Enhancer, Repressor, and Promoter Specificities Combine to Regulate the Rat α-Fetoprotein Gene

Abstract

The upstream transcription control region of the rat alpha-fetoprotein (AFP) gene was analyzed using transient expression of CAT genes in HepG2 cells which express the gene; H4C3 cells which repress the AFP gene but express the albumin gene; and four nonexpressing cell lines. Deletion analysis based on the DNA sequence resolved three upstream enhancers corresponding to the mouse AFP enhancers, but showed additional weak effects from flanking sequences. Quantitative experiments demonstrated that the three enhancers were additive when acting through a single promoter and did not confirm the presence of a distal upstream repressor. All three enhancers stimulated the AFP, albumin, or thymidine kinase (tk) promoter in HepG2, but only the tk and albumin promoters in H4C3. Deletion of a proximal repressor region near the AFP promoter allowed expression in H4C3 cells with the AFP promoter. Thus, the liver-specific developmental repressor is near the AFP promoter, and H4C3 cells provide an in vitro system for analysis of this repressor in transfection assays. The repressor region also blocked expression of the SV40 enhancer through the AFP promoter in hepatic and nonhepatic cell lines, but when this enhancer was combined with an AFP promoter from which the repressor region was deleted, the combination showed expression in all six cell lines studied. AFP expression results from a combination of enhancer, promoter, and repressor activities, and the repressor is functional with a heterologous enhancer in a variety of cells.

Distant Enhancers Stimulate the Albumin Promoter through Complex Proximal Binding Sites

“…This local enhancer makes a considerable contribution to albumin gene expression, equivalent to ϳ40% of the SV40 early enhancer plasmid SV2CAT, and the distant enhancers increase this activity to ϳ240% of SV2CAT. This amounts to 6-fold stimulation, but we have previously shown that in strong enhancer-driven systems, the enhancer contribution is described better as an additive relationship (10). In this paper, comparable enhancer activity stimulated the intact albumin promoter 2.4-fold, Ϫ84 promoter 32-fold, the ⌬4A promoter 48-fold (Fig.…”

“…Albumin Gene Promoter Models-Previous studies have demonstrated that the AFP gene enhancers are strongly active when combined with the albumin gene promoter (10,11,22). Our studies established enhancer stimulation of the promoter from distances of 1.7 kbp or greater, using a rat albumin promoter segment extending from ϩ1 to Ϫ308 bp (22).…”

“…Cloning-Plasmid pAlb(Ϫ175)CAT was constructed from a previously described albumin-promoter construct (10) by combining a segment from the albumin gene promoter from Ϫ175 (an AluI site, blunt, from a partial digest) to ϩ18 (a BglII site, a synthetic linker site replacing a HindIII site in the CAT gene 5Ј-untranslated region) with the CAT gene as a BglII-BamHI segment; these were joined and cloned in pBluescriptII-KSϩ (Stratagene, La Jolla, CA) at SmaI (blunt) and BamHI linker sites. pAlb(Ϫ84)CAT was cloned similarly, but with a promoter segment from Ϫ84 to ϩ18 (NlaIV, blunt, to BglII).…”

“…Ca 3 (PO 4 ) 2 transfection of HepG2 cells was carried out as described previously (10). Individual transfection experiments always consisted of a series of identical plates transfected simultaneously under identical conditions.…”

“…Three strong enhancers lie between the two genes (10,11). These intergenic enhancers are potentially active even when the AFP gene has been developmentally silenced, as demonstrated by studies that combined the enhancers with the albumin promoter.…”