DNA repair

New tales of an old tail

Modifications of DNA-associated histone proteins maintain genome integrity. On damage to DNA, phosphorylation of histone H2A.X determines whether repair is justified or if the damaged cell must die.

Chromosomal DNA wraps around histone proteins to form a complex scaffold called chromatin1. The reorganization of these proteins following DNA damage is crucial for repairing the damage, and so maintaining genomic integrity and reducing the likelihood of cell death or cancer. One such histone modification — known as γ-H2A.X — follows DNA double-strand breaks (DSBs) and involves phosphorylation by the enzyme ATM of serine residue 139, which is located in the carboxy-terminal tail of the histone variant H2A.X (ref. 2). γ-H2A.X generates a chromosomal microenvironment that promotes recruitment of repair proteins3 and facilitates DNA repair to reduce the risk of mutations4. But how this modification is regulated and how it affects cell fate have remained elusive. Two papers5,6, including one on page 591 of this issue, provide insights into these questions.

The discovery of DSB-induced γ-H2A.X sparked enormous efforts to decipher how repair and signalling proteins assemble into foci on chromatin marked by this modification. The search concentrated mainly on identifying repair factors and other histone modifications operating downstream of γ-H2A.X — hence 'moving away' from this priming DSB-associated histone mark. But it emerges that another key chromatin modification in response to DSBs also occurs in the H2A.X tail, just three amino acids away from serine 139 (S139).

Indeed, Xiao et al.5 and Cook et al.6 have now independently discovered that tyrosine residue 142 (Y142) of H2A.X is also phosphorylated (Fig. 1a). Both groups show, however, that unlike S139, Y142 is already phosphorylated in unstressed cells and becomes gradually dephosphorylated after DNA damage. Even more unexpectedly, dephosphorylation of Y142 seems to be a prerequisite for the γ-H2A.X modification, indicating that the phosphorylation status of the Y142 residue of H2A.X regulates what has been considered the main trigger of the entire DSB-induced chromatin pathway. Such a twist in our thinking about genome-maintenance mechanisms clearly deserves a closer look.

Figure 1: A matter of life or death5,6.

a, Normally, the WSTF kinase associates with the carboxy terminus of the histone variant H2A.X and phosphorylates its Y142 residue. Thus, chromatin remains in a 'standby' mode with no unnecessary DNA repair events. b, When DNA double-strand breaks occur after exposure to genotoxic agents, WSTF dissociates and is replaced with the Eya1/3 phosphatases, which dephosphorylate Y142, facilitating S139 phosphorylation (the γ-H2A.X modification) by the ATM enzyme. What happens next depends on whether the damage is repairable. c, If repair is possible, phosphorylated S139 recruits MDC1 and other repair factors. d, If it is not, the γ-H2A.X tail might undergo conformational changes that allow maintenance or re-phosphorylation of Y142. This would prevent retention of repair factors, and instead attract the JNK1 complex, which promotes apoptosis.

The starting point for Xiao et al.5 was the observation that the evolutionarily conserved Y142 in human H2A.X is phosphorylated in vivo. They then found that components of the WICH chromatin-remodelling complex7,8 interact with the carboxy terminus of H2A.X, where Y142 is located. Strikingly, they showed that the WSTF component of WICH has tyrosine-kinase activity, enabling it to phosphorylate Y142. The authors also found that, after DNA damage, WSTF dissociates from chromatin — consistent with a decrease in Y142 phosphorylation — making way for the γ-H2A.X modification (Fig. 1b).

Cook et al.6 observed that, during embryonic development of mouse kidney, deletion of either Eya1 or Eya3 — genes encoding protein-phosphatase enzymes that dephosphorylate tyrosine residues — coincides with increased γ-H2A.X. The authors also found that the Eya1 and Eya3 enzymes bind to and co-localize with γ-H2A.X at foci of DSBs in the nucleus, leading them to consider that H2A.X might be phosphorylated on a tyrosine residue; indeed, they identified Y142 as the target. What's more, in agreement with the observations of Xiao and colleagues, Cook et al. report that after DNA damage there is an Eya1- or Eya3-dependent decrease in tyrosine phosphorylation of H2A.X (Fig. 1b). Reducing Eya levels prevented DNA-damage-induced dephosphorylation of Y142 and the proper interaction of γ-H2A.X with MDC1 — an adaptor protein that senses γ-H2A.X and orchestrates the assembly of repair proteins on the chromatin at DSBs9,10.

