Analysis of the interactions of Nrf-2, PMF-1, and CSN-7 with the 5′-flanking sequence of the mouse 4E-BP1 gene

• Published in: Life sciences (1973) Vol. 79; no. 13; pp. 1221 - 1227
• Main Authors: Stephenson, A.H., Seidel, E.R.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier Inc 22-08-2006
• Subjects: 4E binding protein 1; 5' Flanking Region - genetics; Acetyltransferases - genetics; Acetyltransferases - metabolism; Animals; Arabidopsis; Biogenic Polyamines - physiology; Carrier Proteins - genetics; Carrier Proteins - metabolism; Cell Nucleus - metabolism; Cytosol - metabolism; DNA Primers; Eflornithine - pharmacology; Electrophoretic Mobility Shift Assay; Immunoblotting; Mice; NF-E2-Related Factor 2 - genetics; NF-E2-Related Factor 2 - metabolism; NIH 3T3 Cells; Nuclear Proteins - metabolism; Phosphoproteins - genetics; Phosphoproteins - metabolism; Polyamines; Putrescine; Putrescine - pharmacology; Reverse Transcriptase Polymerase Chain Reaction; Transcription Factors - genetics; Transcription Factors - metabolism; Translation; Translation; Putrescine; Polyamines; 4E binding protein 1
• ISSN: 0024-3205; 1879-0631
• DOI: 10.1016/j.lfs.2006.03.042

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.