Analysis of the interactions of Nrf-2, PMF-1, and CSN-7 with the 5'-flanking sequence of the mouse 4E-BP1 gene.
Nuclear factor erythroid 2-related factor 2 (Nrf-2) binds to a specific polyamine responsive element (PRE) in the promoter region of the spermidine-spermine acetyltransferase (SSAT) gene, a key component of the polyamine catabolic pathway. Regulation of SSAT gene transcription requires the additional interaction of Nrf-2 with polyamine modulated factor 1 (PMF-1). Likewise, transcription of the eukaryotic initiation factor 4E binding protein 1 (4E-BP1) gene is regulated in a polyamine-dependent manner, but the actual mechanism has not previously been determined. Analysis of the 5'-flanking sequence of the murine 4E-BP1 gene indicated the presence of several potential PRE sites, which might be involved in regulating its transcription. Our goal in this research was to determine potential interactions between Nrf-2, PMF-1, the human homologue of the Arabidopsis signalosome complex (CSN-7), and these potential PRE sites. Four PCR fragments containing regions with considerable homology (78%) to the human PRE were generated from the 5'-flanking sequence of the mouse 4E-BP1 gene and the fragments were used in electrophoretic gel mobility shift and supershift assays. Purified Nrf-2 interacted with all four of these fragments, and similar gel shifts were observed with both cytoplasmic and nuclear fractions of NIH-3T3 cells. However, polyamine depletion with difluoromethylornithine (DFMO) eliminated the gel shift. Supershift assays indicated that the shift was due to the binding of Nrf-2, and the binding was competitive with a known Nrf-2 binding sequence. Purified PMF-1 did not bind any of the PCR fragments alone, but when added with Nrf-2, decreased the magnitude of the gel shift for one of the fragments (PRE located at -2060 relative to the transcription start site). CSN-7 did not interact with the sequences, nor did it inhibit protein/DNA interaction. These data indicate a possible mechanism by which polyamines enhance the binding of a Nrf-2/PMF-1 complex to the 5'-flanking region of the 4E-BP1 gene. Since polyamines increase expression of the 4E-BP1 gene, it seems likely that formation of this complex is involved in its transcriptional regulation.
Stephenson
A H
AH
Department of Physiology, Brody School of Medicine, East Carolina University, Greenville, NC 27858, USA.
Seidel
E R
ER
D024506:5' Flanking Region / Q000235:genetics*; D000123:Acetyltransferases / Q000235:genetics / Q000378:metabolism; D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D015317:Biogenic Polyamines / Q000502:physiology; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D018797:Cell Cycle Proteins; D002467:Cell Nucleus / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D017931:DNA Primers; D000518:Eflornithine / Q000494:pharmacology; D024202:Electrophoretic Mobility Shift Assay; D039642:Eukaryotic Initiation Factors; D015151:Immunoblotting; D051379:Mice; D051267:NF-E2-Related Factor 2 / Q000235:genetics* / Q000378:metabolism; D041681:NIH 3T3 Cells; D009687:Nuclear Proteins / Q000378:metabolism; D010750:Phosphoproteins / Q000235:genetics* / Q000378:metabolism; D011700:Putrescine / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D000099228:Diamine N-Acetyltransferase
D000123:Acetyltransferases; D048868:Adaptor Proteins, Signal Transducing; D015317:Biogenic Polyamines; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D017931:DNA Primers; D000518:Eflornithine; D039642:Eukaryotic Initiation Factors; D051267:NF-E2-Related Factor 2; D009687:Nuclear Proteins; D010750:Phosphoproteins; D011700:Putrescine; D014157:Transcription Factors; D000099228:Diamine N-Acetyltransferase; C513682:CSN 7 protein, mouse; C499527:Eif4ebp1 protein, mouse; C495636:Nfe2l2 protein, mouse; C486540:Pmf1 protein, mouse