Chromosomal Translocations in Human Cells Are Generated by Canonical Nonhomologous End-Joining

“…et al., 1998), suggesting that DSBs with blunt DNA ends may not give rise to such translocations. nCas9, a Cas9 nickase due to a D10A mutation (Jinek et al., 2012), can generate DSBs with overhangs if two gRNAs are used that cleave opposite strands (i.e., paired nicks) (Mali et al., 2013a;…”

“We also tested Cas9-dependent induction of DSBs (Cong et al., 2013; Mali et al., 2013b) for translocation formation.”

“Paired nicks generated by nCas9 have the potential to substantially increase cleavage specificity, given that two gRNAs are required per DSB (Mali et al., 2013a; Ran et al., 2013), and we have determined that nCas9-generated paired nicks efficiently induce translocations.”

“To address the role of NHEJ pathways in the joining phase of chromosomal translocation formation, we took advantage of nucleases designed to introduce site-specific DSBs at endogenous loci in human cells (Gaj et al., 2013; Urnov et al., 2010) to induce translocations (Brunet et al.”

“Paired nicks generated by nCas9 have the potential to substantially increase cleavage specificity, given that two gRNAs are required per DSB (Mali et al., 2013a; Ran et al., 2013), and we have determined that nCas9-generated paired nicks efficiently induce translocations.”

“Because mismatches to the gRNA can be tolerated, however, wild-type Cas9 has been associated with off-target mutagenesis (Fu et al., 2013).”

“NALM6 junctions without insertions showed longer microhomology than is typical of wild-type cells, raising the possibility that some of the junctions that did not engage TdT did not join by c-NHEJ; alternatively, TdT may have created microhomology in some of these junctions (Lieber, 2010).”

“To address the role of NHEJ pathways in the joining phase of chromosomal translocation formation, we took advantage of nucleases designed to introduce site-specific DSBs at endogenous loci in human cells (Gaj et al., 2013; Urnov et al., 2010) to induce translocations (Brunet et al.”

“Paired nicks generated by nCas9 have the potential to substantially increase cleavage specificity, given that two gRNAs are required per DSB (Mali et al., 2013a; Ran et al., 2013), and we have determined that nCas9-generated paired nicks efficiently induce translocations.”

“Recurrent reciprocal chromosomal translocations are associated with oncogenesis (Mani and Chinnaiyan, 2010; Mitelman et al., 2007).”

“Proteins that promote alt-NHEJ include the end resection factor CtIP (Bennardo et al., 2008) and LIG3a (Wang et al.”

“…has similarities to what has been termed microhomology-mediated break-induced replication, which has been postulated as a lar Cell 55, 829–842, September 18, 2014 ª2014 Elsevier Inc. 839 mechanism to give rise to copy number variants in the germline (Carvalho et al., 2013; Hastings et al., 2009).”

“…is that loss of the Ku protein is incompatible with human cell survival (Li et al., 2002) due to an essential role in telomere maintenance (Wang et al., 2009), whereas rodent cells and mice are viable in the absence of Ku (Nussenzweig et al., 1996; Taccioli et al., 1994; Zhu et al., 1996).”

“…is that loss of the Ku protein is incompatible with human cell survival (Li et al., 2002) due to an essential role in telomere maintenance (Wang et al., 2009), whereas rodent cells and mice are viable in the absence of Ku (Nussenzweig et al., 1996; Taccioli et al., 1994; Zhu et al., 1996).”

“In contrast, knockdown of c-NHEJ components did decrease androgen-induced translocations, although junction analysis was not reported (Lin et al., 2009).”

“Hypomorphic LIG4 mutations have been reported in patients with a radiosensitive syndrome (O’Driscoll et al., 2001).”

“The canonical pathway of NHEJ (c-NHEJ) is required for cellular resistance to ionizing radiation aswell as for immune system rearrangements and is active throughout the cell cycle (Deriano and Roth, 2013; Goodarzi and Jeggo, 2013; Pannunzio et al., 2014).”

