Cell type-specific expression of the human apoB gene is controlled by two cis-acting regulatory regions.

H K Das,T Leff,J L Breslow

doi:10.1016/s0021-9258(18)37978-x

Abstract

The human apolipoprotein B (apoB) gene codes for two related proteins, apoB-100 and apoB-48. ApoB-100 is synthesized in the liver, is the major protein constituent of low density lipoprotein, and serves as the ligand for the LDL receptor. cis-acting DNA sequence elements required for hepatic specific apoB transcription were identified in hepatoma (HepG2) and epithelial carcinoma (HeLa) cell lines transfected with apoB/CAT (chloramphenicol acetyltransferase) hybrid constructions. HepG2 cells express the transfected apoB constructions at high levels relative to expression in HeLa cells. Mutational analysis of the 5'-flanking region of the apoB gene revealed the presence of positive and negative regulatory regions. The most distal of these regions, located from -261 to -128 (with respect to the start site of transcription), was found to have a roughly equivalent negative activity in both cell types. However, sequences located from -128 to -86 showed a positive activity in HepG2 cells and a negative activity in HeLa cells. Finally, a sequence element located between positions -86 and -70 was found to have a very strong positive effect in HepG2 cells and only a mild positive effect in HeLa cells. These two proximal regions located between -128 and -70 appear to act together to determine the cell type-specific expression of the apoB gene in HepG2 and HeLa cells. Using the gel mobility shift assay and the DNase I footprinting technique, we demonstrated that DNA binding proteins from HepG2 and mouse liver nuclear extracts interact with the crucial positive region located between -86 and -70. This region was also found to contain sequence elements similar to sequences found in the promoters of other apolipoprotein genes, as well as other genes that are expressed in the liver, suggesting that these genes may share some transcriptional regulatory components.

Highlights

The human apolipoprotein B gene codes for processing and catabolism [1,2,3]
Have a very strongpositive effect in HepG2 cells and Elevated apolipoprotein B (apoB) levels are seen in two autosomal dominant only a mild positive effect in HeLa cells. These two genetic disorders associated with premature atherosclerosis, proximal regions located between -128 and -70 ap- familial hypercholesterolemia, and familial combined hyperpear to act together to determine ctehlel type-specific lipoproteinemia. The former is characterized by a decreased expression of the apoB gene in HepG2 and HeLa cells. apoB plasma fractional catabolic rate due to a deficiency of
Clinical disorders binding proteins fromHepG2 and mouse liver nuclear associated with diminished apoB levels called abetalipoproextracts interact with the crucial positive region lo- teinemia andheterozygous and homozygous hypobetalipoprocated between -86 and -70

Summary

RESULTS

-84 (Fig. 1B) were made by ligating the blunt-ended StuI-PuuII (-670 to +122), NcoI-PuuII (-262 to +122), AuaI-PuuII (-86 to +122), and SmaI-PvuII ( - 8 4 to +122) fragments into the SmaI site. Smith) containsaportion of the adenovirus 2 major late promoter (-50 to +33 relative to the start site of major late transcription) and 58 bp of SV40 untranslated leader sequence (SV40 map units 5227-5171) located immediately upstream of the CAT gene in pKT (Fig. 3, see Ref. 31 for construction). ApoB sequences were inserted into the pCTpolylinker immement, replicate Petri dishes of cells were transfected with pKT or with pKTSV (an SV40 early promoter-CAT construction) (Fig. lA). Both the CAT level, expression not seen with the -900 construction This and P-galactosidase activities were measured using the same amount suggests negative elements operating on sequences between of protein. The percentage of CAT substrate converted was divided by the B- ApoB Proximal Positive Region (-128 to -70)-In HepG2 galactosidase activity to normalize for transfection efficiency.

A PO E Gene

DISCUSSION

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