Abstract
In the rat liver, NF1 and CP1 bind to the major P2 promoter of the alpha1B adrenergic receptor gene to generate footprint II. Here we show that, in DDT1MF-2 smooth muscle cells, the major protein bound to footprint II is not NF1 but Sp1, which binds to the 5'-portion of the footprint II sequence (footprint IIb). Mutational analyses demonstrate that the CCCGCG sequence in footprint IIb is critical for Sp1 binding and P2 promoter activity. A second GC box in the P2 promoter also binds the Sp1 protein and contributes to the P2 promoter activity. Gel shift assays indicate that footprint II can bind Sp1, NF1, and CP1, and that the binding of these 3 proteins is mutually exclusive. This is also indicated by the results of functional cotransfection experiments, where transient overexpression of NF1 and Sp1 together caused a similar increase in the activity of a P2/CAT reporter construct as overexpression of either Sp1 or NF1 alone, indicating lack of additivity. The preferential interaction of footprint II with Sp1 in DDT1MF-2 cells and NF1 in liver appears to be due to low levels of NF1 expression in DDT1MF-2 cells and low levels of Sp1 in liver. These observations suggest that NF1 and Sp1 are the major transcription factors involved in controlling the P2 promoter in liver versus DDT1MF-2 cells, respectively, which may be one of the mechanisms responsible for the complex tissue-specific regulation of the expression of the alpha1B adrenergic receptor gene.
Highlights
In the rat liver, NF1 and CP1 bind to the major P2 promoter of the ␣1B adrenergic receptor gene to generate footprint II
The Protein Bound to Footprint II in DDT1MF-2 Cells Is Sp1—Earlier experiments showed that NF1 is the major protein bound to the footprint II sequence in liver, and DDT1MF-2 cells are known for expressing high levels of ␣1BAR protein [11], ␣1BAR mRNA [11], and P2 promoter activity [6]
DNA mobility shift assay (DMSA) showed that this complex was competed away by an Sp1 consensus oligo but not by consensus oligos for AP1, AP2, or C/EBP (Fig. 1B), suggesting that the major protein bound to oligo II in DDT1MF-2 cells was Sp1 or a related protein
Summary
NF1 and CP1 bind to the major P2 promoter of the ␣1B adrenergic receptor gene to generate footprint II. The preferential interaction of footprint II with Sp1 in DDT1MF-2 cells and NF1 in liver appears to be due to low levels of NF1 expression in DDT1MF-2 cells and low levels of Sp1 in liver These observations suggest that NF1 and Sp1 are the major transcription factors involved in controlling the P2 promoter in liver versus DDT1MF-2 cells, respectively, which may be one of the mechanisms responsible for the complex tissue-specific regulation of the expression of the ␣1B adrenergic receptor gene. Since the DDT1MF-2 hamster smooth muscle cell line expresses a similar pattern of three ␣1BAR mRNAs as rat liver [11] and a high level of P2 promoter activity, we wondered whether NF1 has a similar role in transcriptional control in these cells. DNA mobility shift assay (DMSA) using 32Plabeled consensus Sp1 or NF1 oligodeoxyribonucleotides (oligos) showed that rat liver contained high levels of NF1 but low levels of Sp1 binding, whereas the reverse was true in the case of DDT1MF-2 cells
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