Liver-specific knockout of Nrf1 in the mouse leads to spontaneous development of non- alcoholic steatohepatitis with dyslipidemia, and then its deterioration results in hepatoma, but the underlying mechanism remains elusive to date. A similar pathological model is reconstructed here by using human Nrf1α-specific knockout cell lines. Our evidence has demonstrated that a marked increase of the inflammation marker COX2 definitely occurs in Nrf1α^−/− cells. Loss of Nrf1α leads to hyperactivation of Nrf2, which results from substantial decreases in Keap1, PTEN and most of 26S proteasomal subunits in Nrf1α^−/− cells. Further investigation of xenograft model mice showed that malignant growth of Nrf1α^−/−-derived tumors is almost abolished by silencing of Nrf2, while Nrf1α^+/+-tumor is markedly repressed by an inactive mutant (i.e., Nrf2^−/−ΔTA), but largely unaffected by a priori constitutive activator (i.e., caNrf2^ΔN). Mechanistic studies, combined with transcriptomic sequencing, unraveled a panoramic view of opposing and unifying inter-regulatory cross-talks between Nrf1α and Nrf2 at different layers of the endogenous regulatory networks from multiple signaling towards differential expression profiling of target genes. Collectively, Nrf1α manifests a dominant tumor-suppressive effect by confining Nrf2 oncogenicity. Though as a tumor promoter, Nrf2 can also, in turn, directly activate the transcriptional expression of Nrf1 to form a negative feedback loop. In view of such mutual inter-regulation by between Nrf1α and Nrf2, it should thus be taken severe cautions to interpret the experimental results from loss of Nrf1α, Nrf2 or both.

Normal growth and development, as well as adaptive responses to various intracellular and environmental stresses, are tightly controlled by transcriptional networks. The evolutionarily conserved genomic sequences across species highlights the architecture of such certain regulatory elements. Among them, one of the most conserved transcription factors is the basic-region leucine zipper (bZIP) family. Herein, we have performed phylogenetic analysis of these bZIP proteins and found, to our surprise, that there exist a few homologous proteins of the family members Jun, Fos, ATF2, BATF, C/EBP and CNC (cap’n’collar) in either viruses or bacteria, albeit expansion and diversification of this bZIP superfamily have occurred in vertebrates from metazoan. Interestingly, a specific group of bZIP proteins is identified, designated Nach (Nrf and CNC homology), because of their strong conservation with all the known CNC and NF-E2 p45 subunit-related factors Nrf1 and Nrf2. Further experimental evidence has also been provided, revealing that Nach1 and Nach2 from the marine bacteria exert distinctive functions, when compared with human Nrf1 and Nrf2, in the transcriptional regulation of antioxidant response element (ARE)-battery genes. Collectively, further insights into these Nach/CNC-bZIP subfamily transcription factors provide a novel better understanding of distinct biological functions of these factors expressed in distinct species from the marine bacteria to humans.

Transcription factor Nrf2 (nuclear factor-erythroid 2-related factor 2) is a master regulator of antioxidant and/or electrophile response elements (AREs/EpREs)-driven genes involved in homeostasis, detoxification, and adaptation to various stresses. The cytoprotective activity of Nrf2, though being oppositely involved in both cancer prevention and progression, is critically controlled by Keap1 (Kelch-like ECH-associated protein 1), which is an adaptor subunit of Cullin 3-based E3 ubiquitin ligase and also is a key sensor for oxidative and electrophilic stresses. Here, we first report a novel naturally-occurring mutant of Keap1, designated Keap1^ΔC, which lacks most of its C-terminal Nrf2-interacting domain essential for inhibition of the cap’n’collar (CNC) basic-region leucine zipper (bZIP) factor. This mutant Keap1^ΔC is yielded by translation from an alternatively mRNA-spliced variant lacking the fourth and fifth exons, but their coding sequences are retained in the wild-type Keap1 locus (with no genomic deletions). Although this variant was found primarily in the human highly-metastatic hepatoma (MHCC97H) cells, it was widely expressed at very lower levels in all other cell lines examined. Such Keap1^ΔC retains no or less ability to inhibit Nrf2, so that it functions as a dominant-negative competitor of Keap1 against its inhibition of Nrf2 due to its antagonist effect on Keap1-mediated turnover of Nrf2 protein.

Among multiple distinct isoforms, Nrf1D is synthesized from a de novo translation of an alternatively-spliced transcript of Nrf1 mRNA, as accompanied by a naturally-occurring deletion of its stop codon-flanking 1466 nucleotides. This molecular event leads to the generation of a reading frameshift mutation, which results in a constitutive substitution of the intact Nrf1’s C-terminal 72 amino acids (aa, covering the second half of the leucine zipper motif to C-terminal Neh3L domain) by an additional extended 80-aa stretch to generate a unique variant Nrf1D. The C-terminal extra 80-aa region of Nrf1D was herein identified to be folded into a redox-sensitive transmembrane domain, enabling it to be tightly integrated within the endoplasmic reticulum (ER) membranes. Notably, the salient feature of Nrf1D enables it to be distinguishable from prototypic Nrf1, such that Nrf1D is endowed with a lesser ability than wild-type Nrf1 to mediate target gene expression. Further evidence has also been presented revealing that both mRNA and protein levels of Nrf1D, together with other isoforms similar to those of Nrf1, were detected to varying extents in hemopoietic and somatic tissues. Surprisingly, we found the existence of Nrf1D-derived isoforms in blood plasma, implying that it is a candidate secretory transcription factor, albeit its precursor acts as an integral transmembrane-bound CNC-bZIP protein that entails dynamic topologies across membranes, before being unleashed from the ER to enter the blood.