Defective DSB repair correlates with abnormal nuclear morphology and is improved with FTI treatment in Hutchinson-Gilford progeria syndrome fibroblasts

Abstract

Impaired DSB repair has been implicated as a molecular mechanism contributing to the accelerating aging phenotype in Hutchinson-Gilford progeria syndrome (HGPS), but neither the extent nor the cause of the repair deficiency has been fully elucidated. Here we perform a quantitative analysis of the steady-state number of DSBs and the repair kinetics of ionizing radiation (IR)-induced DSBs in HGPS cells. We report an elevated steady-state number of DSBs and impaired repair of IR-induced DSBs, both of which correlated strongly with abnormal nuclear morphology. We recreated the HGPS cellular phenotype in human coronary artery endothelial cells for the first time by lentiviral transduction of GFP-progerin, which also resulted in impaired repair of IR-induced DSBs, and which correlated with abnormal nuclear morphology. Farnesyl transferase inhibitor (FTI) treatment improved the repair of IR-induced DSBs, but only in HGPS cells whose nuclear morphology was also normalized. Interestingly, FTI treatment did not result in a statistically significant reduction in the higher steady-state number of DSBs. We also report a delay in localization of phospho-NBS1 and MRE11, MRN complex repair factors necessary for homologous recombination (HR) repair, to DSBs in HGPS cells. Our results demonstrate a correlation between nuclear structural abnormalities and the DSB repair defect, suggesting a mechanistic link that may involve delayed repair factor localization to DNA damage. Further, our results show that similar to other HGPS phenotypes, FTI treatment has a beneficial effect on DSB repair.

Introduction

Hutchinson-Gilford progeria syndrome (HGPS) is a rare genetic disorder that causes rapid premature aging shortly after birth, recapitulating multiple pathologies associated with aging and resulting in a median lifespan of 13 years (reviewed in Pollex and Hegele [1]). The disease is caused by de novo mutations within exon 11 of the LMNA gene which partially activates a cryptic splice donor site, resulting in deletion of 50 amino acids from exon 11, with subsequent production of a “truncated” form of lamin A termed, progerin or lamin AΔ50 [2], [3]. LMNA encodes the A-type nuclear lamins, primarily lamins A and C. Along with the primary B-type lamins, lamin B1 and lamin B2, the A-type lamins form the nuclear lamina [4], [5], a scaffold-like structure that lines the inner nuclear membrane and also contributes to the nuclear matrix [6]. Multiple LMNA mutations produce nuclear structural irregularities, demonstrating that A-type lamins are intricately involved in nuclear structural organization (reviewed in Dechat et al. [7]). Furthermore, there are multiple lines of evidence indicating a central role for A-type lamins in chromatin organization (reviewed in Dechat et al. [7]).

Accordingly, HGPS cells exhibit altered nuclear structural characteristics and chromatin organization. Nuclear structural irregularities include changes in the spatial distribution of nuclear pore complexes [8] as well as modifications of the nuclear lamina [8], [9] that are likely responsible for the abnormal nuclear morphology observed in primary HGPS cells [2], [3], [8]. These structural irregularities are accompanied by functional changes, including reduced deformability of the nuclear lamina [9], increased nuclear stiffness and sensitivity to mechanical stress [10], and mitotic defects, including abnormal chromosome segregation, delays in cytokinesis, nuclear reassembly, and binucleation [11], [12]. Multiple chromatin organization changes have been described as well. Particularly, HGPS cells exhibit loss of peripheral heterochromatin [8], [13] that may be due to epigenetic changes, including up-regulation of H3K9me3 and H4K20me3, which define constitutive heterochromatin, and downregulation of H3K27me3, which defines facultative heterochromatin [13], [14], [15]. In addition to epigenetic changes, HP1α, which is usually associated with H3K9me3, is downregulated and partially dissociated [14], [15]. It is likely that these abnormalities influence nuclear biological processes that are dependent on proper nuclear structure and chromatin organization.

It has been demonstrated that the nuclear morphological changes and altered chromatin characteristics are not caused by lamin A haploinsufficiency but by the presence of progerin in a dominant gain-of-function fashion, possibly through its accumulation at the inner nuclear membrane (INM) [8], [14], where it appears to alter nuclear lamina structure [9]. Accordingly, reduction of the farnesylated form of progerin using farnesyl transferase inhibitors (FTIs) [16], [17], [18], [19], [20], [21], [22] and reduction of progerin using antisense morpholinos [14] improve nuclear abnormalities in HGPS cells as well as various disease phenotypes in HGPS mouse models. The success of FTIs in these studies and the current lack of any other therapeutic approach for HGPS have lead to a current phase II clinical trial examining the beneficial effect of FTIs in HGPS patients (ClinicalTrials.gov identifier: NCT00425607).

Interestingly, recent studies suggest that DSB accumulation due to impaired DSB repair is one of the mechanisms leading to the accelerated aging phenotype [19], [23], [24], [25]. DSB accumulation appears to be due at least in part to impaired localization of DSB repair factors including Rad51 and Rad50 [24]. It has also been shown that xeroderma pigmentosum group A (XPA), a nucleotide excision repair protein (NER), aberrantly localizes to a subset of DSBs in HGPS cells through interaction with chromatin and inhibits the localization of Rad51 and Rad50, perhaps through steric hindrance [24]. There is evidence to suggest that XPA does not localize to camptothecin (CPT)-induced DSBs, indicating that XPA-localized DSBs may be functionally different in origin or repair [24]. However, XPA-mediated interference with DSB repair does not fully explain the DSB repair problem in HGPS cells since repair of CPT-induced DSBs in HGPS cells is slower compared to wild type cells [24].

In this study, we performed a quantitative analysis of the steady-state number of DSBs and the repair kinetics of IR-induced DSBs in HGPS fibroblasts. We also examined whether the observed deviations from wild type for these characteristics correlated with abnormal nuclear morphology and whether they were caused by progerin. Further, we examined whether FTI treatment can decrease the steady-state DSB level and improve the repair kinetics of IR-induced DSBs in HGPS cells. Lastly, to expand on previous studies that showed that localization of repair factors to DSBs is impaired, we performed a quantitative analysis of the localization kinetics of the MRN repair complex factors, phospho-NBS1 and MRE1, to γ-irradiation-induced DSBs.