Regulation of Rat Ornithine Decarboxylase Promoter Activity by Binding of Transcription Factor Sp1

Abstract

Ornithine decarboxylase (ODC) is the rate-limiting enzyme of polyamine biosynthesis. We investigated the transcriptional regulation of the rat ODC gene using transient expression assays. The 5'-flanking region (-1156 to +13) of the ODC gene was sufficient to mediate strong basal expression of a luciferase reporter. Sequences between -345 and -93 contributed to basal promoter activity. This region, containing five potential Sp1 binding sites, was analyzed by electrophoretic mobility shift assays. Three specific DNA-protein complexes were identified using H35 nuclear extracts and the -345/-93 ODC probe. Binding to all three was eliminated by competition with an oligonucleotide containing an Sp1 binding site, but not by a mutant Sp1 oligonucleotide. Preincubation with an antibody against Sp1 supershifted complexes associated with one or more of Sp1 binding sites 1-4 as well as with site 5. DNase I footprinting revealed two protected regions: PR-I (-92 to -130) and PR-II (-304 to -332). PR-I contains a putative binding site for Sp1 that was protected by recombinant Sp1 protein. Transfection studies in Schneider SL2 cells demonstrated that the ODC promoter is transactivated up to 350-fold by Sp1 and that this transactivation is dependent on the presence of Sp1 binding sites 1-4. Thus, although the ODC promoter binds multiple nuclear proteins, Sp1 or a related protein appears to be a critical determinant of ODC transcription, possibly through cooperative interactions between Sp1 and additional transcription factors.

The human hepatitis B virus transactivator X gene product regulates Sp1 mediated transcription of an insulin-like growth factor II promoter 4

“…Sp1 is a ubiquitous mammalian transcription factor belonging to a member of the C 2 -H 2 zinc-®nger family, thought to be involved in the regulation of a large number of genes (Kadonaga and Tjian, 1986), including genes involved in proliferative responses (Lu et al, 1994), extracellular matrix protein genes (Tamaki et al, 1995), house-keeping genes (Kumar et al, 1995) and a number of growth factor genes (Chen et al, 1992). Sp1 has also been implicated in the function of retinoblastoma gene (Rb) product which is known to regulate cell cycle progression and repress cell growth and dierentiations (Hagen et al, 1992;Horowitz et al, 1990), suggesting that Sp1 may be involved in modulating transcription in response to the growth state of the cell.…”

Acceleration of Thrombomodulin Gene Transcription by Retinoic Acid

“…Probes were labeled with [␣-32 P]dCTP by using the random primer extension labeling system. Nuclear run-on study was performed using [␣- 32 P]dUTP-labeled RNA of nuclear fraction of cells treated or not treated with 10 M t-RA for 6 h. The labeled RNA probe was purified by DNase I and proteinase K treatments, then absorbed on a nitrocellulose filter, and further purified with trichloroacetic acid washing and additional DNase I treatment. The probe eluted by SDS treatment was recovered by ethanol precipitation.…”

“…Recombinant human Sp1 and AP2, and affinity-purified rabbit polyclonal antibodies against RARs, RXR␣, and Sp1 were purchased from Santa Cruz. [␣- 32 P]dCTP, the random primer extension labeling system and Renaissance Western blot Chemiluminescence Reagent were obtained from PerkinElmer Life Sciences. ISOGEN, t-RA, 9C-RA, Ch55, T7 RNA polymerase, and RNase inhibitor were from Wako Pure Chemical, Osaka, Japan.…”

“…ISOGEN, t-RA, 9C-RA, Ch55, T7 RNA polymerase, and RNase inhibitor were from Wako Pure Chemical, Osaka, Japan. Rabbit reticulocyte lysate system, [␥- 32 P]ATP, [␣-32 P]UTP, and nitrocellulose paper were obtained from Amersham Pharmacia Biotech. Biotinylated anti-rabbit IgG and horseradish peroxidase-conjugated streptavidin were obtained from Vector Laboratories.…”

“…Darrow et al (31) indicated that RA-induced expression of the tissue plasminogen activator gene during F9 teratocarcinoma cell differentiation might involve Sp1. The binding abilities and functional significance of Sp1 for the promoters of various genes, such as murine ornithine decarboxylase (32), tissue factor (33), interleukin-6 (34), and prolactin receptor (35) genes, have been reported. However, there is no direct evidence concerning interactions between Sp1, RA receptor proteins, and the four Sp1 sequences in the TM gene, especially in the case of HUVE cells treated with retinoids.…”