Chromatin modulation and DNA damage response A- De visu: chromatin decondensation in mammalian cells B- Chromatin modulation 1- Covalent histones modifications 2- Histone exchange 3- ATP-dependent chromatin remodeling C- Models of dynamic chromatin remodeling at sites of DNA damage

Nucleosome and chromatin structure Costelloe et al. Exp. Cell.Res :

chromatin decondensation in mammalian cells Kruhlak et al. JCB :

H2B-PAGFP H3-PAGFP GR-PAGFP

Covalent histones modifications in DNA repair - Histones are rich in polar aminoacids: Lysine, Arginine, Serine, Threonine -> located on the exposed exterior surface of histone octamers. - Differences in electrostatic properties between the modified and unmodified forms of these residues can significantly affect the interaction between histones and DNA or other proteins. - Four modifications: Phosphorylation (kinase/phosphatase), acetylation (HAT/HDAC), methylation (methyl transferase/demethylase), ubiquitylation (ubiquitin ligase/deubiquitinase)

Main phosphorylation event:  -H2AX in DSB - ATM/ATR-dependent (Tel1/Mec1 in yeast) - Occurs early after DSB induction - Spreads over 1 Mb in mammals (100 Kb in yeast) - Acts as a docking platform for the recruitment of DNA repair proteins

Methylation: H3K79 - constitutive on undamaged euchromatin in mammalian cells - Buried in higher-order chromatin - Interacts with hydrophobic residues of the chromodomain of 53BP1 (Rad9 in Yeast). - model: DSB causes a passive relaxation of higher-order chromatin surrounding the break -> allows 53BP1 to interact with H3K79 specifically at the break site. - Similar mechanism proposed for H4K20me / Crb2 in yeast

Acetylation - Acetylation of lysines in the N-terminal tails of H3 and H4 removes the positive charges on the side chain -> destabilizes higher-order chromatin structure. - Acetylated lysines can be recognized and recruit bromodomain-containing proteins such as Tip60 - Regulated by histone acetyltransferases (HATs) and histone deacetylases (HDACs)

Composition of the yeast SWR1, INO80, NuA4 and human TIP60 chromatin remodeling/histone acetyltransferase complexes involved in DNA repair (Chromatin remodeling) (HAT) (Chromatin remodeling/HAT) Yeast SWR1, INO80 and NuA4 complexes share many subunits with human TIP60. It is postulated that TIP60 is a hybrid of at least two and possibly all three yeast complexes. (Chromatin remodeling) : Tip60

Proposed role for Tip60 and the NuA4 complex in chromatin remodeling during DNA repair Squatrito et al., Trends Cell Biol. 2006, 16: Tip60-dependent acetylation/ phosphorylation of ATM Recruitment of MRX to the DSB Phosphorylation of NBS1, H2AX  H2AX acts as a docking platform, recruits DDR proteins (eg MDC1) and spreads the signal further downstream from the DSB NuA4 might recognize  H2AX, facilitating Tip60 acetylation of Histones H4 and  H2AX P400 might catalyze histone exchange between  H2AX and unmodified H2AZ.

Histone exchange at a DSB Papamichos-Chronakis et al. Genes Dev : Yeast  -H2Av Tip60 Ac H2Av p400 Drosophila Kusch et al. Science : histone exchange

Carrozza, Kush and Workman, Nat. Struct. Biol Chromatin remodelling- principles

Composition of the yeast SWR1, INO80, NuA4 and human TIP60 chromatin remodeling/histone acetyltransferase complexes involved in DNA repair (Chromatin remodeling) (HAT) (Chromatin remodeling/HAT) Yeast SWR1, INO80 and NuA4 complexes share many subunits with human TIP60. It is postulated that TIP60 is a hybrid of at least two and possibly all three yeast complexes. (Chromatin remodeling) : Tip60

Studies in yeast: the MAT/HO system Papamichos-Chronakis et al. Genes Dev : Southern blot Homologous recombination

NHEJ Papamichos-Chronakis et al. Genes Dev :

a, A DSB was induced at MAT in strains expressing Flag–H2B or Flag–H3, and ChIP was done with antibodies against -H2A or Flag. DNA was analysed by real-time PCR using primers corresponding to sequences on the left (- ) or the right (+ ) side of the DSB (0), and results were normalized to the ratio of immunoprecipitation (IP) to input DNA at time 0. Data are the mean s.e.m. b, Nuclei were prepared after DSB induction, and chromatin was digested with MNase and subjected to Southern blot analysis using a probe for MAT DNA. The triangles denote increasing times of MNase digestion. M indicates a 1-kb DNA ladder. Figure 1: Chromatin changes at the MAT DSB. Kb Wt: loss of histones and nucleosome integrity near the DSB Tsukuda et al. Nature :

a, A DSB was induced at MAT, and ChIP was done with antibodies against Flag in an mre11::Kan-MX strain expressing Flag–H2B and an hta1/hta2-S129* strain expressing Flag–H3. DNA was analysed by real- time PCR on both the left (- ) and the right (+ ) side of the DSB (0). Data are the mean s.e.m. b, MNase analysis was done on nuclei isolated from the mre11::Kan-MX strain by Southern blot analysis as described in Fig. 1b. Figure 2: MRX is involved in histone loss at the MAT DSB. Tsukuda et al. Nature :

a, A DSB was induced at MAT in an arp8::Kan-MX mutant expressing Flag–H3, and ChIP was done with antibodies against Flag. Precipitated DNA was quantified as described in Fig. 1a. b, ChIP was done with antibodies against Myc in a wild-type strain that contained Ino80–Myc before (- Gal) and after (+ Gal) DSB induction. Ino80–Myc association was normalized to histone H3 occupancy. c, MNase digestion was done on nuclei isolated from an arp8::Kan-MX mutant as described in Fig. 1b. Data in a and b are the mean s.e.m. Figure 3: The INO80 complex is required for histone eviction at the MAT DSB. FLAG-H3 Tsukuda et al. Nature :

Chromatin remodeling factors in non-DSB repair Blue: ATP-dependent chromatin remodelers Histone modifying enzymes: yellow Chromatin assembly factors: magenta Ataian and Krebs, Biochem. Cell. Biol. 2006, 84:

Model of DSB repair by HR in the HO system in yeast MRX binds to the DNA ends, recruits Tel1 (ATM). Tel1 and Mec1 (ATM/ATR) phosphorylate H2A:  H2A  H2A acts as a platform for the recruitment of additional repair proteins. Mre11 recruits RSC. INO80-dependent nucleosome eviction occurs at the break site. RSC may facilitate loading of cohesin (rings) near the break site. Cohesin binds up to 100 kb from the DSB, dependent of Mre11 and  H2A. NuA4 (HAT) is recruited through  H2A and acetylates H4 (H4K8).  H2A and H4K8 recruit a new pool of INO80. A pool of INO80 involved in transcription resides at the MAT locus. SWI-SNF is recruited and might facilitate recruitment of Rad51 and Rad52 to homologous donor sequences. H4 acetylation (H4K16) is reduced by the histone deacetylase Rpd3 Chromatin reassembly by CAF occurs either concurrently with repair or after completion. Dephosphorylation of  H2A by Pph3 occurs probably after histones or nucleosomes have been displaced Wong et al., J Mol Histol. 2006, 37: