SUMMARY Defective DNA repair by homologous recombination (HR) is thought to be a major contributor to tumorigenesis in individuals carrying Brca1 mutations. Here we show that DNA breaks in Brca1-deficient cells are aberrantly joined into complex chromosome rearrangements by a process dependent on the non-homologous end joining (NHEJ) factors, 53BP1 and DNA Ligase 4. Loss of 53BP1 alleviates hypersensitivity of Brca1 mutant cells to PARP inhibition and restores error-free repair by HR. Mechanistically, 53BP1 deletion promotes ATM-dependent processing of broken DNA ends to produce recombinogenic single-stranded DNA competent for HR. In contrast, Lig4 deficiency does not rescue the HR defect in Brca1 mutant cells, but prevents the joining of chromatid breaks into chromosome rearrangements. Our results illustrate that HR and NHEJ compete to process DNA breaks that arise during DNA replication, and that shifting the balance between these pathways can be exploited to selectively protect or kill cells harboring Brca1 mutations.

Brca1- and Brca2-deficient cells have reduced capacity to repair DNA double-strand breaks (DSBs) by homologous recombination (HR) and consequently are hypersensitive to DNA damaging agents, including cisplatin and poly(ADP-ribose) polymerase (PARP) inhibitors. Here we show that loss of the MLL3/4 complex protein, PTIP, protects Brca1/2-deficient cells from DNA damage and rescues the lethality of Brca2-deficient embryonic stem cells. However, PTIP deficiency does not restore HR activity at DSBs. Instead, its absence inhibits the recruitment of the MRE11 nuclease to stalled replication forks, which in turn protects nascent DNA strands from extensive degradation. More generally, acquisition of PARPi and cisplatin resistance is associated with replication fork (RF) protection in Brca2-deficient tumor cells that do not develop Brca2 reversion mutations. Disruption of multiple proteins, including PARP1 and CHD4, leads to the same end point of RF protection, highlighting the complexities by which tumor cells evade chemotherapeutic interventions and acquire drug resistance.

H4K20 methylation is a broad chromatin modification that has been linked with diverse epigenetic functions. Several enzymes target H4K20 methylation, consistent with distinct mono-, di-, and trimethylation states controlling different biological outputs. To analyze the roles of H4K20 methylation states, we generated conditional null alleles for the two Suv4-20h histone methyltransferase (HMTase) genes in the mouse. Suv4-20h-double-null (dn) mice are perinatally lethal and have lost nearly all H4K20me3 and H4K20me2 states. The genome-wide transition to an H4K20me1 state results in increased sensitivity to damaging stress, since Suv4-20h-dn chromatin is less efficient for DNA double-strand break (DSB) repair and prone to chromosomal aberrations. Notably, Suv4-20h-dn B cells are defective in immunoglobulin class-switch recombination, and Suv4-20h-dn deficiency impairs the stem cell pool of lymphoid progenitors. Thus, conversion to an H4K20me1 state results in compromised chromatin that is insufficient to protect genome integrity and to process a DNA-rearranging differentiation program in the mouse.[Keywords: H4K20 methylation; Suv4-20h enzymes; DNA repair; genome integrity; B-cell differentiation; class-switch recombination] Supplemental material is available at http://www.genesdev.org. Received February 18, 2008; revised version accepted May 30, 2008. Histone lysine methylation is a central epigenetic modification in eukaryotic chromatin. Five major positions for lysine methylation exist in the histone N termini, each with distinct regulatory functions. The repressive methyl marks H3K9, H3K27, and H4K20 are involved in constitutive heterochromatin formation and gene repression, X inactivation, and Polycomb silencing, and in DNA damage repair, mitotic chromosome condensation, and gene regulation (Allis et al. 2007). Additional complexity arises through the fact that histone methylation can be present in three distinct states (mono, di, or tri), which may have different biological readouts depending on the association with specific binding partners. Although there has been significant insight in histone lysine methylation pathways, we still know very little about how the diverse methylation states affect chromatin biology.H4K20 methylation is evolutionarily conserved from Schizosaccharomyces pombe to man . In mammalian cells, H4K20me1 is exclusively induced by the PrSet7/KMT5A histone methyltransferase (HMTase) (Fang et al. 2002;Nishioka et al. 2002), where it has been linked with transcriptional repression (Karachentsev et al. 2005) and X inactivation (Kohlmaier et al. 2004). More recently, genome-wide profiling of H4K20me1 also revealed enrichment of this mark across actively transcribed genes (Papp and Muller 2006;Vakoc et al. 2006). H4K20me1 is very dynamic throughout the cell cycle and becomes highly enriched during S phase (Jorgensen et al. 2007;Tardat et al. 2007;Huen et al. 2008