The C-Terminal Domain of Nrf1 Negatively Regulates the Full-Length CNC-bZIP Factor and Its Shorter Isoform LCR-F1/Nrf1β; Both Are Also Inhibited by the Small Dominant-Negative Nrf1γ/δ Isoforms that Down-Regulate ARE-Battery Gene Expression

The C-terminal domain (CTD, aa 686-741) of nuclear factor-erythroid 2 p45-related factor 1 (Nrf1) shares 53% amino acid sequence identity with the equivalent Neh3 domain of Nrf2, a homologous transcription factor. The Neh3 positively regulates Nrf2, but whether the Neh3-like (Neh3L) CTD of Nrf1 has a similar role in regulating Nrf1-target gene expression is unknown. Herein, we report that CTD negatively regulates the full-length Nrf1 (i.e. 120-kDa glycoprotein and 95-kDa deglycoprotein) and its shorter isoform LCR-F1/Nrf1 beta (55-kDa). Attachment of its CTD-adjoining 112-aa to the C-terminus of Nrf2 yields the chimaeric Nrf2-C112(Nrf1) factor with a markedly decreased activity. Live-cell imaging of GFP-CTD reveals that the extra-nuclear portion of the fusion protein is allowed to associate with the endoplasmic reticulum (ER) membrane through the amphipathic Neh3L region of Nrf1 and its basic c-tail. Thus removal of either the entire CTD or the essential Neh3L portion within CTD from Nrf1, LCR-F1/Nrf1 beta and Nrf2-C112(Nrf1), results in an increase in their transcriptional ability to regulate antioxidant response element (ARE)-driven reporter genes. Further examinations unravel that two smaller isoforms, 36-kDa Nrf1 gamma and 25-kDa Nrf1 delta, act as dominant-negative inhibitors to compete against Nrf1, LCR-F1/Nrf1 beta and Nrf2. Relative to Nrf1, LCR-F1/Nrf1 beta is a weak activator, that is positively regulated by its Asn/Ser/Thr-rich (NST) domain and acidic domain 2 (AD2). Like AD1 of Nrf1, both AD2 and NST domain of LCR-F1/Nrf1 beta fused within two different chimaeric contexts to yield Gal4D:Nrf1 beta(607) and Nrf1 beta:C270(Nrf2), positively regulate their transactivation activity of cognate Gal4- and Nrf2-target reporter genes. More importantly, differential expression of endogenous ARE-battery genes is attributable to up-regulation by Nrf1 and LCR-F1/Nrf1 beta and down-regulation by Nrf1 gamma and Nrf1 delta.