title: MCRS2 represses the transactivation activities of Nrf1.
abstract: BACKGROUND
  Nrf1 [p45 nuclear factor-erythroid 2 (p45 NF-E2)-related factor 1], a member of the CNC-bZIP (CNC basic region leucine zipper) family, is known to be a transcriptional activator by dimerization with distinct partners, such as Maf, FosB, c-Jun, JunD, etc. The transcriptional roles of CNC-bZIP family are demonstrated to be involved in globin gene expression as well as the antioxidant response. For example, CNC-bZIP factors can regulate the expression of detoxification proteins through AREs, such as expression of human gamma-glutamylcysteine synthetases (GCS), glutathione S-transferases (GST), UDP-glucuronosyl transferase (UDP-GT), NADP (H) quinone oxidoreductase (NQOs), etc. To further explore other factor(s) in cells related to the function of Nrf1, we performed a yeast two-hybrid screening assay to identify any Nrf1-interacting proteins. In this study, we isolated a cDNA encoding residues 126-475 of MCRS2 from the HeLa cell cDNA library. Some functions of MCRS1 and its splice variant-MSP58 and MCRS2 have been previously identified, such as transforming, nucleolar sequestration, ribosomal gene regulation, telomerase inhibition activities, etc. Here, we demonstrated MCRS2 can function as a repressor on the Nrf1-mediated transactivation using both in vitro and in vivo systems.
  
  
  RESULTS
  To find other proteins interacting with the CNC bZIP domain of Nrf1, the CNC-bZIP region of Nrf1 was used as a bait in a yeast two-hybrid screening assay. MCRS2, a splicing variant of p78/MCRS1, was isolated as the Nrf1-interacting partner from the screenings. The interaction between Nrf1 and MCRS2 was confirmed in vitro by GST pull-down assays and in vivo by co-immunoprecipitation. Further, the Nrf1-MCRS2 interaction domains were mapped to the residues 354-447 of Nrf1 as well as the residues 314-475 of MCRS2 respectively, by yeast two-hybrid and GST pull-down assays. By immunofluorescence, MCRS2-FLAG was shown to colocalize with HA-Nrf1 in the nucleus and didn't result in the redistribution of Nrf1. This suggested the existence of Nrf1-MCRS2 complex in vivo. To further confirm the biological function, a reporter driven by CNC-bZIP protein binding sites was also shown to be repressed by MCRS2 in a transient transfection assay. An artificial reporter gene activated by LexA-Nrf1 was also specifically repressed by MCRS2.
  
  
  CONCLUSIONS
  From the results, we showed MCRS2, a new Nrf1-interacting protein, has a repression effect on Nrf1-mediated transcriptional activation. This was the first ever identified repressor protein related to Nrf1 transactivation.
authors.affiliation: IBMS, Academia Sinica, Taipei 11529, Taiwan, ROC. chaperon123@gmail.com
authors.forename: Jia-Long
authors.initials: JL
authors.lastname: Wu
authors.forename: Young-Sun
authors.initials: YS
authors.lastname: Lin
authors.forename: Chi-Chiang
authors.initials: CC
authors.lastname: Yang
authors.forename: Yu-Jen
authors.initials: YJ
authors.lastname: Lin
authors.forename: Shan-Fu
authors.initials: SF
authors.lastname: Wu
authors.forename: Ying-Ting
authors.initials: YT
authors.lastname: Lin
authors.forename: Chien-Fu
authors.initials: CF
authors.lastname: Huang
mesh_terms: D002478:Cells, Cultured; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D017930:Genes, Reporter; D006367:HeLa Cells; D006801:Humans; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D051266:Nuclear Respiratory Factor 1 / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation; D014162:Transfection
chemical_list: D018076:DNA, Complementary; C113292:MCRS1 protein, human; D009687:Nuclear Proteins; D051266:Nuclear Respiratory Factor 1; D016601:RNA-Binding Proteins; D012097:Repressor Proteins; D015534:Trans-Activators