Neuronal accumulation of unrepaired DNA in a novel specific chromatin domain: structural, molecular and transcriptional characterization

Cellular DNA damage response (DDR) is a molecular signaling pathway that is strongly induced by cytotoxic DNA lesions, such as double strand breaks (DSBs) which are produced by endogenous or exogenous genotoxic agents. In neurons, the DDR is mediated by the kinase ATM, which phosphorylates crucial protein partners in this pathway [1]. Mammalian neurons are highly vulnerable to DNA damage due to their high metabolic rate for energy production, generating cytotoxic reactive oxygen species (ROS) that can produce oxidative DNA damage. In addition, the relaxed chromatin configuration (euchromatin) of the majority of neuronal populations facilitates genotoxic agents gaining access to DNA and disrupting its structure [2, 3]. Cytotoxic DSBs can be derived from the conversion of single-strand breaks (SSBs) into DSBs or can be induced by environmental agents, such as ionizing radiation (IR), including X-rays, and chemotherapeutic drugs. They are especially detrimental for neurons as they affect genome integrity and global transcriptional activity [3‐5]. Moreover, DSBs can produce energy starvation given that the DDR is a very high ATP consuming process [6]. Since post-mitotic neurons lack sister chromatids that serve as a template to ensure “error-free” repair by homologous recombination (HR), DSBs need to be repaired by non-homologous end joining (NHEJ) [7]. Due to the fact that DSB ends need to be processed before religation, errors can be introduced in NHEJ repair, resulting in neuronal dysfunction which ultimately contribute to neurodegeneration [8].

In addition to diseases with neurological manifestations caused by mutations in DNA repair factors, there is increasing evidence that defective DNA repair with an accumulation of DNA lesions and loss of genome stability underlies aging and many neurodegenerative disorders in human patients and animal experimental models [6, 9‐12]. It has recently been reported that accumulated DNA damage can produce a deregulated DDR, leading to a senescence-like phenotype in neurons [9].

DDR occurs in the context of chromatin and requires elaborated epigenetic changes of histones in DNA damaged sites and flanking regions to stabilize broken DNA ends, in order to facilitate access of repair factors to damaged sites [8, 13]. Notwithstanding the extensive evidence of DNA damage-associated changes in the epigenome, it still remains unclear how neurons tolerate the accumulation of DNA lesions and how neurons process DNA damage in chromatin compartments and sequestrated unrepaired DNA in a few persistent DNA damage foci (PDDF) with a non-random spatial organization.

In a previous study, using an experimental model of DNA damage in rat sensory ganglion neurons (SGNs) with IR (4 Gy), we demonstrated that the neuronal DDR includes the formation of two categories of DNA-damage chromatin compartments [3]. The first are transient small and very numerous foci, which disappear within the first day post-IR, reflecting an effective DNA repair. The second consist of a few PDDF where unrepaired DNA is accumulated and remained at 15 days post-IR. Furthermore this neuronal DDR does not induce apoptosis but triggers G0-G1 cell cycle transition [3]. In this context, SGNs exposed to IR provide an excellent experimental system for investigating the nuclear organization and fate of unrepaired DNA accumulated in PDDF. Moreover, IR with a sub-lethal dose (4 Gy) in rodents triggers DDR, prevents neuronal apoptosis and makes it possible to study the long-term compartmentalization and dynamic of unrepaired DNA and its relationships with specific chromatin modifications and transcription rate. Finally, due to the absence of blood brain barrier in peripheral ganglia, the DNA damage-induced dysfunction of SGNs is a main component in peripheral neuropathies caused by cancer chemotherapy [14, 15]. In this study we analyze the following in irradiated SGNs i) the spatiotemporal organization of PDDF, ii) the molecular composition of these foci, iii) the ultrastructural compartmentalization of PDDF in cleared chromatin domains with unfolded chromatin fibers, iv) the transcriptional activity of PDDF and flanking chromatin domains, and v) the specific spatial association of PDDF with nuclear compartments which promotes gene silencing.

