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. 2000 May 15;28(10):2091-8.
doi: 10.1093/nar/28.10.2091.

Functional interactions between an atypical NF-kappaB site from the rat CYP2B1 promoter and the transcriptional repressor RBP-Jkappa/CBF1

S H Lee  1 , X Wang, J DeJong
Affiliations

Functional interactions between an atypical NF-kappaB site from the rat CYP2B1 promoter and the transcriptional repressor RBP-Jkappa/CBF1

S H Lee et al. Nucleic Acids Res. .

Abstract

The phenobarbital-inducible rat cytochrome P450 (CYP) 2B1 and 2B2 proteins are encoded by homologous genes whose promoters contain a mammalian-apparent long terminal repeat retrotransposon (MaLR). An NF-kappaB-like site within the MaLR forms multiple protein-DNA complexes with rat liver and HeLa cell nuclear extracts. Using antibody supershift assays, we have identified these complexes as NF-kappaB and RPB-Jkappa/CBF1. Competition assays using a series of single site mutant oligonucleotides reveal that the recognition sites for these two factors overlap. We also show that the CYP2B1/2 NF-kappaB element, but not the Igkappa NF-kappaB element, can repress transcription in vitro when positioned upstream of the heterologous adenovirus major late core promoter. In addition, RBP-Jkappa over-expressed in COS-7 cells repressed expression in vivo from an SV40-luciferase reporter construct that contained the CYP2B1/2 NF-kappaB element. Finally, we observe similar levels of NF-kappaB and RBP-Jkappa binding activities in nuclear extracts prepared from control and phenobarbital-induced rat livers. The results suggest that RBP-Jkappa/CBF1 binds an atypical NF-kappaB site in the CYP2B1/2 promoters and may help to maintain a low level of expression in the absence of inducer.

