Deficiency in the nuclear-related factor erythroid 2 transcription factor (Nrf1) leads to genetic instability

Abstract

Nuclear factor erythroid-derived 2-related factor 1 (Nrf1) regulates cellular stress response genes, and has also been suggested to play a role in other cellular processes. We previously demonstrated that hepatocyte-specific deletion of Nrf1 in mice resulted in spontaneous apoptosis, inflammation, and development of liver tumors. Here, we showed that both fibroblasts derived from Nrf1 null mouse embryos and fibroblasts expressing a conditional Nrf1 allele showed increased micronuclei and formation of abnormal nuclei. Lentiviral shRNA-mediated knockdown of Nrf1 in SAOS-2 cells also resulted in increased micronuclei, abnormal mitosis and multi-nucleated cells. Metaphase analyses showed increased aneuploidy in Nrf1(-/-) embryonic fibroblasts. Nuclear defects in Nrf1-deficient cells were associated with decreased expression of various genes encoding kinetochore and mitotic checkpoint proteins. Our findings suggest that Nrf1 may play a role in maintaining genomic integrity, and that Nrf1 dysregulation may induce tumorigenesis.

Figure 1. Nrf1 deficient cells exhibit abnormal nuclei

(A) Micrograph of DAPI stained wild type and Nrf1-/- mouse embryonic fibroblasts. (C) Micrograph of DAPI stained DMSO- or 4OHT-treated Nrf1flox/flox;Cre-ERT2 fibroblasts. Note aberrant shaped nuclei and micronuclei (arrowheads) in Nrf1-/- cells and 4OHT-treated Nrf1flox/flox;Cre-ERT2 fibroblasts. (B, D) Histograms show mean ± standard deviation of micronuclei in control and Nrf1 knockout cells. Significant P values are indicated *(P < 0.05).

Figure 2. Knockdown of Nrf1 leads to abnormal nuclear shape and micronuclei formation

(A) Protein extracts of SAOS cells transduced with scramble shRNA, or Nrf1 shRNA were subjected to western blotting using the antibodies indicated. (B) Representative immunofluorescence analysis of SAOS cells transduced with scramble shRNA, or Nrf1 shRNA. Note multinuclei, donut-shaped and aberrant shaped nuclei in Nrf1 knockdown cells. Arrowhead indicates micronuclei. (C) Histograms show mean ± standard deviation of micronuclei in control and Nrf1 knockdown SAOS cells. Significant P values are indicated *(P < 0.05). (D) Protein extracts of HCT116 cells transduced with scramble shRNA, or Nrf1 shRNA were subjected to western blotting using the antibodies indicated. (E) Histograms show mean ± SD of abnormal and micronuclei in control and Nrf1 knockdown HCT116 cells. Significant P values are indicated *(P < 0.05).

Figure 3. Knockdown of Nrf1 in SAOS cells leads to abnormal chromosome segregation

Micrographs of DAPI stained SAOS cells transduced with Nrf1 shRNA showing atypical anaphase figures (multipolar centrosome, abnormal chromosome segregation and anaphase bridge). Histogram shows percentage of abnormal mitotic figures in control and Nrf1 knockdown cells.

Figure 4. Nrf1^-/- cells exhibit increased chromosomal instability

(A) Representative Giemsa-stained metaphase spread of wild type MEF showing normal (40) number of chromosomes. (B) Representative Giemsa-stained metaphase spread of Nrf1-/- MEF showing aneuploidy. (C) Analysis of the percentage of aneuploid cells in MEF cultures. Values are from three independent wild type and Nrf1^-/- MEF cultures. 95 metaphases were counted for each MEF line. (D) Distribution of chromosome numbers of wild type and Nrf1^-/- MEF lines. Data shown are combined values from two independent MEF lines per genotype.

Figure 5. Nrf1-deficient cells have abnormal expression of kinetochore and spindle assembly checkpoint genes

Comparison of mRNA encoding kinetochore and spindle assembly checkpoint genes in (A) wild type and Nrf1^-/- MEF cells, (B) DMSO and 4-OHT treated Nrf1flox/flox;Cre-ERT2 fibroblasts by real time RTPCR analysis. Expression levels of genes were quantitated relative to endogenous beta-tubulin-5 (Tubb5) levels as an internal reference, and calculated as 2^{(Ct test gene-Ct Tubb5)}. Fold change in expression was determined from the difference between the averaged expression levels relative to control. Mean values ± SD of each genotype are shown, P-values less than 0.05 were considered statistically significant. (C) Whole cell lysates of 2 independent cultures of wild type and Nrf1^-/- MEFs were immunoblotted with anti-NDC80 antibody, and beta-Actin was used as loading control.