Biochemical Society Annual Symposium No. 76 The impact of heterochromatin on DSB repair Aaron A. Goodarzi, Angela T. Noon and Penny A. Jeggo 1 Genome Damage and Stability Centre, University of Sussex, Brighton BN1 9RQ, U.K. Abstract DNA NHEJ (non-homologous end-joining) is the major DNA DSB (double-strand break) repair pathway in mammalian cells. Although NHEJ-defective cell lines show marked DSB-repair defects, cells defective in ATM (ataxia telangiectasia mutated) repair most DSBs normally. Thus NHEJ functions independently of ATM signalling. However, ∼15 % of radiation-induced DSBs are repaired with slow kinetics and require ATM and the nuclease Artemis. DSBs persisting in the presence of an ATM inhibitor, ATMi, localize to heterochromatin, suggesting that ATM is required for repairing DSBs arising within or close to heterochromatin. Consistent with this, we show that siRNA (small interfering RNA) of key heterochromatic proteins, including KAP-1 [KRAB (Kr ¨ uppel-associated box) domain-associated protein 1], HP1 (heterochromatin protein 1) and HDAC (histone deacetylase) 1/2, relieves the requirement for ATM for DSB repair. Furthermore, ATMi addition to cell lines with genetic alterations that have an impact on heterochromatin, including Suv39H1/2 (suppressor of variegation 3–9 homologue 1/2)-knockout, ICFa (immunodeficiency, centromeric region instability, facial anomalies syndrome type a) and Hutchinson–Guilford progeria cell lines, fails to have an impact on DSB repair. KAP-1 is a highly dose-dependent, transient and ATM-specific substrate, and mutation of the ATM phosphorylation site on KAP-1 influences DSB repair. Collectively, the findings show that ATM functions to overcome the barrier to DSB repair posed by heterochromatin. However, even in the presence of ATM, γ -H2AX (phosphorylated histone H2AX) foci form on the periphery rather than within heterochromatic centres. Finally, we show that KAP-1’s association with heterochromatin is diminished as cells progress through mitosis. We propose that KAP-1 is a critical heterochromatic factor that undergoes specific modifications to promote DSB repair and mitotic progression in a manner that allows localized and transient chromatin relaxation, but precludes significant dismantling of the heterochromatic superstructure. Introduction ATM (ataxia telangiectasia mutated) is a PIKK (phosphoinositide 3-kinase-like kinase) that lies at the heart of a signal transduction response to DNA DSBs (double- strand breaks) [1]. The very existence of a highly complex DSB signalling response attests to the significance of DSBs as lesions promoting genomic instability and/or cell death. The dramatic clinical features caused by loss of ATM in A-T (ataxia telangiectasia) patients serves further to demonstrate the importance of an appropriate response to DSB formation [2]. ATM signalling activated by DSB formation promotes cell-cycle-checkpoint arrest, apoptosis and also influences DSB repair. Whereas apoptosis functions to remove damaged cells from the cycling population, cell-cycle-checkpoint arrest can both enhance the opportunity for repair and serve as an alternative to apoptosis to limit the proliferative Key words: ataxia telangiectasia, chromatin structure, damage-response signalling, double- strand break repair, heterochromatin, radiosensitivity. Abbreviations used: A-T, ataxia telangiectasia; ATM, ataxia telangiectasia mutated; ATMi, ATM inhibitor; ATR, ATM- and Rad3-related; DAPI, 4 ′ ,6-diamidino-2-phenylindole; DSB, double-strand break; EC-DSB, DSB located within euchromatic DNA; γ -H2AX, phosphorylated histone H2AX; HC-DSB, DSB located within or close to heterochromatic DNA; HDAC, histone deacetylase; H3K9ac, histone H3 acetylated at Lys 9 ; H3K9me, histone H3 methylated at Lys 9 ; H3K9me3, histone H3 trimethylated at Lys 9 ; HP1, heterochromatin protein 1; IR, ionizing radiation; KAP-1, KRAB (Kr ¨ uppel-associated box) domain-associated protein 1; NHEJ, non-homologous end-joining; PFGE, pulsed-field gel electrophoresis; siRNA, small interfering RNA; Suv39H1/2, suppressor of variegation 3–9 homologue 1/2. 1 To whom correspondence should be addressed (email p.a.jeggo@sussex.ac.uk). capacity of damaged cells. DNA NHEJ (non-homologous end-joining) functions as the major DSB-rejoining pathway in mammalian cells [3]. A-T cell lines rejoin the majority of DSBs with normal kinetics, demonstrating that most NHEJ occurs independently of ATM signalling [4,5]. However, the rejoining of ∼15 % of IR (ionizing radiation)-induced DSBs require ATM and additional proteins that function in the ATM signal transduction process, including γ -H2AX (phosphorylated histone H2AX) and 53BP1 (p53-binding protein 1) [5]. Previous studies have demonstrated that, although the majority of DSBs are repaired with fast kinetics in mammalian cells, a subset of breaks is rejoined with slower kinetics [6]. Strikingly, ATM is required for the slow component of DSB repair, which represents a ∼15 % subset of X- or γ -ray-induced DSBs [5]. ATM-dependent DSB repair also requires the nuclease Artemis [5]. Artemis nuclease activity has the capacity to cleave hairpin-ended DSBs and to remove 3 ′ - or 5 ′ -single-stranded overhangs following remodelling of the DNA ends by the DNA-PK (DNA- dependent protein kinase) [7,8]. Furthermore, Artemis is an ATM-dependent substrate after radiation [5,9–11]. For this and other reasons, it was suggested that ATM may function to promote Artemis-dependent end processing before rejoining by NHEJ [5]. However, in a more recent study, a closer look at the ATM-dependency for DSB repair following exposure to a range of agents producing DNA ends of differing complexity suggested that there was only a weak correlation between end Biochem. Soc. Trans. (2009) 37, 569–576; doi:10.1042/BST0370569 C  The Authors Journal compilation C  2009 Biochemical Society 569