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Figures

Figure 1
Figure 1
(A) Schematic diagram of the rat CYP450 2B1 and 2B2 promoters. Core promoter binding factors, including TBP, C/EBP and Sp1, are indicated at the far right. CA refers to a repetitive CA sequence of five (2B1) or 19 (2B2) reiterations. The black arrow (–1242 to –739) pointing away from the core promoter indicates a solitary LTR from a family of mammalian apparant LTR retrotransposons (MaLR). The predicted boundaries between the U3, R and U5 regions of the LTR are shown in the expansion below, along with the position of putative NF-κB and polyadenylation (AATAAA) sites. GRE indicates the position of a glucocorticoid response element. The dark box centered around –2300 (PBRE) represents a distal enhancer that confers responsiveness to PB. Sequences further upstream consist of distinct regions of LINE-1 elements, as well as reiterated GA dinucleotide sequences. See text for references. (B) Comparison of selected sequences within the MaLR from the rat CYP2B1 and 2B2 and mouse CYP2b10 promoters. The rat CYP2B1 and 2B2 sequences are identical within the region shown and include a direct repeat (DR) of 12 nt. The putative NF-κB and RBP-Jκ sites are shaded and indicated with arrows. The MaLR element from the mouse CYP2b10 promoter lacks the DR, but contains NF-κB and RBP-Jκ sites.
Figure 2
Figure 2
(A) The NF-κB element from the rat CYP2B1/2 MaLR LTR forms several shifted complexes in rat liver nuclear extracts. Band shift reactions contain probes for NF-κB Igκ (lanes 1–3) or NF-κB 2B1/2 (lanes 4–6), as shown. One major complex (C1) and up to three minor complexes (C2–C4) were observed. Addition of unlabeled oligonucleotide competitors NF-κB Igκ (lanes 2 and 5) and NF-κB 2B1/2 (lanes 3 and 6) indicate that the C1 complex is competed by the NF-κB 2B1/2 site, but not the NF-κB Igκ site. (B) C1 complex formation is specific for the NF-κB 2B1/2 element. Reactions were performed using the NF-κB 2B1/2 probe as described in (A). Oligonucleotide competitors include none (lane 1), 2B1/2 core –43 to –13 (lane 2), NF-κB 2B1/2 (lane 3), NF-κB Igκ (lane 4), 2B1/2 DR (lane 5), C/EBP (lane 6), AP-1 (lane 7), Sp1 (lane 8), AdML TATA (lane 9), AP-1-like site A (lane 10), AP-1-like site B (lane 11) and an AP-1 mutant (lane 12). The upper C2–C4 complexes are competed by both the NF-κB 2B1/2 and NF-κB Igκ oligonucleotides (lanes 3 and 4), while competition for the C1 complex is seen only with NF-κB 2B1/2 (lane 3).
Figure 3
Figure 3
Localization and inducibility of protein–DNA complexes formed with the NF-κB 2B1/2 element. Extracts were prepared from rat liver nuclei (lanes 1–7) and cytoplasm (lanes 8–14) and tested for complex formation using the NF-κB 2B1/2 element as probe. Inducibility of complex formation was tested by the addition of DOC, formamide or NP-40, as indicated. The C1 complex (lanes 1–7) was restricted to nuclear extracts and was unaffected by treatment with dissociating reagents.
Figure 4
Figure 4
Supershift assays using antibodies against NF-κB subunits p50, p52 and p65 and against RBP-Jκ. Band shift reactions contain HeLa cell nuclear extract (N.E., lanes 1–10) and a kinase-labeled NF-κB 2B1/2 probe. Reactions were carried out in the absence (lanes 1–5) or presence (lanes 6–14) of DOC and NP-40. Lanes 11–14 contain antibody alone. The results indicate that Ab-p50 (lanes 2 and 7) and Ab-p65 (lanes 3 and 8) supershift the upper complex, while Ab-RBP-Jκ (lanes 5 and 10) supershifts the lower complex. The positions of RBP-Jκ, NF-κB and supershifted complexes are indicated to the left.
Figure 5
Figure 5
(A) Sequences of CYP2B1/2 NF-κB mutant oligonucleotides. Sequences (top strand) of NF-κB 2B1/2 (oligo 1) and NF-κB Igκ (oligo 2) oligonucleotides, and various point substitutions (oligos 3–12) are aligned. The relative ability of these oligos to compete for NF-κB and RBP-Jκ binding activity is indicated to the right (100%, +++++; 80–100%, ++++; 60–80%, +++; 40–60%, ++; 20–40%, +; <20%, –/+; no binding, –). (B) Competition for NF-κB and RBP-Jκ complex formation using mutant oligonucleotides. Oligonucleotides 1–12 were used as competitors in band shift reactions containing HeLa cell nuclear extracts and a labeled NF-κB 2B1/2 probe (lanes 1–12). Lane C shows a control experiment performed in the absence of competitor.
Figure 6
Figure 6
(A) The NF-κB element from the CYP2B1/2 promoter represses transcription from the adenovirus major late promoter in vitro. Adenovirus major late (AdML) promoter–G-free cassette in vitro transcription templates containing two NF-κB Igκ (pMLG4G-Igκ) (lanes 1 and 3) or NF-κB 2B1/2 (pMLG4G-2B1/2) (lanes 2 and 4) sites were tested for activity in in vitro transcription experiments using HeLa cell nuclear extracts. Reactions were performed in the absence (lanes 1 and 2) or presence (lanes 3 and 4) of the NF-κB 2B1/2 oligonucleotide competitor. (B) A myc-tagged RBP-Jκ construct (pCMV-RBP-Jκ) overexpresses RBP-Jκ in COS-7 cells. RBP-Jκ protein (68 kDa) was detected in extracts prepared 48 h post-transfection using anti-myc antibodies. (C) Overexpressed RBP-Jκ protein reduces expression from an SV40 promoter construct that contains the CYP2B1/2 NF-κB site. The left panel shows that a control vector (pGL3-Pro) and an IgK NF-κB site vector (pGL3-Pro-Igκ) are not affected by co-transfection and overexpression of RBP-Jκ in vivo, whereas a construct with the CYP2B1/2 NF-κB site (pGL3-Pro-CYP2B1/2) is reduced. The right panel shows that repression of pGL3-Pro-CYP2B1/2 is dependent on the amount of co-transfected pCMV-RBP-Jκ.
Figure 7
Figure 7
Formation of NF-κB, RBP-Jκ and several other protein–DNA complexes does not differ in rat hepatic extracts prepared from control (C) or PB-induced animals (I). Oligonucleotides spanning the CYP2B1/2 core (–45 to –13) (lanes 1 and 2) and the AdML TATA element (lanes 3 and 4) were used to normalize activity of control (C) and PB-induced (I) extracts with respect to TATA element binding activities. Oligos tested include NF-κB Igκ (lanes 5 and 6), NF-κB 2B1/2 (lanes 7 and 8), AP-1 (lanes 9 and 10), Sp1 (lanes 11 and 12) and C/EBP (lanes 13 and 14).
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