Histone H1 couples initiation and amplification of ubiquitin signalling after DNA damage

Abstract

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.

USP51 deubiquitylates H2AK13,15ub and regulates DNA damage response

“…Our result that USP51 overexpression reduces H2AK15ub but has little effect on the formation of RNF168 foci appears to contradict this observation. One possible explanation is that USP51 overexpression may not completely remove H2AK15ub at IRinduced DNA damage sites, and the residual H2AK15ub along with histone H1 ubiquitylation catalyzed by RNF8 (Thorslund et al 2015) can still promote the formation of RNF168 foci.…”

“…RNF8 ubiquitylates histones H1 (Thorslund et al 2015), and ubiquitylated histone H1 products help recruit RNF168, another E3 ubiquitin ligase that is mutated in RIDDLE syndrome, to the chromatin surrounding DNA damage sites (Doil et al 2009;Stewart et al 2009). The primary target of RNF168 is Lys13 and Lys15 of histone H2A (H2AK13,15) (Gatti et al 2012;Mattiroli et al 2012).…”

Yeast HMO1: Linker Histone Reinvented

“…In contrast, Ku, which is integral to DSB repair by nonhomologous end joining, has been reported to readily displace human H1 from DNA ends (87). In human cells, ubiquitylation of H1 at DSB sites was also recently implicated in the recruitment of repair factors, adding to the collection of histone marks that contribute to repair events (88).…”

H1 provides the missing link

“…Notably, although both UDMs were each endowed with ubiquitin-binding properties, they were unique in having evolved to target specialized ubiquitin structures at DSBs. Indeed, while UDM2 specifically interacted with ubiquitylated H2A histones, division of labor has it that UDM1 was important in bringing RNF168 to DSBs via an hitherto unknown factor [9].Notwithstanding the formidable nature in singling out this factor amongst the multiplicity of ubiquitin structures at DSBs, the Mailand team embarked on the mission to tease out exactly how RNF168 migrates to DSBs [11]. Because RNF168 is recruited to DSBs via an RNF8-UBC13-dependent ubiquitylation process [6,7], and the E3-E2 pair encoded the major K63-based ubiquitylating activity at DSBs, the authors took an unbiased proteomics approach and quantitatively compared the abundance of K63-ub conjugates in control cells with those that have been exposed to ionizing radiation (IR).…”

“…Notwithstanding the formidable nature in singling out this factor amongst the multiplicity of ubiquitin structures at DSBs, the Mailand team embarked on the mission to tease out exactly how RNF168 migrates to DSBs [11]. Because RNF168 is recruited to DSBs via an RNF8-UBC13-dependent ubiquitylation process [6,7], and the E3-E2 pair encoded the major K63-based ubiquitylating activity at DSBs, the authors took an unbiased proteomics approach and quantitatively compared the abundance of K63-ub conjugates in control cells with those that have been exposed to ionizing radiation (IR).…”