Our results in healthy SGNs exposed to sub-lethal doses of IR for inducing DSBs demonstrate that unrepaired DNA is retained for extended periods of time, up to 45 days in our study, in a few PDDF. The number and size of these foci is dependent on the total dose of IR, indicating a direct relationship between DNA damage accumulation and PDDF formation. PDDF are not simply static DNA damage “scars” of the genome, but in fact dynamic and specifically compartmentalized chromatin domains that recruit essential factors for signaling and repair DNA, such as γH2AX, pATM, Ub-H2A and WRAP53. Furthermore, the progressive reduction of SGNs carrying PDDF over time of post-IR reported here supports that a slower DNA repair is still ongoing in some foci. This is consistent with the permanent enrichment of DNA damage signaling and repair factors in PDDF, independently of the post-IR time and the total dose of IR administered. Our study provides the first analysis on the organization and dynamics of persistent foci of damaged DNA in neurons.

Regarding the possible pathophysiological implications of the accumulation of unrepaired DNA damage in neuronal PDDF, the loss of genomic integrity may contribute to both cognitive ageing and the pathogenesis of neurodegenerative diseases [5, 12, 34]. An important challenge in this context is to understand how neurons can tolerate the accumulation of DNA damage and what is the threshold in DNA lesions that trigger neurodegeneration. Neurons are particularly vulnerable to DNA damage because their high metabolic rate which generates ROS. Moreover, it is believed that neurons have a decreased ratio of anti-oxidant to pro-oxidant enzymes that may potentially result in a state of elevated oxidative stress and DNA damage [35]. Importantly, recent studies demonstrate that the high transcriptional activity in a subset of genes that govern crucial neuronal functions, such as early-response genes, can trigger the formation of DSBs [34, 36]. The generation of DSBs is mediated by the activity of topoisomerase IIβ, an enzyme which is robustly expressed in neurons and linked to transcription-related functions [37]. The transcription activity-induced DSBs in a physiological context raises the question of whether the accumulation of unrepaired or erroneously repaired DSBs could result in the formation of PDDF, and potentially contribute to the pathogenesis of neurological diseases [36]. In support of this notion, we have found PDDF in approximately 5 % of non-irradiated SGNs. In this vein, the normal production of DSBs under physiological conditions is exacerbated by the neuronal accumulation of β-amyloid in a murine model of Alzheimer disease [34].

In the case of SGNs, increased vulnerability to DNA damage occurs due to the absence of blood brain barrier in peripheral ganglia that facilitates the access of genotoxic agents, including drugs used in cancer chemotherapy [38‐40]. Indeed, DNA damage in SGN seems to be an important component in the long-term peripheral neurotoxicity of anticancer chemotherapy [15, 41].

An important aspect of our results is the neuronal asymmetry in the kinetics of DDR. Indeed, PDDF appear in approximately 30 % of the global population of SGNs at 15 days post-IR while the majority of neurons lack PDDF, indicating effective DNA repair. Different lines of evidence suggest that the kinetics of DDR is related to the individual pattern of transcriptional activity. For example, active neurons break and repair their DNA more often than their less active, “resting”, neighbours, resulting in broad differences in accumulated DNA lesions which will become progressively apparent in the long term [5].

Concerning the structural organization of PDDF reported here, it is well-known in non-neuronal cultured cell lines that DDR occurs in the chromatin landscape. Thus, the DDR requires local modifications in chromatin structure that first become more decondensed and accessible to DNA repair factors, followed by restoration of chromatin organization upon completion of DNA repair [1, 13, 42, 43]. Essential factors for these chromatin modifications are posttranslational modifications of histones, including phosphorylation of the H2AX and ubiquitylation of H2A and H2B, as well as chromatin chaperones and ATP-dependent remodeling factors [44]. Our electron microscopy findings in SGNs allowed us to characterize chromatin modifications in neuronal PDDF. Important hallmarks of PDDF are the super-relaxation of chromatin, which appears as cleared nuclear areas, the sharp boundary between damaged chromatin and the adjacent euchromatin, and the complete transcriptional silencing. The existence of cleared nuclear areas was initially reported in the classical study of Cavanagh et al. [38] in rat SGNs treated with adriamycin, a genotoxic drug used in chemotherapy, although the authors did not relate this finding with DNA damage. Interestingly, the large-scale chromatin decompaction in PDDF occurs in an euchromatic environment, while heterochromatin clumps lack immunolabeling for DNA damage and repair factors. This indicates the higher vulnerability to DNA damage of the euchromatin, which is transcriptionally active or poised for activation [45]. The fine structure of PDDF is characterized by a loose network of fibers ranging from 30 nm chromatin fibers [46] to 11 nm. This finding supports the notion that unfolded 30 nm fiber intermediates provide a structural scaffold which is accessible for signaling and repair factors, as the selective and strong 53BP1 immunogold labeling of the fibers reflects. Therefore, the relaxed state of chromatin in PDDF fits the requirement for DNA repair process, which takes place on exposed DNA [47]. Furthermore, two of the factors enriched in neuronal PDDF, WRAP53 and Ub-H2A, have been implicated in DNA repair in cellular models of DNA damage. Mechanistically, WRAP53 targets the ubiquitin E3 ligase RNF8 to DSBs where ubiquitylated histones H2A and H2AX promote the assembly of repair factor such as 53BP1 [22, 24].

