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. 2014 Feb 6;94(2):246-56.
doi: 10.1016/j.ajhg.2014.01.007.

ERCC6L2 mutations link a distinct bone-marrow-failure syndrome to DNA repair and mitochondrial function

Hemanth Tummala  1 , Michael Kirwan  1 , Amanda J Walne  1 , Upal Hossain  2 , Nicholas Jackson  3 , Corinne Pondarre  4 , Vincent Plagnol  5 , Tom Vulliamy  6 , Inderjeet Dokal  2
Affiliations

ERCC6L2 mutations link a distinct bone-marrow-failure syndrome to DNA repair and mitochondrial function

Hemanth Tummala et al. Am J Hum Genet. .

Abstract

Exome sequencing was performed in three index cases with bone marrow failure and neurological dysfunction and whose parents are first-degree cousins. Homozygous truncating mutations were identified in ERCC6L2 in two of the individuals. Both of these mutations affect the subcellular localization and stability of ERCC6L2. We show here that knockdown of ERCC6L2 in human A549 cells significantly reduced their viability upon exposure to the DNA-damaging agents mitomycin C and Irofulven, but not etoposide and camptothecin, suggesting a role in nucleotide excision repair. ERCC6L2-knockdown cells also displayed H2AX phosphorylation, which significantly increased upon genotoxic stress, suggesting an early DNA-damage response. Intriguingly, ERCC6L2 was seen to translocate to the mitochondria and the nucleus in response to DNA damage, and ERCC6L2 knockdown induced intracellular reactive oxygen species (ROS). Treatment with the ROS scavenger N-acetyl cysteine attenuated the Irofulven-induced cytotoxicity in ERCC6L2-knockdown cells and abolished ERCCGL2 traffic to the mitochondria and nucleus in response to this DNA-damaging agent. Collectively, these observations identify a distinct bone-marrow-failure syndrome due to mutations in ERCC6L2, a gene implicated in DNA repair and mitochondrial function.

