Neil2-null Mice Accumulate Oxidized DNA Bases in the Transcriptionally Active Sequences of the Genome and Are Susceptible to Innate Inflammation

Abstract

Why mammalian cells possess multiple DNA glycosylases (DGs) with overlapping substrate ranges for repairing oxidatively damaged bases via the base excision repair (BER) pathway is a long-standing question. To determine the biological role of these DGs, null animal models have been generated. Here, we report the generation and characterization of mice lacking Neil2 (Nei-like 2). As in mice deficient in each of the other four oxidized base-specific DGs (OGG1, NTH1, NEIL1, and NEIL3), Neil2-null mice show no overt phenotype. However, middle-aged to old Neil2-null mice show the accumulation of oxidative genomic damage, mostly in the transcribed regions. Immuno-pulldown analysis from wild-type (WT) mouse tissue showed the association of NEIL2 with RNA polymerase II, along with Cockayne syndrome group B protein, TFIIH, and other BER proteins. Chromatin immunoprecipitation analysis from mouse tissue showed co-occupancy of NEIL2 and RNA polymerase II only on the transcribed genes, consistent with our earlier in vitro findings on NEIL2's role in transcription-coupled BER. This study provides the first in vivo evidence of genomic region-specific repair in mammals. Furthermore, telomere loss and genomic instability were observed at a higher frequency in embryonic fibroblasts from Neil2-null mice than from the WT. Moreover, Neil2-null mice are much more responsive to inflammatory agents than WT mice. Taken together, our results underscore the importance of NEIL2 in protecting mammals from the development of various pathologies that are linked to genomic instability and/or inflammation. NEIL2 is thus likely to play an important role in long term genomic maintenance, particularly in long-lived mammals such as humans.

Keywords: DNA damage; DNA damage and repair; DNA enzyme; DNA glycosylase; NEIL2; gene knockout; inflammation; knock-out animals; reactive oxygen species; transcription-coupled repair.

FIGURE 1.

Neil2 gene targeting. A, Neil2 wild-type allele (WT), knock-out (KO) vector, conditional allele (floxed), and knock-out/null allele (KO) are shown. The red bar below the WT allele represents the 5′-flanking probe for a diagnostic Southern blot. EcoRI sites relevant to the Southern blot are shown. The arrowheads indicate PCR primers for genotyping. The size of each PCR amplicon is shown next to the reverse primer. Cre loxP, Cre-loxP recombination; neo, neomycin-resistance gene; HSV-tk, herpes simplex virus-thymidine kinase gene; HR, homologous recombination. B, genotyping of the mice. The upper panels show Southern blot data. The DNA was digested with EcoRI and separated in 1% agarose/TAE gels. The filters were hybridized with the 5′-flanking probe. The WT band (4.3 kb), floxed allele band (5.9 kb), and KO allele band (4.1 kb) are shown. The extra bands (highlighted by asterisks) likely occurred due to incomplete digestion of genomic DNA. The lower panels show PCR data. The mutant line with the floxed allele was genotyped by primers shown as blue arrowheads in A; the line with the KO allele was genotyped by primers shown as green arrowheads in A. C, expression of Neil2 transcript. Mock (−RT) and RT-PCR amplification profile of a segment of Neil2 (exon 4) mRNA from kidney tissue in WT, Neil2 heterozygous (KO^/+), and Neil2-null mice (KO/KO) at 24 months. RT +/− indicates samples with or without treatment with RT. Mouse Gapdh was used as a loading control to confirm the integrity and equal loading of RNA in each lane. D, expression profile of NEIL2 protein as shown by Western blotting, using rabbit polyclonal anti-NEIL2 antibody. WT, KO^/+, and KO/KO represent protein samples from kidney tissue of WT, Neil2 heterozygous, and Neil2-null mice, respectively. Purified NEIL2 (25 ng) was used as a positive control. GAPDH was used as a loading control to confirm equal loading of protein from each sample.

FIGURE 2.