The sharp ultrastructural boundary between the damaged chromatin of PDDF and adjacent euchromatin supports that unrepaired genes congregate and isolate in these damage/repair foci to reduce genome instability and preserve global transcription in undamaged euchromatin. We hypothesized that the sequestration of DNA lesions in one or two individual PDDF allows the neuron to tolerate the accumulation of unrepaired DNA without triggering apoptotic pathways. The molecular barriers between damaged and undamaged chromatin in PDDF are unknown. Interestingly, a recent molecular study, using a new cell-based DSB inducible system to characterize the chromatin landscape around DSB, demonstrates that the recruitment of cohesin prevents γH2AX spreading [48]. The authors propose that, in addition to the function in chromatin architecture, cohesin helps to isolate active genes from damaged ones carrying DSBs. The compartmentalization of neuronal DNA damage might involve DSB-containing chromosome domains moving over relatively large distances to be clustered in PDDF. This interpretation is consistent with previous observations in cell lines revealing increased mobility of the break sites upon IR treatment [49, 50]. Accordingly, in yeast and mammalian cell lines exposed to IR, the formation of DSBs in the nucleolus and heterochromatin results in the movement and relocalization of breaks to the periphery of these nuclear structures [51, 52].

Our results provide the first demonstration of a complete transcriptional silencing at DNA damage foci in neurons. Three lines of evidence support gene silencing within PDDF: i) the total absence of 5’-FU incorporation, a precursor for RNA synthesis, demonstrated with the high resolution of the immunogold electron microscopy, ii) the absence of immunogold labeling for the RNA polymerase II phosphorylated on Ser 2, a well established marker of the elongation phase of transcription [29, 53], and iii) the lack of concentration of RNAs, as indicated the preferential cytochemical staining for ribonucleoproteins [25]. Moreover, transcriptional repression corresponds exactly to the local distribution of γH2AX, which spreads on the entire PDDF but does not propagate on active genes in adjacent euchromatin. Therefore, the expression of γH2AX can be used as a reliable marker of gene silencing in DNA damaged neurons. Transcriptional silencing at PDDF can be essential to reduce genome instability by preventing the synthesis of aberrant mRNA and protein products encoded by damaged genes. In accordance with the PDDF enrichment in p-ATM and Ub-H2A, recent molecular studies have reported an ATM-dependent transcriptional repression at DSBs mediated by H2A ubiquitylation, whereas deubiquitylation of H2A restores transcription [23].

An intriguing aspect of PDDF is their preferential spatial association with the nucleolar and less frequently, nuclear periphery. Interestingly, both nuclear domains are enriched in constitutive heterochromatin, characterized by the abundance of repeated DNA sequences and the binding of HP1 proteins, and correlates with transcriptional silencing [54, 55]. Positioning of PDDF at these repressive nuclear environments may facilitate transcriptional silencing of damaged genes and contribute to maintain genomic stability within PDDF. Alternatively, but not mutually exclusive, nucleolus- and heterochromatin-associated PDDF may represent repair centers for damaged ribosomal genes and repeated DNA sequences of heterochromatin, respectively. In this vein, in non-neuronal culture cell lines exposed to IR, the relocation of DSBs have been reported from the initial induction site in the nucleolus or heterochromatin to the periphery of both structures [51, 52, 56, 57]. Further ChIP-seq analysis of the γH2AX-binding DNA will be necessary to determine what genes are enriched in neuronal PDDF, and particularly if they contain damaged ribosomal genes and repeated sequences of heterochromatin.

In conclusion, our results strongly indicate that unrepaired DNA in neurons is sequestrated in one to three discrete PDDF of transcriptionally silent chromatin. This transcriptional silencing can be essential to preserve genome stability and prevent the synthesis of aberrant mRNA and protein products encoded by damaged genes. Moreover, the expression of γH2AX can be used as a reliable marker of gene silencing in DNA damaged neurons.