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Figures

Figure 1
Figure 1
Truncating Mutations in ERCC6L2 (A and B) Shown are two families in which ERCC6L2 mutations segregate as an autosomal-recessive trait. A Sanger sequencing trace and the genotype of each individual are given; inferred genotypes are in parentheses. (B) For family 2, a plus sign (+) indicates the WT allele and a minus sign (−) indicates the mutant allele. The normal sequencing trace to the left of family 2 comes from an unrelated individual. (C) The identified ERCC6L2 alterations leading to premature truncation are indicated on a diagram of the protein; functional domains are also annotated. (D and E) H&E staining of bone marrow trephine biopsies from an unrelated control sample and the index case (case 1) from family 1 reveals the degree of hypoplasia in this individual. (F and G) Immunostaining on these bone marrow trephine sections revealed the presence of ERCC6L2 in a normal unrelated control, but it was not detected in the affected individual. (H and I) Positive control staining for GAPDH antigen was observed in both sections. Figures are representative of different images taken from different fields of view. Magnification is 40×. The scale bar represents 50 μm.
Figure 2
Figure 2
Truncating Mutations Affect the Localization and Degradation of ERCC6L2 (A) Confocal-microscopy images demonstrate predominant cytoplasmic and nuclear localization of WT GFP-ERCC6L2 in A549 cells. (B and C) Both truncating variants of ERCC6L2 formed aggregates. (E–L) Colocalization in yellow was observed for both of the altered ERCC6L2 proteins with BiP antibody, which binds to the ER protein BiP (E and F), LC3β antibody, which localizes to autophagic vacuoles (H and I), and LysoTracker, which stains lysosomes (K and L). The colocalization observed was not an artifact of cross-channel noise or bleed from a compliment channel. The Pearson correlation coefficients for colocalization revealed r2 > 0.5 when measured for the entire cell with ZEN software (Zeiss). (D, G, and J) No colocalization was observed between WT ERCC6L2 and any of these organelle markers (Pearson correlation coefficients r2 < 0.08). (M–O) Neither the WT nor altered forms of ERCC6L2 showed any colocalization with ubiquitin, as evidenced by the green staining pattern (Pearson correlation coefficient r2 < 0.03). All panels are representative of images taken from different fields of view in three separate experiments. Images display DAPI (blue), GFP-tagged ERCC6L2 (green), and organelle-labeling markers (red). The scale bar represents 30 μm.
Figure 3
Figure 3
ERCC6L2 Plays a Role in the DNA-Damage-Response Pathway (A and B) Compared to nontarget-siRNA-transfected cells, ERCC6L2-knockdown cells showed reduced survival after 48 hr treatment with MMC and Irofulven in a dose-dependent manner. Error bars represent the SEM obtained from three independent experiments (one-way ANOVA with Tukey’s test). (C and D) Compared to nontarget-siRNA-transfected cells, ERCC6L2-knockdown cells revealed γH2AX foci at basal level. (E and F) γH2AX foci were higher in number in ERCC6L2-knockdown cells than in nontarget-siRNA-transfected cells after treatment with Irofulven. Panels are representative of images taken from different fields of view displaying γH2AX (green) and DAPI (blue). The scale bar represents 5 μm. (G) The bar graph represents the quantified γH2AX fluorescence value obtained from each individual field of view and normalized to the number of cells stained by DAPI (n > 50) at each individual time point. Error bars represent the SEM (Mann-Whitney test), derived from data obtained from five fields of view at each individual time point from two independent experiments.
Figure 4
Figure 4
ERCC6L2 Traffics to the Mitochondria and Nucleus after Genotoxic Stress (A) Immunoblotting was performed on subcellular fractionated protein lysates after MMC and Irofulven treatment with ERCC6L2 antibody. Abbreviations are as follows: Cy, cytosolic; N, nuclear; and M, membraneous. Subcellular fractionation was verified by GAPDH as a cytoplasmic marker and BiP as a membrane marker. (B) The increased membraneous and nuclear localization of ERCC6L2 is represented graphically by extrapolation of the values acquired from densitometry analysis. (C–Q) Confocal images show ERCC6L2 localization to the mitochondria and nucleus after genotoxic stress. Nuclear localization of ERCC6L2 (white arrows) showed a correlation with DAPI staining after MMC (I and K) and Irofulven (N and P) treatment but to a lesser extent with DMSO (D and F). Colocalization of ERCC6L2 to the mitochondria is represented in yellow for MMC (J and L) and Irofulven (O and Q) treatment, but not DMSO (E and G) treatment. The colocalization observed was not an artifact of cross-channel noise or bleed from a compliment channel. For colocalization analysis, the Pearson correlation coefficients (r2) of the relative distribution of the two channels in mitochondrial regions of the cells revealed r2 > 0.482 after MMC treatment, r2 > 0.739 after Irofulven treatment, and r2 < 0.03 after DMSO treatment. Images display MitoTracker (red), ERCC6L2 (green), and DAPI (blue). Panels are representative of images taken from different fields of view in three separate experiments. The scale bar represents 30 μm.
Figure 5
Figure 5
The Relationship among ERCC6L2, ROS, and Genotoxic Stress (A) Compared to cells transfected with nontarget siRNA, A549 cells transfected with ERCC6L2 siRNA showed an increase in intracellular ROS levels. Error bars represent the SEM of three independent experiments performed in triplicate (∗∗p < 0.01). (B) Changes in ROS levels upon Irofulven stimulation were monitored over time. Error bars represent the SEM of eight readings in three independent experiments. (C) Analysis with fluorescence-activated cell sorting revealed reduced cytotoxicity of Irofulven in ERCC6L2-knockdown cells in the presence of NAC in a concentration-dependent manner. Error bars represent the SEM from three independent experiments. (D) Immunocytochemistry on A549 cells treated with Irofulven (650 nM) in the presence of NAC (10 mM). Images display MitoTracker (red), ERCC6L2 (green), and DAPI (blue). Panels are representative of images taken from different fields of view in three separate experiments. The scale bar represents 30 μm.
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