Histone H1 couples initiation and amplification of ubiquitin signalling after DNA damage.
DNA double-strand breaks (DSBs) are highly cytotoxic DNA lesions that trigger non-proteolytic ubiquitylation of adjacent chromatin areas to generate binding sites for DNA repair factors. This depends on the sequential actions of the E3 ubiquitin ligases RNF8 and RNF168 (refs 1-6), and UBC13 (also known as UBE2N), an E2 ubiquitin-conjugating enzyme that specifically generates K63-linked ubiquitin chains. Whereas RNF168 is known to catalyse ubiquitylation of H2A-type histones, leading to the recruitment of repair factors such as 53BP1 (refs 8-10), the critical substrates of RNF8 and K63-linked ubiquitylation remain elusive. Here we elucidate how RNF8 and UBC13 promote recruitment of RNF168 and downstream factors to DSB sites in human cells. We establish that UBC13-dependent K63-linked ubiquitylation at DSB sites is predominantly mediated by RNF8 but not RNF168, and that H1-type linker histones, but not core histones, represent major chromatin-associated targets of this modification. The RNF168 module (UDM1) recognizing RNF8-generated ubiquitylations is a high-affinity reader of K63-ubiquitylated H1, mechanistically explaining the essential roles of RNF8 and UBC13 in recruiting RNF168 to DSBs. Consistently, reduced expression or chromatin association of linker histones impair accumulation of K63-linked ubiquitin conjugates and repair factors at DSB-flanking chromatin. These results identify histone H1 as a key target of RNF8-UBC13 in DSB signalling and expand the concept of the histone code by showing that posttranslational modifications of linker histones can serve as important marks for recognition by factors involved in genome stability maintenance, and possibly beyond.
Ubiquitin Signaling Group, Protein Signaling Program, The Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, DK-2200 Copenhagen, Denmark.
Tina
T
Thorslund
Ubiquitin Signaling Group, Protein Signaling Program, The Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, DK-2200 Copenhagen, Denmark.
Anita
A
Ripplinger
Ubiquitin Signaling Group, Protein Signaling Program, The Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, DK-2200 Copenhagen, Denmark.
Saskia
S
Hoffmann
Proteomics Program, The Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, DK-2200 Copenhagen, Denmark.
Thomas
T
Wild
Division of Biochemistry, Cancer Genomics Center, Netherlands Cancer Institute, 1066 CX Amsterdam, the Netherlands.
Michael
M
Uckelmann
Ubiquitin Signaling Group, Protein Signaling Program, The Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, DK-2200 Copenhagen, Denmark.
Bine
B
Villumsen
Proteomics Program, The Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, DK-2200 Copenhagen, Denmark.
Takeo
T
Narita
Division of Biochemistry, Cancer Genomics Center, Netherlands Cancer Institute, 1066 CX Amsterdam, the Netherlands.
Titia K
TK
Sixma
Proteomics Program, The Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, DK-2200 Copenhagen, Denmark.
Chunaram
C
Choudhary
Ubiquitin Signaling Group, Protein Signaling Program, The Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, DK-2200 Copenhagen, Denmark.
Simon
S
Bekker-Jensen
Ubiquitin Signaling Group, Protein Signaling Program, The Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, DK-2200 Copenhagen, Denmark.
Niels
N
Mailand
D002843:Chromatin / Q000378:metabolism; D053903:DNA Breaks, Double-Stranded; D004249:DNA Damage; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000378:metabolism; D006657:Histones / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008239:Lysine / Q000378:metabolism; D017434:Protein Structure, Tertiary; D015398:Signal Transduction; D025801:Ubiquitin / Q000378:metabolism*; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry / Q000378:metabolism; D054875:Ubiquitination
D002843:Chromatin; D004268:DNA-Binding Proteins; D006657:Histones; C486521:RNF8 protein, human; D025801:Ubiquitin; C471607:UBE2N protein, human; D044763:Ubiquitin-Conjugating Enzymes; C535210:RNF168 protein, human; D044767:Ubiquitin-Protein Ligases; D008239:Lysine