Identification and characterization of transcriptional regulatory regions associated with expression of the human...

Multiple cis-acting regulatory elements have been mapped within a 1-kilobase fragment spanning nucleotides -651 through +356 of the human apolipoprotein E gene using a transient expression system based on the chloramphenicol acetyltransferase gene as well as DNase I footprinting techniques. A 651-base pair 5'-flanking region of the human apolipoprotein E gene was capable of directing chloramphenicol acetyltransferase gene expression over a 48-fold range among the various cultured cell lines tested. Deletion analysis of this 651-base pair upstream region linked to either the chloramphenicol acetyltransferase gene or the intact apolipoprotein E structural sequences revealed at least three regulatory domains within the proximal 383 nucleotides. One of these domains contained a GC box transcriptional control element. Further analysis demonstrated that the other two domains contained enhancer-like activity. These enhancer-like elements were located within the nucleotides spanning -366 to -246 and -193 to -124. A third enhancer element was identified in the first intron, within nucleotides +44 to +262. Changing the distance of the three enhancer elements from the transcription start site and reversing their orientation did not significantly alter their effects on transcription rates. However, enhancer activity was influenced by the promoter and cell line that were used. DNase I footprinting assays showed that specific sequences within two of these elements (-193 to -124 and +44 to +262) bind proteins in nuclear extracts from HepG2 and Chinese hamster ovary cells. A protein footprint also was identified for a GC box element at nucleotides -59 to -45. Thus, control of apolipoprotein E gene expression is the result of a complex interaction of several different regulatory elements.