“…is that loss of the Ku protein is incompatible with human cell survival (Li et al., 2002) due to an essential role in telomere maintenance (Wang et al., 2009), whereas rodent cells and mice are viable in the absence of Ku (Nussenzweig et al., 1996; Taccioli et al., 1994; Zhu et al., 1996).”

“Mice with mutations in c-NHEJ components develop RAG recombinase-mediated pro-B lymphomas on a p53-deficient background with complex Myc translocations involving microhomology (Zhu et al., 2002).”

“However, it is consistent with c-NHEJ protection of DNA ends and suggests that c-NHEJ is not an inherently error-prone process (Bétermier et al., 2014).”

“(Grawunder et al., 1998), as have 411BR primary skin fibroblasts (O’Driscoll et al., 2001).”

“Although NHEJ components are conserved, human cells have substantially higher DNA-PK activity than rodent cells (Finnie et al., 1995; Beamish et al., 2000; Meek et al., 2001).”

“…performed in mouse cells, in particular in lymphoid cells involving programmed double-stranded breaks (DSBs) and in embryonic stem cells using I-SceI or zinc finger nuclease (ZFN)-generated DSBs (Boboila et al., 2012; Nussenzweig and Nussenzweig, 2010; Weinstock et al., 2007; Simsek et al., 2011).”

“Proteins that promote alt-NHEJ include the end resection factor CtIP (Bennardo et al., 2008) and LIG3a (Wang et al., 2005).”

“Most studies analyzing translocation formation have been performed in mouse cells, in particular in lymphoid cells involving programmed double-stranded breaks (DSBs) and in embryonic stem cells using I-SceI or zinc finger nuclease (ZFN)-generated DSBs (Boboila et al., 2012; Nussenzweig and Nussenzweig, 2010; Weinstock…”

“Molecu Cas9 has also been used in HEK293 cells to induce a translocation found in lung tumors (Choi and Meyerson, 2014).”

“Thus, as with repair of a single DSB, intrachromosomal repair of two DSBs in human cells requires the LIG4:XRCC4 complex, similar to rodent cells (Guirouilh-Barbat et al., 2007; Simsek and Jasin, 2010).”

“…performed in mouse cells, in particular in lymphoid cells involving programmed double-stranded breaks (DSBs) and in embryonic stem cells using I-SceI or zinc finger nuclease (ZFN)-generated DSBs (Boboila et al., 2012; Nussenzweig and Nussenzweig, 2010; Weinstock et al., 2007; Simsek et al., 2011).”

“For CtIP knockdown experiments, cells were transfected using LipofectamineTM RNAiMax (Invitrogen) with 40 nmol siRNA following the manufacturer’s protocol, and the level of knockdown was evaluated by western blotting.”

“The canonical pathway of NHEJ (c-NHEJ) is required for cellular resistance to ionizing radiation aswell as for immune system rearrangements and is active throughout the cell cycle (Deriano and Roth, 2013; Goodarzi and Jeggo, 2013; Pannunzio et al., 2014).”

“Junctions that form by alt-NHEJ have more microhomology and longer deletions than junctions formed by c-NHEJ (Fattah et al., 2010; Guirouilh-Barbat et al., 2007; Kabotyanski et al., 1998; Oh et al., 2013; Simsek and Jasin, 2010; Smith et al., 2003).”

“Junctions that form by alt-NHEJ have more microhomology and longer deletions than junctions formed by c-NHEJ (Fattah et al., 2010; Guirouilh-Barbat et al., 2007; Kabotyanski et al., 1998; Oh et al., 2013; Simsek and Jasin, 2010; Smith et al., 2003).”

“Similarly, c-NHEJ mutant B cells undergoing class switching have increased Myc translocations, implying that alt-NHEJ is a robust mechanism for generating translocations (Boboila et al., 2010).”

