Peroxiredoxin 1 safeguards the nucleolar genome from oxidative damage

Abstract

Peroxiredoxin 1 (PRDX1) is a highly conserved, thiol-dependent peroxidase that rapidly scavenges reactive oxygen species to modulate redox signaling. PRDX1-null mice exhibited genomic instability, shortened lifespan, and accelerated tumorigenesis, including development of lymphomas, sarcomas, and carcinomas. Despite extensive characterization of these phenotypes, the molecular mechanism by which PRDX1 loss causes genomic instability remains poorly understood. Here we show that PRDX1 deficiency alters nucleolar morphology, impairs RNA Polymerase I (POL-I)-dependent transcription of pre-ribosomal RNAs and triggers nucleolar genomic instability. This oxidative stress-induced nucleolar dysfunction promotes the stability of secondary DNA structures, such as RNA-DNA hybrids and G-quadruplex DNA, contributing to nucleolar genomic instability. We demonstrate that PRDX1 loss reduces nascent rRNA levels and impairs rRNA processing, further affecting ribosome biogenesis. Mechanistically, we established that PRDX1 loss triggers activation of the nucleolar DNA damage response including activation of DNA repair kinase ATM and the nucleolar factor TCOF1 within the nucleolus, and recruitment of the MRE11-RAD50-NBS1 (MRN) complex subunit NBS1 to ribosomal DNA (rDNA) loci. NBS1 accumulation correlates with the repression of rDNA transcription by POL-I, potentially delaying rRNA synthesis, and safeguarding the nucleolar genome from further oxidative damage. Collectively, these findings uncover a previously unrecognized, but critical role, for PRDX1 in maintaining nucleolar integrity and ribosomal biogenesis through redox-dependent regulation of rDNA transcription and processing machinery.

Figure 1:. Loss of PRDX1 induces nucleolar abnormalities

(A) Western blot analysis of human non–small cell lung cancer H1299 cells transfected with either an empty vector (EV) or a wild-type PRDX1-Flag expression vector (PRDX1-Flag). Cells were transfected 24 hours prior to lysate collection for cellular fractionation. NCL, DDX21, and FBL serve as nucleolar protein markers, while GAPDH indicates enrichment of cytosolic and nucleoplasmic fractions. (B) Western blot analysis of human non–small cell lung cancer A549 parental cells and PRDX1 KO cells. Tubulin was used as loading control. (C) Confocal microscopy images of A549 parental cells and PRDX1 knockout (KO) cells. Oxidative stress was induced by treatment with 200 μM H₂O₂ for 30 minutes. Nuclei and nucleoli were visualized using DAPI (blue) and an anti-Nucleolin (NCL) antibody (red) respectively. Lower magnification images of each condition and a selected nucleus at higher magnification are shown (a-d). (D) Comparison of nucleolar shapes. In each condition, nucleoli were classified as canonical nucleoli (irregular shape), round-shaped, or cap-like shaped, and percentages were compared. Statistical analysis was assessed by two-way ANOVA. *P < 0.05, **P < 0.01, **P < 0.0001, ns, not significant. (E) Representative images showing the three distinct nucleolar morphologies observed in parental and PRDX1 KO cells.

Figure 2:. Loss of PRDX1 induces both nucleoplasmic and nucleolar genomic instability

(A) Images of A549 cells costained with S9.6 for anti–R-loops detection (green), anti-Nucleolin (red), and DAPI (blue) and analyzed by immunofluorescence as described in Methods. A representative image of independent experiments is shown. At least 100 cells were scored for each condition. (B) Quantification of images shown in (A). Statistical significance was assessed by two-way ANOVA. **P < 0.01, ***P < 0.001. (C) Cells costained with anti-G4 (red), and DAPI (blue) and revealed by immunofluorescence. A representative image of independent experiments is shown. At least 100 cells were scored for each condition. (D) Quantification of images shown in (C). Statistical significance was assessed by two-way ANOVA. ****P < 0.0001, ns, not significant. (E) Cells were costained with anti-FANCD2 (green), and DAPI (blue), and revealed by immunofluorescence. A representative image of independent experiments is shown. At least 100 cells were scored for each condition. (F) Quantification of images shown in (E). Statistical significance was assessed by two-way ANOVA. ****P < 0.0001. (G) Cells costained with anti-gamma-H2AX (green), anti-Fibrillarin (red), and DAPI (blue) and analyzed using immunofluorescence. A representative image of independent experiments is shown. At least 100 cells were scored for each condition. (H) Quantification of images shown in (G). Statistical significance was assessed by two-way ANOVA. *P < 0.05, **P < 0.01, ns, not significant. Note. For each set of staining (A-F), lower magnification images of each condition and a selected nucleus at higher magnification are shown (a-d).

