Exposure of living organisms to IR leads to multiple types of DNA lesions including the double-stranded breaks (DSB), which are a dangerous insult for the stability of the genome. Formation of DSB in the genome leads to activation of a tightly regulated cascade of events termed DNA damage response (DDR), which controls the cellular response to the damage. In the DDR, the ternary protein complex MRN is the first recruit to the damage site. It facilitates the recruitment and activation of the major transducer of the damage signal, the ataxia-telangiectasia mutated (ATM) kinase. In parallel MDC1 and 53BP1, which have a role in the activation of ATM, are also recruited to the damage site. Another hallmark of the damage site is phosphorylation of the histone H2A variant H2AX on Ser-139 (in human) to form the γH2AX [29
]. Phosphorylation of H2AX is mediated by the kinases ATM and DNA-PK following exposure to IR. Activated ATM phosphorylates numerous additional substrates and by that regulates their activity. The substrates of ATM are involved in all cellular aspects relevant to DNA damage response including sensing the damage, repair of DNA lesions, control of cell cycle progression, apoptosis, regulation of gene expression and more [31
The cellular response to IR includes major changes in the organization of the chromatin both locally (at the damage site), as well as globally. Among the local changes are de-condensation of the chromatin [33
] possibly by recruitment of chromatin re-modeling complexes such as SWI/SNF [34
], phosphorylation of histones H2AX and H2B, acetylation of histone H4 on multiple sites [36
], ubiquitylation of histone H2A and H2B [37
], incorporation of Lys-56 acetylated histone H3 to the chromatin [40
] and changes in the organization of the heterochromatin localized protein HP1 [41
]. In addition, global de-condensation of the chromatin was found to occur through phosphorylation of KAP-1 by ATM [43
]. At later stages of the repair process, specific chromatin remodeling complexes, such as INO80, are recruited to the damage site to reverse the damage-induced changes [36
]. Considering that substantial chromatin reorganization occurs following exposure to IR, it would be expected that ubiquitous chromatin architectural proteins, such as the HMGNs, would be also involved in the cellular response to IR.
The importance of HMGN1 for the cellular response to IR was first identified by the hyper-sensitivity of Hmgn1−/−
mice to IR; 12 months following exposure to IR the mortality rate of Hmgn1−/−
mice was more than twice the mortality rate of their Hmgn1+/+
littermates. The higher death rate of the Hmgn1−/−
mice was associated with high incidence of lymphomas. Similar hyper-sensitivity to IR was found also in primary cells, which were prepared from these mice. The hyper-sensitive phenotype of the Hmgn1−/−
cells was rescued by ectopic expression of wild-type HMGN1. In contrast, ectopic expression of a point-mutated HMGN1, which cannot bind nucleosomes, failed to rescue the hyper-sensitive phenotype [44
]. This indicates that the ability of HMGN1 to support cell survival following exposure to IR is mediated by its interaction with chromatin. The defective ability of the Hmgn1−/−
cells to stop at the G2/M checkpoint [44
] suggested that HMGN1 may have a role in the activation of the ATM pathway, which controls the G2/M checkpoint response.
Indeed, recently we were able to show that HMGN1 is required for the activation of ATM following IR treatment. Activation of the ATM kinase was measured by the autophosphorylation levels of ATM as well as by the phosphorylation levels of several of its substrates. These two assays revealed a 2–3 fold reduction in ATM activation in Hmgn1−/−
cells in comparison to their Hmgn1+/+
littermates. The faulty activation of ATM in the Hmgn1−/−
cells was rescued by ectopic expression of wild-type HMGN1, but not by ectopic expression of a point mutated HMGN1, which cannot bind nucleosomes [45
]. Thus, in a similar manner to the IR hypersensitivity phenotype of the Hmgn1−/−
], the ability of HMGN1 to support activation of ATM is mediated by the interaction of HMGN1 with chromatin. The DSB sensing factors 53BP1, MDC1 and the MRN complex, which are involved in the activation of ATM, were activated correctly in the Hmgn1−/−
cells, suggesting that HMGN1 affects the ATM itself.
More detailed analysis of ATM activation revealed that HMGN1 also modulates the binding of ATM to chromatin [45
]. Induction of ATM binding to chromatin after formation of DSB is well established and is associated with its activation [46
]. ATM retention to chromatin was compared between Hmgn1−/−
cells and Hmgn1+/+
cells using biochemical and in situ
assays, which were developed by Andegeko et al.
]. With the biochemical assay, the ATM was extracted in a step-wise manner using increasing buffer stringency to distinguish between chromatin unbound protein and chromatin bound protein. In the in situ
assay, the free ATM was washed away from the nucleus by detergents before the fixation and only then the chromatin-bound ATM, which was left inside of the cells, was immunostained. Significantly, ATM binding to chromatin in Hmgn1−/−
cells was 3-fold higher than in the Hmgn1+/+
cells both before and after exposure to IR [45
]. It seems that in the absence of HMGN1, ATM interacts with chromatin incorrectly prior to any induction of DSB, and to a greater degree following induction of DSB. This faulty interaction of ATM with chromatin prior to the IR-induced damage appears to inhibit the activation of ATM following DSB formation.
In search for the mechanism by which HMGN1 affects the activation of ATM, evidences for direct interaction between the two proteins were not found. HMGN1 is not recruited to the damaged areas and does not co-localize with ATM. However, ATM activation is dependent on HMGN1 ability to bind chromatin. Therefore, we explored a possible indirect mechanism, in which HMGN1 affects the activation of ATM through modulation of chromatin organization [45
]. HMGN1 was previously shown to induce acetylation of Lys-14 in histone H3 (H3K14Ac) [9
] and to enhance the activity of PCAF, the major HAT that acetylates histone H3 on Lys-14 [10
]. Therefore, the possibility that HMGN1 affects ATM through modulation of histone acetylation was tested. Indeed, exposure to IR leads to the activation of PCAF [48
] and to a global increase in the levels of H3K14Ac, in a HMGN1 dependent manner [45
]. In order to establish the importance of HMGN1-dependent histone acetylation to the activation of ATM, histone deacetylase (HDAC) inhibitors were used. Addition of HDAC inhibitors to cells reduces the deacetylation rate of histones, and by that leads to an increase in the levels of histone acetylation. Treatment with histone deacetylase (HDAC) inhibitors reduced the chromatin retention of ATM in Hmgn1−/−
cells and increased the activation of ATM in these cells to similar level as in the Hmgn1+/+
]. Taken together, it appears that HMGN1 modulates the levels of H3K14Ac, both before and following exposure to IR possibly through enhancement of PCAF activity. Through this mechanism, it regulates the nuclear organization of ATM. Moreover, the HMGN1-dependent nuclear organization of ATM prior to induction of DSB is crucial for proper activation of ATM and for the survival of the cells ().
Fig. 2 The involvement of HMGN1 in response to DSB. A. The importance of HMGN1 to the nuclear organization of ATM under steady state conditions. Prior to any damage, the HMGN1 affects the nuclear organization of the ATM through modulation of the chromatin structure (more ...)
The notion that chromatin organization plays a role in the activation process of ATM was initially based on experiments, in which increased auto-phosphorylation levels of ATM were induced by interference with the structure of the chromatin fiber without breaking it [50
]. In these experiments the structure of the chromatin fiber was modified either by salt, by the intercalating agent chloroquine or by a HDAC inhibitor. Only recently a mechanistic insight into this process has been gained through the HMGN1 knock-out mice [45
]. In this mechanism, regulation of histone modifications by HMGN1 along with modifying enzymes dictates the interaction of ATM with the chromatin fiber and the potential of ATM activation following DSB formation.