“Similarly, cancer and model translocations induced by nucleases in several human cell lines also show little or no microhomology at translocation junctions (Brunet et al., 2009; Piganeau et al., 2013).”

“Another striking difference is that loss of the Ku protein is incompatible with human cell survival (Li et al., 2002) due to an essential role in telomere maintenance (Wang et al.”

“LIG4-null (L4 / ) cells are deficient in LIG4 but maintain XRCC4 expression; XRCC4 mutant (X4 / ) cells are deficient in both XRCC4 and LIG4, because LIG4 is unstable in the absence of XRCC4 (Bryans et al., 1999) (Figure 1A).”

“Junctions that form by alt-NHEJ have more microhomology and longer deletions than junctions formed by c-NHEJ (Fattah et al., 2010; Guirouilh-Barbat et al., 2007; Kabotyanski et al., 1998; Oh et al., 2013; Simsek and Jasin, 2010; Smith et al., 2003).”

“Loss of c-NHEJ components does not, however, completely abrogate NHEJ (Delacôte et al., 2002; Kabotyanski et al., 1998; Liang and Jasin, 1996), suggesting that there are alternative ways to join ends, referred to as alt-NHEJ.”

“…performed in mouse cells, in particular in lymphoid cells involving programmed double-stranded breaks (DSBs) and in embryonic stem cells using I-SceI or zinc finger nuclease (ZFN)-generated DSBs (Boboila et al., 2012; Nussenzweig and Nussenzweig, 2010; Weinstock et al., 2007; Simsek et al., 2011).”

“, 2002) due to an essential role in telomere maintenance (Wang et al., 2009), whereas rodent cells and mice are viable in the absence of Ku (Nussenzweig et al.”

“Recurrent reciprocal chromosomal translocations are associated with oncogenesis (Mani and Chinnaiyan, 2010; Mitelman et al., 2007).”

“…has similarities to what has been termed microhomology-mediated break-induced replication, which has been postulated as a lar Cell 55, 829–842, September 18, 2014 ª2014 Elsevier Inc. 839 mechanism to give rise to copy number variants in the germline (Carvalho et al., 2013; Hastings et al., 2009).”

“Similarly, cancer and model translocations induced by nucleases in several human cell lines also show little or no microhomology at translocation junctions (Brunet et al., 2009; Piganeau et al., 2013).”

“Loss of c-NHEJ components does not, however, completely abrogate NHEJ (Delacôte et al., 2002; Kabotyanski et al., 1998; Liang and Jasin, 1996), suggesting that there are alternative ways to join ends, referred to as alt-NHEJ.”

“…performed in mouse cells, in particular in lymphoid cells involving programmed double-stranded breaks (DSBs) and in embryonic stem cells using I-SceI or zinc finger nuclease (ZFN)-generated DSBs (Boboila et al., 2012; Nussenzweig and Nussenzweig, 2010; Weinstock et al., 2007; Simsek et al., 2011).”

“Ionizing radiation, a potent inducer of translocations in rodent cells, does not significantly induce translocations in a LIG4 mutant human cell line (Soni et al., 2014).”

“The canonical pathway of NHEJ (c-NHEJ) is required for cellular resistance to ionizing radiation aswell as for immune system rearrangements and is active throughout the cell cycle (Deriano and Roth, 2013; Goodarzi and Jeggo, 2013; Pannunzio et al., 2014).”

“…of patient-derived translocations (Gillert et al., 1999; Langer et al., 2003; Reichel et al., 1998; Weinstock et al., 2006; Zhang et al., 2002; Zucman-Rossi et al., 1998), suggesting that cancer translocations may also arise from some other type of DNA end structure. nCas9-induced nicks…”

“…of Cas9-induced DSBs means that the translocation junctions often do not exhibit the small deletions typical of patient-derived translocations (Gillert et al., 1999; Langer et al., 2003; Reichel et al., 1998; Weinstock et al., 2006; Zhang et al., 2002; Zucman-Rossi et al., 1998), suggesting…”

“The remaining LIG4 in these cells has residual catalytic activity (O’Driscoll et al., 2001), and it may also promote DNAPKcs bridging activity (Cottarel et al., 2013) to protect ends from extensive degradation.”

