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 This organization of chromatin in an intact nucleus is microscopically apparent as the darker and lighter staining regions. Heterochromatin stains more darkly and is often located around the nuclear periphery, while lighter colored euchromatin punctuates the interior. DNA is compacted at the first level by wrapping 147 bp around a protein core called a nucleosome, which is a complex of 8 small basic proteins called histones: two copies of H2A two of H2B, two copies of H3 and two of H4. This produces a beads-on-a-string configuration in its most open conformation, or a more compact 11 nm fiber, which is visible by electron microscopy. Linker Histone H1 binds outside of the nucleosome, and interactions between adjacent H1s further compact the chromatin into 30 nm fibers, in a solenoid structure,

Chromatin condenses into chromosomes during mitosis In mitosis, chromatin compacts even further to allow the cells to accurately divide the chromosomes into the daughter cells,. Further compaction at the centromere provides greater strength to counteract the pulling forces generated by the mitotic spindle. In the electron micrograph, you can make out the condensed fibers that are looped onto a scaffold to make up the familiar mitotic chromosome. Properly condensed chromosomes can neatly align along the metaphase plate, and the mitotic checkpoint machinery ascertains that all chromosomes are attached to the spindle before beginning separation.

Histone Octamer Crystal Structure H3 tail H4 tail Nucleosomes are the basic unit of chromatin, and besides simply wrapping the DNA , histones have long N-terminal tails that protrude from the nucleosomal core, here you can see the tails in the crystal structure. H2B tail H2A tail Luger et al., Nature 1997

Histone tails are heavily modified Phosphorylation Methylation Acetylation Ubiquitylation Sumoylation Ribosylation H3 K9 methyl SUV3-9 H3 K14 Acetyl BRG1 SWI/SNF ATP-driven nucleosome remodeling bromo HP1 chromo These tails can undergo a variety of PTMs such as: P, Me, Ac, Ub, SUMO, ribosylation. Histone modifications can modulate chromatin directly by changing the nucleosomal structure, loosening or tightening the structure, or promote association of chromatin modifying proteins. Here are two notable examples. Methylation of H3 at lysine 9 by the SUVAR 3-9 methyltransferase is bound by heterochromatin HP1 through its chromodomain which is a binding domain that specifically recognizes methylated residues; acetylated residues of histone H3 recruits nucleosome remodeling complexes through their bromodomain. Chromatin opening is catalyzed by ATP-driven nucleosome sliding and eviction. euchromatin heterochromatin

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 The are other chromatin marks regulate cellular processes, one of the best examples is in gene expression. Certain histone marks like H3 lysine 4 methylation is found at the promoters of active genes, while other marks such as H3 lysine 27 represses gene expression and transcription.

Can chromatin control the cellular response to DNA damage? Ionizing radiation e.g. X-rays, gamma rays UV light Replication errors We want to know if chromatin structure can control or change how the cell responds to DNA damage. Although tightly packed chromatin limits access to DNA, it that doesn’t mean it is impervious to being damaged. It has been estimated that the DNA in a single human cell endures well over 10,000 lesions in day. These insults can come from many sources- from endogenous sources, such as errors in DNA replication or from the action of reactive molecules created by cellular metabolism. The exogenous sources of damage are ionizing radiation, UV light from the sun, or chemical carcinogens in the environment or drugs such as those used as chemotherapy. . Chemical carcinogens & Chemotherapeutics Cellular metabolites e.g. reactive oxygen species

Cell system for visualizing a specific genomic location LacI mCherry LacO array x256 mCherry-lacI U2OS cells with 2 LacO integrations into the genome Interphase nucleus Mitotic Chromosomes mCherry-lacI DAPI-stained DNA Dig-labelled LacO probe DAPI-stained DNA To study the link between chromatin and DNA damage response directly, we have created a human cell line that harbors two lac operator sequence arrays integrated into the chromosomes. The lac repressor is a protein that binds to the lac operator sequence, so when the lacR is tagged with a Cherry-red fluorescent protein, we can visualize the location of chromosome carrying the lac operator array in the nucleus. Next to the lac operator is a nuclease site, so when we express the nuclease in the cells, we are effectively visualizing the site of DNA damage.

