Class switching and meiotic defects in mice lacking the E3 ubiquitin ligase RNF8.
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.
Santos
Margarida Almeida
MA
Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Huen
Michael S Y
MS
Jankovic
Mila
M
Chen
Hua-Tang
HT
López-Contreras
Andrés J
AJ
Klein
Isaac A
IA
Wong
Nancy
N
Barbancho
Juan L R
JL
Fernandez-Capetillo
Oscar
O
Nussenzweig
Michel C
MC
Chen
Junjie
J
Nussenzweig
André
A
D000818:Animals; D002843:Chromatin / Q000378:metabolism; D002868:Chromosomal Proteins, Non-Histone; D053903:DNA Breaks, Double-Stranded; D004268:DNA-Binding Proteins; D017578:Immunoglobulin Class Switching / Q000235:genetics / Q000502:physiology*; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism; D008231:Lymphopenia / Q000235:genetics / Q000503:physiopathology; D008540:Meiosis / Q000235:genetics / Q000502:physiology*; D051379:Mice; D018345:Mice, Knockout; D012333:RNA, Messenger / Q000235:genetics; D011995:Recombination, Genetic; D014158:Transcription, Genetic; D000071857:Tumor Suppressor p53-Binding Protein 1; D044767:Ubiquitin-Protein Ligases / Q000172:deficiency / Q000235:genetics / Q000502:physiology*
D002843:Chromatin; D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; D012333:RNA, Messenger; C498302:Trp53bp1 protein, mouse; D000071857:Tumor Suppressor p53-Binding Protein 1; C549817:Rnf8 protein, mouse; D044767:Ubiquitin-Protein Ligases
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