“…junctions often do not exhibit the small deletions typical of patient-derived translocations (Gillert et al., 1999; Langer et al., 2003; Reichel et al., 1998; Weinstock et al., 2006; Zhang et al., 2002; Zucman-Rossi et al., 1998), suggesting that cancer translocations may also arise…”

“Thus, in mouse cells, DSB repair leading to translocations differs from intrachromosomal repair of a single DSB (Simsek and Jasin, 2010; Weinstock et al., 2006), whereas in human cells both types of repair are similar and mediated by c-NHEJ.”

“Although NHEJ components are conserved, human cells have substantially higher DNA-PK activity than rodent cells (Finnie et al., 1995; Beamish et al., 2000; Meek et al., 2001).”

“Junctions that form by alt-NHEJ have more microhomology and longer deletions than junctions formed by c-NHEJ (Fattah et al., 2010; Guirouilh-Barbat et al., 2007; Kabotyanski et al., 1998; Oh et al., 2013; Simsek and Jasin, 2010; Smith et al., 2003).”

“…lengths of microhomology lar Cell 55, 829–842, September 18, 2014 ª2014 Elsevier Inc. 829 (legend continued on next page) 830 Molecular Cell 55, 829–842, September 18, 2014 ª2014 Elsevier Inc. (Gillert et al., 1999; Langer et al., 2003; Zucman-Rossi et al., 1998; Mattarucchi et al., 2008).”

“Although NHEJ components are conserved, human cells have substantially higher DNA-PK activity than rodent cells (Finnie et al., 1995; Beamish et al., 2000; Meek et al., 2001).”

“HCT116 LIG3flox/ cells containing a conditional LIG3 allele and a deletion allele (Oh et al., 2014) were further engineered to express human LIG1 with a mitochondrial leader sequence (MtLIG1).”

“…lengths of microhomology lar Cell 55, 829–842, September 18, 2014 ª2014 Elsevier Inc. 829 (legend continued on next page) 830 Molecular Cell 55, 829–842, September 18, 2014 ª2014 Elsevier Inc. (Gillert et al., 1999; Langer et al., 2003; Zucman-Rossi et al., 1998; Mattarucchi et al., 2008).”

“In patient 411BR cells, the translocation frequency was not affected, consistent with a report that low levels of LIG4 are sufficient for DSB repair in vitro (Windhofer et al., 2007), but deletion lengths were intermediate between control cells and other LIG4 mutants.”

“Junctions that form by alt-NHEJ have more microhomology and longer deletions than junctions formed by c-NHEJ (Fattah et al., 2010; Guirouilh-Barbat et al., 2007; Kabotyanski et al., 1998; Oh et al., 2013; Simsek and Jasin, 2010; Smith et al., 2003).”

“However, unlike mouse cells, a significant fraction was also derived from other chromosomes, as reported for other human translocations (Piganeau et al., 2013; Sobreira et al., 2011; Zucman-Rossi et al., 1998).”

“These results suggest locus effects in the ability to switch from c-NHEJ to alt-NHEJ; alternatively, the oncogenic NPM-ALK fusion protein formed by the translocation could provide a slight growth advantage (Zhang et al., 2013).”

“TdT is required for diversification of antigen receptors, and TdT-generated insertions at these and other DSBs are dependent on c-NHEJ components (Boubakour-Azzouz et al., 2012; Smith et al., 2003).”

“In addition, alt-EJ is mainly mediated by the CtIP/ MRN complex.(319) Furthermore, Polθ can indicate the microhomologous DNA ends to support the joining of DNA ends.”

“Hypothesis-driven in vitro studies are now beginning to reveal some of the mechanistic processes generating these structures 9–13 , and generate further predictions that can be assessed in the genomics data.”