NRF2, a member of the NFE2 family of transcription factors, is not essential for murine erythropoiesis, growth, and development

Abstract

The locus control region of the beta-globin gene is composed of four erythroid-specific hypersensitive sites. Hypersensitive site 2 has been shown to be a powerful enhancer and contains a tandem repeat sequence for the transcription factors AP1 and NFE2 (activating protein 1 and nuclear factor erythroid 2, respectively). The human NRF2 (NFE2 related factor 2) has been isolated by bacterial expression screening using this core sequence as a probe. p45-NFE2, NRF1, and NRF2 belong to the CNC ("cap 'n' collar") subfamily of the basic region-leucine zipper transcription factors, which exhibits strong homology at specific regions such as the "CNC" and the DNA binding and leucine zipper domains. Although the erythroid-specific p45-NFE2 has been implicated in globin gene regulation, p45-NFE2 null mice succumb to bleedings due to lack of platelets and those that survive exhibit only a mild anemia. To determine the function of NRF2, which we found to be widely expressed in vivo, we have characterized the genomic structure of the mouse NRF2 gene, disrupted the Nrf2 gene by homologous recombination in mouse embryonic stem cells (ES cells), and generated NRF2-/- mice. Homozygous mutant mice developed normally, were not anemic, reached adulthood, and reproduced. Our studies indicate that NRF2 is dispensable for mouse development.

Figure 1

Sequence of NRF2. (A) Comparison of the putative translation start site of NRF2. The translation of the murine cDNA at the exon 1 and exon 2 junction is compared with the translation of the human NRF2 sequence T39964T39964 found in the EST data base. The extra amino acids are italicized, the methionine described in previous publications is in boldface type, and the G nucleotide underlined is missing in entry U20532 (GenBank) (31). (B) Alignment of the 5′ ends of the cDNAs (Nrf2cDNA and U20532) and genomic (Nrf2GEN) sequences of the first exon of mNrf2. The numbers above the cDNA represent the number of RACE clones that start at that base. ∗, Putative translation start sites derived from our sequence; •, the published translation start site (31). The sequence of intron 1 is between parentheses with gt representing the splice donor junction; sequence differences are underlined; the missing nucleotide G in U20532 is in boldface italicized type in our cDNA and genomic sequences.

Figure 2

Expression of the mNRF2. Adult mouse multiple tissue RNA blots (Left) and mouse embryos RNA blots (Right) hybridized with mNRF2 and β-actin.

Figure 3

Localization of NRF2 mRNA in the mouse embryo. In situ hybridization was performed on neighboring sections using antisense (Left) and sense (Right) probe. Two saggital sections are shown under darkfield illumination. Li, liver; Lu, lung; K, kidney; I, intestines; S, stomach; Oe, esophagus entrance (nasopharynx); C, choroid plexus; Sm, submandibular gland; O, Olfactory epithelium; T, testis; Bf, brown fat; > points to the thyroid.

Figure 4

Localization of Nrf2 mRNA in the day 16 embryo developing stomach (A), intestine (B), kidney (C), and lung (D) under darkfield illumination. m, Muscle; l, lumen; k, kidney; a, adrenal gland; b, bronchus; v, blood vessel. (Bar = 100 μm.)

Figure 5

(A) Genomic sequence and targeting constructs with the resultant NRF2 knockout gene. (B) Southern blot analysis of ES cells. An external probe XbaI–HindIII fragment gave a 15.5-kb band for the wild-type and 16.5-kb band for the homologous recombinant. (C) Southern blot analysis of F₂ offspring. A StuI–EcoRV probe gave a 2.4-kb band for the wild type and a 1.5-kb band for the recombinant.

Figure 6

Northern blot analysis of RNA from wild-type, NRF2^+/−, and NRF2^−/− mice. The NRF2 message is 2.5 kb, and the NRF2-LacZ chimeric message is 4 kb.