Cell system for creating localized chromatin domains LacI mCherry Normal LacO array x256 mCherry-lacI Heterochromatin factor Expanded Condensed LacI mCherry Euchromatin factor Lac repressor (LacI) fusions to chromatin proteins mCherry-lacI We are using this scheme further, and creating localized chromatin changes at the lac array, by fusing different chromatin modifying proteins to the mCherry-lacR, for targeting to this site. We have created a large assortment of chromatin modifier fusions to different heterochromatin proteins to create condensed chromatin and euchromatin proteins to open the chromatin.

The cellular response to DNA double-strand breaks (DDR) Double-strand break ends M R N Damage recognition and ATM activation M R N Mre11-Rad50-Nbs1 Ku70/80 ATM P DNA-PK M R N Signal amplification and transduction by mediators g-H2AX domain P P P P M R N P MDC1 P M R N P Single-strand DNA P P ATM ATR 53BP1 P P Effector kinase activation We are beginning to test whether our chromatin environment can change the way cells deal with DSBs, to begin, since it is a well-understood pathway. once a DSB is bound by sensors and signaling molecules are activated the downstream responses can vary from repair, via different pathways. To apoptotic cell death. Knowing what signals lead to one fate or the other could help us understand how to modulate the cancer cell response to DNA damaging chemotherapy, and try to increase the amount of cancer cell death. CHK1 CHK2 Cell cycle checkpoint activation Apoptosis DNA repair (NHEJ, HR) Downstream effectors SMC1 CDC25 p53 PML BRCA1 BRCA2

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? 3D migration – define. Another line of investigation is whether nuclear changes of migrating cells affects their capacity to repair DNA damage

Closely-spaced “bed of nails” 5 m high 1 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 with 8 m pillar spacing More widely-spaced “forest” 8 m 12 m Cells migrating through a tight matrix (like metastatic cancer cells do) undergo some dramatic changes to the nucleus. The size and relative stiffness of the nucleus becomes a significant barrier to migration through narrow openings < ~10 m (ECM, basement membranes). Nuclear deformations are a rate-limiting step for cell movements through micron-sized pores, and regulation of invasive migration 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 (A) Schematic representation of a non-migrating cell in which chromatin is organized in highly condensed heterochromatin regions and non-condensed euchromatin regions. (B) Schematic representation of a fibroblast-like tumor cell migrating through fibers of the extracellular matrix towards the right side of the figure. The migration process requires reshaping of the nucleus and “squeezing” it through the narrow opening in the extracellular matrix. Increased heterochromatinization in the migrating cell may contribute to decreased nuclear size, increased nuclear stiffness and better nuclear reshaping in the following ways: i) Condensed chromatin can pull the nuclear envelope “inward” to support reduction in nuclear size; ii) Condensed chromatin can increase the stiffness of the nucleus making it easier for the cytoskeleton to move the nucleus inside the cell; iii) Condensed chromatin can form stronger anchoring points for the LINC complex (SUN and KASH domain proteins), strengthening the interaction between the cytoskeleton and the nucleus. Dynein motor complexes anchored to the nuclear envelope by the LINC complex slide along microtubules towards the centrosome while pulling the nucleus towards the leading edge of the cell. At the rear of the cell, myosin II-dependent contraction of actin filaments, which could be anchored to the nuclear envelope by the LINC complex, contract the nuclear diameter and push the nucleus forward. From: Gerlitz and Bustin, Trends Cell Biol. 2011

U2OS cells 8 m pillar spacing HeLa cells Acts as a system to mimic the restrictive matrix that metastasizing cells must traverse when they invade tissues. 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. So understanding the DNA damage response in migrating cells can also help us understand physiological mechanisms of genome maintenance in normal tissues.