkinase activation CHK1 CHK2 P Cell cycle checkpoint activation Apoptosis DNA repair (NHEJ, HR) Downstream effectors SMC1 CDC25 p53 PML BRCA1 BRCA2

  11. Cells undergo many changes during 3D migration • Cytoskeleton reorganization • Adhesion complexes • Signaling molecules • Endocytic pathways • Nuclear changes 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?

  12. Closely-spaced “bed of nails” More widely-spaced “forest” 20 m high, 10 m diameter with 8 m pillar spacing 5 m high1 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

  13. 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

  14. U2OS cells 8 m pillar spacing HeLa cells DAPI 53BP1

  15. 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.

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