TY - JOUR
T1 - Class switching and meiotic defects in mice lacking the E3 ubiquitin ligase RNF8
AU - Santos, Margarida Almeida
AU - Huen, Michael S Y
AU - Jankovic, Mila
AU - Chen, Hua-Tang
AU - López-Contreras, Andrés J
AU - Klein, Isaac A
AU - Wong, Nancy
AU - Barbancho, Juan L R
AU - Fernandez-Capetillo, Oscar
AU - Nussenzweig, Michel C
AU - Chen, Junjie
AU - Nussenzweig, André
PY - 2010/5/10
Y1 - 2010/5/10
N2 - 53BP1 is a well-known mediator of the cellular response to DNA damage. Two alternative mechanisms have been proposed to explain 53BP1's interaction with DNA double-strand breaks (DSBs), one by binding to methylated histones and the other via an RNF8 E3 ligase-dependent ubiquitylation pathway. The formation of RNF8 and 53BP1 irradiation-induced foci are both dependent on histone H2AX. To evaluate the contribution of the RNF8-dependent pathway to 53BP1 function, we generated RNF8 knockout mice. We report that RNF8 deficiency results in defective class switch recombination (CSR) and accumulation of unresolved immunoglobulin heavy chain-associated DSBs. The CSR DSB repair defect is milder than that observed in the absence of 53BP1 but similar to that found in H2AX(-/-) mice. Moreover, similar to H2AX but different from 53BP1 deficiency, RNF8(-/-) males are sterile, and this is associated with defective ubiquitylation of the XY chromatin. Combined loss of H2AX and RNF8 does not cause further impairment in CSR, demonstrating that the two genes function epistatically. Importantly, although 53BP1 foci formation is RNF8 dependent, its binding to chromatin is preserved in the absence of RNF8. This suggests a two-step mechanism for 53BP1 association with chromatin in which constitutive loading is dependent on interactions with methylated histones, whereas DNA damage-inducible RNF8-dependent ubiquitylation allows its accumulation at damaged chromatin.
AB - 53BP1 is a well-known mediator of the cellular response to DNA damage. Two alternative mechanisms have been proposed to explain 53BP1's interaction with DNA double-strand breaks (DSBs), one by binding to methylated histones and the other via an RNF8 E3 ligase-dependent ubiquitylation pathway. The formation of RNF8 and 53BP1 irradiation-induced foci are both dependent on histone H2AX. To evaluate the contribution of the RNF8-dependent pathway to 53BP1 function, we generated RNF8 knockout mice. We report that RNF8 deficiency results in defective class switch recombination (CSR) and accumulation of unresolved immunoglobulin heavy chain-associated DSBs. The CSR DSB repair defect is milder than that observed in the absence of 53BP1 but similar to that found in H2AX(-/-) mice. Moreover, similar to H2AX but different from 53BP1 deficiency, RNF8(-/-) males are sterile, and this is associated with defective ubiquitylation of the XY chromatin. Combined loss of H2AX and RNF8 does not cause further impairment in CSR, demonstrating that the two genes function epistatically. Importantly, although 53BP1 foci formation is RNF8 dependent, its binding to chromatin is preserved in the absence of RNF8. This suggests a two-step mechanism for 53BP1 association with chromatin in which constitutive loading is dependent on interactions with methylated histones, whereas DNA damage-inducible RNF8-dependent ubiquitylation allows its accumulation at damaged chromatin.
KW - Animals
KW - Chromatin
KW - Chromosomal Proteins, Non-Histone
KW - DNA Breaks, Double-Stranded
KW - DNA-Binding Proteins
KW - Immunoglobulin Class Switching
KW - Intracellular Signaling Peptides and Proteins
KW - Lymphopenia
KW - Meiosis
KW - Mice
KW - Mice, Knockout
KW - RNA, Messenger
KW - Recombination, Genetic
KW - Transcription, Genetic
KW - Ubiquitin-Protein Ligases
U2 - 10.1084/jem.20092308
DO - 10.1084/jem.20092308
M3 - Journal article
C2 - 20385748
SN - 0022-1007
VL - 207
SP - 973
EP - 981
JO - The Journal of Experimental Medicine
JF - The Journal of Experimental Medicine
IS - 5
ER -