RT-PCR profiling of various mouse genes. RT-PCR-mediated expression profiling of pol β, β-globin, NeuroD, and NanoG in various tissues (kidney, lung, liver, and whole brain, as indicated) of wild-type mice. RT + and − represent samples with and without treatment with RT. Amplification of constitutively expressing mouse Hprt was used as control. N/A means not applicable, as the assay was not carried out for the respective genes from the corresponding tissues.

FIGURE 3.

Age-dependent accumulation of oxidized DNA bases in the transcribed versus nontranscribed genome of Neil2-null mice. A, LA-qPCR was used to evaluate oxidized base-specific genomic DNA damage levels in kidney tissue of WT versus Neil2-null mice of different age groups (2, 8, and 24 months old). Representative gels showing PCR-amplified fragments encompassing mouse pol β/β-globin and NeuroD/NanoG as transcribed and nontranscribed gene pairs, respectively. Amplification of each large fragment (upper panels) was normalized to that of a small fragment of the corresponding gene (lower panels), and the data were expressed as lesion frequency/10 kb of DNA as described under “Experimental Procedures.” Histograms represent the DNA damage quantitation for WT versus Neil2-null mice in each case (n = 3, **, p < 0.01). Error bars indicate standard error of the mean. B, LA-qPCR for DNA damage analysis for two additional tissue samples (lung and liver) from WT and Neil2-null mice of the 24-month age group (n = 3, **, p < 0.01). C, analysis of the accumulation of oxidized DNA bases in whole brain tissue samples from WT and Neil2-null mice with NeuroD as the transcribing gene and NanoG as the nontranscribed gene (n = 3, **, p < 0.01).

FIGURE 4.

Co-IP analysis. Nuclear extracts (1 mg, benzonase and/or EtBr-treated) prepared from fresh liver tissue of WT or Neil2-null mice were immunoprecipitated with anti-NEIL2 (A) or Lig IIIα (B) or RNAP II (C) and tested for the presence of associated proteins with specific Abs.

FIGURE 5.

Quantitative ChIP assays. A, ChIP/Re-ChIP analysis (first IP with RNAP II Ab and the second IP with NEIL2 Ab or control IgG) of liver tissue of WT mice, showing co-occupancy of RNAP II and NEIL2 on the transcribed genome. B, Q-ChIP analysis of liver tissue of WT versus Neil2 KO mice showing differential association of RNAP II with the transcribed genome. C, Q-ChIP analysis involving NEIL2 and liver tissue of WT mice showing preferential association of NEIL2 with the transcribed genes. Mouse Hprt/pol β and NanoG/NeuroD were used as transcribed and nontranscribed gene pairs, respectively. For Q-ChIP analysis, genes of interest were amplified from immunoprecipitated DNA by SYBR Green-based qPCR. Each sample was assayed in triplicate, and the amount of immunoprecipitated DNA was calculated as the percentage of input sample. (*, p < 0.05; **, p < 0.01). Other details are provided under “Experimental Procedures.”

FIGURE 6.

Detection of chromosomal abnormalities. A, Giemsa-stained metaphase spread of Neil2^+/+ (panels a and b) and Neil2^−/− (panels c and d) mouse embryonic fibroblast cells. Note radials and chromosome gaps as indicated by arrows. B, segments of metaphases from Neil2^+/+ (panels a and b) and Neil2^−/− (panel c and d) cells showing telomere FISH signals. Metaphases were analyzed by FISH with a telomere-specific probe. Note the higher frequency of telomere signal loss in Neil2^−/− cells, as indicated by arrows.

FIGURE 7.

Increased susceptibility of Neil2 KO mice to inflammation. Wild-type and Neil2-null mice (8 months old) were challenged intranasally with LPS (100 ng/lung) or TNF-α (20 ng/lung) or GOx (1 milliunit/lung). A, 16 h after challenge, bronchoalveolar lavage fluid was derived, and inflammatory cells were counted and expressed as the number of cells/ml. B, representative microscopic images of cells visible in saline-challenged (left panels) and LPS-challenged mouse lung tissue (right panels). Magnification: upper panels, ×32; lower panels, ×134 (representing blue-boxed portions of the upper panel).