Figure 3:. Loss of PRDX1 alters chromatin structure of rDNA and ribosome biogenesis

(A) Schematic diagram of the human ribosomal DNA (rDNA) unit and primers used for qPCR. Primer sets include H0 and H42.9 for the promoter region, P1 for 18S ribosomal RNA (rRNA), P2 for 28S rRNA, and P3/P4 for the intergenic spacer (IGS) region. Primers sequence can be found in Materials and Methods (B and C) Chromatin immunoprecipitation (ChIP) assay showing the enrichment of rDNA in RNA Polymerase I (POL-I) (B), and Histone H3 trimethyl-Lysine 4 (H3K4me3) (C) immunoprecipitates. Statistical analysis was performed using two-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, not significant. (D-F) Representative fluorescence microscopy images showing EU (green) incorporation in A549 parental and PRDX1 KO cells. DAPI was used to counterstain nuclei (D). (E) Statistical analysis of EU intensity was performed using two-way ANOVA (****P < 0.0001). (F) Representative images showing EU (green) and Nucleolin (red) staining in A549 parental and PRDX1 KO cells. Nucleolin serves as a nucleolar marker, and DAPI (blue) was used for nuclear counterstaining. The overlap between EU and Nucleolin signals indicates sites of rRNA synthesis. (G) Polysome profiling in A549 parental and PRDX1 KO cells.

Figure 4:. Loss of PRDX1 induces nucleolar DNA damage response via MRN recruitment and NBS1 enrichment at the rDNA loci.

(A, B) Western blot analysis of human non–small cell lung cancer A549 parental cells and PRDX1 KO cells (A) and human non–small cell lung cancer H1299 control and cells infected with short hairpin RNA interference targeting PRDX1 (B), following cell fractionation. Fibrillarin (FBL), and DDX21 serve as nucleolar protein markers, while GAPDH indicates enrichment of cytosolic and nucleoplasmic fractions. Note: P, parental, KO, PRDX1 KO, C, control shRNA, KD, shPRDX1. WCE, whole cell extract; Cyto, cytoplasmic fraction; N-Plasm, nucleoplasmic fraction; N-olar, nucleolar fraction. (C) Representative confocal microscopy images of A549 parental and PRDX1 KO cells stained with antibodies against TCOF1 (green) and Nucleolin (NCL) (red) with or without H₂O₂ treatment (200μM, 30min). Lower magnification images of each condition and a selected nucleus at higher magnification are shown (a-d). (D) Quantification of TCOF1 signal intensity per nucleus. (E) ChIP assay of A549 parental and PRDX1 KO cells using an anti-NBS1 antibody. The primer sets H0 and H42.9 are located in the promoter region, and P4 is in the intergenic spacer region (see Figure 3A). Statistical analysis was performed using two-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns; not significant.

Figure 5:. PRDX1 ectopic expression reverses the nucleolar DDR and restores POL-I activity.

(A) Comparison of ROS levels in A549 parental, PRDX1-KO, PRDX1-KO Rescue (PRDX1-KO complemented with wild type PRDX1), and Mutant (PRDX1-KO complemented with CPRS mutated PRDX1) cells (see Figure S1A) following H₂O₂ treatment (200 μM for 30 min). Oxidative stress was detected by CellRox Deep Red and monitored by flow cytometry. The response to H₂O₂ treatment was compared to the baseline where endogenous reactive oxygen species (ROS) were detected in non-treated (NT) parental cells. Loss of PRDX1 induces more ROS when treated with H₂O₂ (PRDX1 KO-H₂O₂), while Rescue-H₂O₂ cells resist the induction of ROS levels as parental cells. Mutant cells phenocopied PRDX1 KO cells. (B) Western blot analysis of human non–small cell lung cancer A549 parental, PRDX1 KO, and PRDX1 rescue cells treated with H₂O₂ (200 μM) for 30 min, followed by cell fractionation. Fribrillarin (FBL) serves as nucleolar protein marker, while GAPDH indicates enrichment of cytosolic and nucleoplasmic fractions. Note: Par, parental cells, KO, PRDX1 KO cells, and Res, PRDX1 wild type-rescued cells. (C) ChIP assay showing the enrichment of rDNA in NBS1 immunoprecipitates with or without H2O2 treatment (200 μM, 30 min). The primer sets H0 and H42.9 are located in the promoter region, P1 is in the coding region, and P4 is in the intergenic spacer region (see Figure 3A). Note that NBS1 binding was reduced in Rescue cells (PRDX1-KO complemented with wild type PRDX1) similar to parental cells. (D) ChIP assay showing the enrichment of rDNA in RNA Polymerase I (POL-I) immunoprecipitates with or without H₂O₂ treatment (200 μM, 30 min). Primer sets P1 and P2 are located in 18S and 28S ribosomal RNA, respectively, and P3/P4 are in the intergenic spacer region (see Figure 3A). Note that POL-I binding in Rescue cells returned to levels observed in parental cells. Statistical analysis was performed using two-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns; not significant. (E) In PRDX1-proficient cells (green), reactive oxygen species (ROS) are efficiently scavenged to prevent aberrant Pol I-dependent rDNA transcription, thereby maintaining nucleolar homeostasis. Loss of PRDX1 (pink) resulted in accrued ROS level potentially triggering activation of the DNA damage response kinase ATM through autophosphorylation and activation of nucleolar DNA damage response via recruitment and enrichment of the MRN complex, and TCOF1 at rDNA loci, particularly at the pre-rRNA promoter region. Accumulation of NBS1 restricts POL-I-dependent transcription at rDNA coding regions, including 18S, 5.8S, and 28S, potentially delaying rRNA synthesis and protecting the nucleolar genome from further oxidative damage. Elevated DNA breaks caused by ROS accumulation following PRDX1 loss may trigger a comparable response.