Together, these findings5,6make a compelling case for Y142 phosphorylation as a new modification of H2A.X and suggest that a balance between the kinase activity of WSTF and the phosphatase activity of Eya proteins regulates both the formation of γ-H2A.X-marked chromatin and the recruitment of repair factors to DSBs. And, besides uncovering another dimension of the chromatin response to genotoxic stress, each paper provides other surprising results.

First, the WAC catalytic domain that Xiao and colleagues5 identified in the amino terminus of WSTF shares no sequence similarity with other known kinase enzymes4 — an intriguing finding, the significance of which extends beyond DNA repair. WSTF probably also phosphorylates substrates other than H2A.X, and the identification of these might help explain the clinical symptoms associated with Williams–Beuren syndrome, a neurodevelopmental disorder linked to deletions of the WSTF gene. Furthermore, other proteins might contain a WAC domain, and a search for such hitherto unrecognized tyrosine kinases could be rewarding.

Second, Cook et al.6 report that peptides derived from the carboxy-terminal tail of H2A.X that were phosphorylated on both S139 and Y142 did not bind MDC1, consistent with the fact that Y142 dephosphorylation is required for γ-H2A.X–MDC1 interaction. What was unexpected, however, was that the doubly phosphorylated H2A.X peptide binds the protein kinase JNK1 — an established inducer of programmed cell death (apoptosis). It seems, therefore, that phosphorylated Y142 might function as a decision-maker, determining cell fate after DNA damage. When repair is possible, Y142 is dephosphorylated, allowing the γ-H2A.X modification and the recruitment of repair factors (Fig. 1c). Otherwise, Y142-phosphorylated H2A.X persists, recruiting the JNK1 complex to 'switch' to the pro-apoptotic mode, and eliminate cells with irreversibly damaged genomes from the organism (Fig. 1d).

As with all inspiring discoveries, the work of Xiao et al.5 and Cook et al.6 raises yet more questions. As WSTF is the kinase responsible for Y142 phosphorylation — and could thus be viewed as a negative regulator of γ-H2A.X — one would predict that reducing WSTF levels could facilitate γ-H2A.X formation. In fact, the opposite happens: in the absence of WSTF, γ-H2A.X and focus formation cannot be sustained, and MDC1 recruitment to DSBs is inhibited5. To explain this conundrum, Xiao et al.5 propose that WSTF might also help adjust local chromatin structure for maintenance of γ-H2A.X. This is plausible, as the WICH complex also has chromatin-remodelling activity during DNA replication7,8.

The main conceptual issue arising from Cook and colleagues' results6 is the proposed role of phosphorylated Y142 in promoting cell death. On one hand, the authors provide evidence for increased H2A.X–JNK1 interaction in cells exposed to high doses of radiation. This indeed supports the switch model, as such Y142-mediated recruitment of JNK to sites of DSBs helps direct cells towards apoptosis as a last resort. On the other hand, they show that Y142 is dephosphorylated after DNA damage, resulting in the loss of the 'docking site' for JNK1. At first glance at least, this finding does not fit the switch model, calling for more work to reconcile it with the observed pro-apoptotic effects of Y142 phosphorylation. It may be, however, that Y142 is re-phosphorylated after futile attempts to repair excessive DNA damage.

Clearly, the issue of the efficiency of DSB repair and the role of posttranslational chromatin modifications in this process is here to stay. Nevertheless, the two papers5,6 provide a fresh conceptual framework and tools to tackle this challenge, which should enable us to better understand the genesis of major genome-instability diseases, including cancer, premature ageing and neurodegeneration.


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Lukas, J., Bartek, J. New tales of an old tail. Nature 458, 581–583 (2009). https://doi.org/10.1038/458581a

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