It is now well established that chromatin plays a central role in the cellular response to ionizing radiation and in DSB repair (1
). Here, we show that HMGN1, a structural protein known to affect chromatin condensation (14
) and histone modifications (26
), plays a role in the ionizing radiation response. The link between HMGN1 and the ionizing radiation response is supported by several observations: First, the survival of ionizing radiation–treated Hmgn1−/−
mice is lower than that of their Hmgn1+/+
littermates; second, Hmgn1−/−
MEFs are more sensitive to ionizing radiation than Hmgn1+/+
MEFs; and third, expression of HMGN1, but not S20,24E-HMGN1 mutant protein, in Hmgn1−/−
MEFs increases their ionizing radiation resistance. Thus, HMGN1 can be considered as an additional chromatin binding protein that affects the repair of DSBs.
DSB repair involves changes in chromatin structure and in posttranslational modifications in histone tails (1
). HMGN1 affects both the levels of histone posttranslational modification (26
) and the stability of the higher-order chromatin structure (14
) and, therefore, it could affect one or more key steps in the DSB repair processes. The phosphorylation of H2ax at and near DSBs triggers the accumulation of various types of histone modifications that lead to changes in chromatin condensation that are necessary for subsequent DSB repair (28
). Although we have not detected significant differences in the levels of γ-H2AX between Hmgn1+/+
cells, loss of HMGN1 could affect some of the other histone modifications associated with DSB repair (28
). It may be relevant that in both H2ax−/−
) and Hmgn1−/−
() cells, the G2
-M checkpoint is impaired. Both of these cells were less sensitive to ionizing radiation treatment and exhibited a significantly higher threshold than normal, before a significant number of cells arrested before entry into M. For the H2ax−/−
cells, it was proposed that below a certain threshold of DNA damage, lack of H2AX phosphorylation disrupts the accumulation of factors necessary to activate the G2
-M checkpoint. Just like H2AX, HMGN1 may be necessary to efficiently activate the G2
-M threshold at low, but not at high, levels of DSB (30
). The failure of both H2ax−/−
cells to activate the G2
-M checkpoint at low ionizing radiation doses strengthens the notion that the structure of chromatin plays an important role in this process. However, the phenotype of the two cell types is distinct in many aspects, indicating distinct ionizing radiation response pathways involving chromatin structure. We suggest that HMGN1, and perhaps other members of the HMGN protein family, facilitate the formation of the chromatin structures that ensure efficient ionizing radiation response and proper DSB repair.
Our finding that the tumor incidence of aged mice lacking HMGN1 protein is almost twice that of wild-type mice is in agreement with a possible role for the protein in ensuring the fidelity of the G2
-M checkpoint. The G2
-M checkpoint arrest of ionizing radiation–irradiated cells serves to ensure the fidelity of DSB repair before entry into mitosis (13
). Faulty repair may lead to mutation and increase tumor frequency. Cells taken from aged mice have significantly more DSBs than cells taken from young mice, an indication of spontaneous DSB occurrences during their life span (32
). Thus, faulty G2
-M arrest and increased mutation frequency could be the underlying cause for the increased tumor burden in Hmgn1−/−
mice. Because HMGN1 is expressed in most tissues, it can be expected that the tumors would be found in various tissues.
Primary mouse cells usually require introduction of two “activated” oncogenes for transformation, unless certain key growth control or oncogenic genes are already disrupted (33
). Our finding that a single transformation with SV40 large T antigen was sufficient to change the basic properties of the primary cells indicate that the absence of HMGN1 is sufficient to disrupt cellular events that control cell proliferation and growth. The growth control mechanisms disrupted by loss of HMGN1 protein may have rendered the animals and the MEFs more susceptible to additional events that ultimately lead to malignant transformations. The interaction of HMGN1 protein with nucleosomes alters the structure of chromatin and modulates various DNA-related nuclear processes including transcription (18
). Thus, the increased tumor burden and tumorigenicity of Hmgn1−/−
mice and MEFs could be due not only to an impaired G2
-M checkpoint but also to indirect effects that lead to alteration in the cellular transcription profile.
Our findings reemphasize the importance of chromatin in the cellular response to ionizing radiation damage and identify HMGN1 as an additional chromatin regulatory element involved in carcinogenesis.