An enhancer element in the 5' flanking region of the human apolipoprotein E gene, known as upstream regulatory element 1 (URE1), has previously been implicated in the expression of this gene. The URE1 element, which spans nucleotides -193 to -124 of the 5' -flanking region of the human apolipoprotein E gene, contains two sequences that bind to nuclear proteins, as determined by the DNase I footprinting assay. In the present study of URE1, we have characterized these sequences further. Deletion of one of the footprint sequences, at nucleotides -161 to -141, reduced URE1 enhancer activity substantially. A 30-base pair oligonucleotide that included this protein-binding sequence was able, by itself, to act as an enhancer. This sequence, termed the positive element for transcription (PET), was demonstrated by gel retention analysis to bind at least two protein factors, one of which is the transcription factor Sp1. Sp1 appeared to be the only protein required for the enhancer activity of PET to be manifested. In vitro transcription assays showed that the PET sequence was necessary for efficient transcription directed by the apoE promoter and that the PET sequence was the dominant regulatory element in the apoE promoter, Gel filtration chromatography and PET oligonucleotide-affinity chromatography were used to isolate a second PET-binding factor, a Mr = 55,000 protein, from HeLa cell nuclear extracts. It appeared to compete with Sp1 for a common binding site in the PET sequence, but it was not required for enhancer activity. The second footprint sequence in URE1, at nucleotides -184 to -173, also bound Sp1, but it was not required for enhancer activity. A third Sp1-binding region was located at a proximal GC box element (nucleotides -54 to -45). This region had no enhancer activity, but it was required for maximum transcriptional activity of the apoE promoter. Thus, the regulation of apoE gene expression is influenced by different protein-binding sequences, with trancription factor Sp1 playing major roles in both basal promoter activity and enhancer activity.
|Number of pages||9|
|Journal||Journal of Biological Chemistry|
|Publication status||Published - 1990 Jun 5|
All Science Journal Classification (ASJC) codes
- Molecular Biology
- Cell Biology