Complexity of CNC transcription factors as revealed by gene targeting of the Nrf3 locus

Abstract

Cap'n'collar (CNC) family basic leucine zipper transcription factors play crucial roles in the regulation of mammalian gene expression and development. To determine the in vivo function of the CNC protein Nrf3 (NF-E2-related factor 3), we generated mice deficient in this transcription factor. We performed targeted disruption of two Nrf3 exons coding for CNC homology, basic DNA-binding, and leucine zipper dimerization domains. Nrf3 null mice developed normally and revealed no obvious phenotypic differences compared to wild-type animals. Nrf3(-/-) mice were fertile, and gross anatomy as well as behavior appeared normal. The mice showed normal age progression and did not show any apparent additional phenotype during their life span. We observed no differences in various blood parameters and chemistry values. We infected wild-type and Nrf3(-/-) mice with acute lymphocytic choriomeningitis virus and found no differences in these animals with respect to their number of virus-specific CD8 and CD4 T cells as well as their B-lymphocyte response. To determine whether the mild phenotype of Nrf3 null animals is due to functional redundancy, we generated mice deficient in multiple CNC factors. Contrary to our expectations, an absence of Nrf3 does not seem to cause additional lethality in compound Nrf3(-/-)/Nrf2(-/-) and Nrf3(-/-)/p45(-/-) mice. We hypothesize that the role of Nrf3 in vivo may become apparent only after appropriate challenge to the mice.

FIG. 1.

Example of FISH mapping results for Nrf3. The left panel shows the FISH signals on the mouse chromosome, and the right panel shows the same mitotic figure stained with DAPI to identify mouse chromosome 6.

FIG. 2.

Generation of Nrf3-deficient mice. (A) Replacement of two exons of the Nrf3 gene, comprising the sequences coding for the bZIP domain, by the neomycin-cytosine deaminase cassette of the targeting vector. Nrf3 gene exons are represented by open boxes, and the exon encoding the Nrf3 bZIP domain is shown. The targeting vector contains 7.5 and 3.2 kb of Nrf3 gene homologous genomic sequences on the 5′ and 3′ sides of the neomycin-cytosine deaminase cassette, respectively. The probe used for Southern analysis and the expected fragments from EcoRI/SpeI digests of wild-type and knockout genomic DNA are indicated. tk, thymidine kinase; neo, neomycin; cda, cytosine deaminase. (B) Generation of Nrf3^−/− mice. Southern blot analysis of Nrf3^+/− mouse matings yielding offspring with homozygous wild-type (wt) and heterozygous and homozygous mutant (null) genotypes. ko, knockout.

FIG. 3.

Nrf3 transcript analysis of wild-type and gene-targeted mice. Northern blot analysis showing Nrf3 mRNA levels in various tissues in wild-type and Nrf3 null mice was done. Ten micrograms of total RNA was used per lane. The same blot was probed with actin- and GAPDH (glyceraldehyde-3-phosphate dehydrogenase gene)-specific control probes.

FIG. 4.

Response of wild-type and Nrf3 null mice to LCMV infection. Nrf3^−/− mice and control mice have comparable T- and B-cell responses to LCMV. IFN-γ production by virus-specific CD8 (A) and CD4 (B) T cells was determined on days 8 and 30 postinfection. Spleen cells from day 8 or day 30 infected mice were stimulated in vitro for 5 h with either MHC class I-restricted epitope NP396-404 (A) or MHC class II-restricted epitope GP61-80 (B). The number of LCMV-specific ASC was determined at day 8 (C) and day 30 (D) in the spleen and on day 30 in the bone marrow (E). LCMV-specific IgG, IgG2a, and IgG1 was determined by an enzyme-linked immunospot assay. The data shown represent a mean of four mice per group. Wt, wild type.

FIG. 5.

Generation of compound Nrf3^−/−/Nrf2^−/− and Nrf3^−/−/p45^−/− null mice. Southern blot analyses of Nrf3^+/−/Nrf2^+/− mice matings (A) and Nrf3^−/− and p45^+/− mice matings (B) are shown. Double knockout (ko) animals are indicated in boldface type. wt, wild type.