Rat Alpha-fetoprotein Research Articles

ZBTB20 is a sequence-specific transcriptional repressor of alpha-fetoprotein gene.

Alpha-fetoprotein (AFP) represents a classical model system to study developmental gene regulation in mammalian cells. We previously reported that liver ZBTB20 is developmentally regulated and plays a central role in AFP postnatal repression. Here we show that ZBTB20 is a sequence-specific transcriptional repressor of AFP. By ELISA-based DNA-protein binding assay and conventional gel shift assay, we successfully identified a ZBTB20-binding site at −104/−86 of mouse AFP gene, flanked by two HNF1 sites and two C/EBP sites in the proximal promoter. Importantly, mutation of the core sequence in this site fully abolished its binding to ZBTB20 in vitro, as well as the repression of AFP promoter activity by ZBTB20. The unique ZBTB20 site was highly conserved in rat and human AFP genes, but absent in albumin genes. These help to explain the autonomous regulation of albumin and AFP genes in the liver after birth. Furthermore, we demonstrated that transcriptional repression of AFP gene by ZBTB20 was liver-specific. ZBTB20 was dispensable for AFP silencing in other tissues outside liver. Our data define a cognate ZBTB20 site in AFP promoter which mediates the postnatal repression of AFP gene in the liver.

Differences in the binding of thyroid hormones and indoles by rat alpha 1-fetoprotein and serum albumin.

The transport of small molecules in the blood, normally assured by serum albumin in the adult, is not well known in the fetus since the albumin concentration is low in fetal serum and inversely related to the alpha 1-fetoprotein concentration. In order to investigate whether rat alpha 1-fetoprotein might be a fetal counterpart to albumin, the binding properties of these two proteins have been compared with respect to a series of molecules of biological importance, especially during fetal development: thyroid hormones and indole analogues. Though high-affinity binding of thyroxine was found with both rat alpha 1-fetoprotein and albumin, a significant difference in the number of binding sites for this hormone was found with the two proteins. Further, while rat serum albumin strongly bound L-tryptophan and indolyl-3-acetic acid (Ka approximately equal to 10(5) M-1), rat alpha 1-fetoprotein did not bind any of the indoles tested. These results are discussed with respect to the physiological and pharmacological significance of the transport role of these proteins.

Hepatoma cell-specific ganciclovir-mediated toxicity of a lentivirally transduced HSV-TkEGFP fusion protein gene placed under the control of rat alpha-fetoprotein gene regulatory sequences.

Suicide gene therapy combining herpes simplex virus thymidine kinase gene transfer and ganciclovir administration can be envisioned as a powerful therapeutical approach in the treatment of hepatocellular carcinoma; however, safety issues regarding transgene expression in parenchyma cells have to be addressed. In this study, we constructed LATKW, a lentiviral vector expressing the HSV-TkEGFP gene placed under the control of the promoter elements that control the expression of the rat alpha-fetoprotein, and assayed its specific expression in vitro in hepatocarcinoma and nonhepatocarcinoma human cell lines, and in epidermal growth factor stimulated human primary hepatocytes. Using LATKW, a strong expression of the transgene was found in transduced hepatocarcinoma cells compared to a very low expression in nonhepatocarcinoma human cell lines, as assessed by Northern blot, RT-PCR, FACS analysis and ganciclovir-mediated toxicity assay, and no expression was found in lentivirally transduced normal human hepatocytes. Altogether, these results demonstrate the possibility to use a lentivirally transduced expression unit containing the rat alpha-fetoprotein promoter to restrict the HSV-TK-mediated induced GCV sensitivity to human hepatocarcinoma cells.

Regulation of alpha-fetoprotein expression by Nkx2.8.

The alpha-fetoprotein (AFP) gene is an important model of developmental gene silencing and neoplastic gene reactivation. Nkx2.8 is a divergent homeodomain factor originally cloned through its binding to the promoter-coupling element (PCE), a regulatory region upstream of the AFP promoter that mediates stimulation by distant enhancers. Nkx2.8 is the only developmentally regulated factor that has been associated with AFP gene expression. Fetoprotein transcription factor, an orphan nuclear receptor, has also been shown to bind the PCE but is not developmentally regulated. The binding specificities of both families of transcription factor were determined, and overlapping sites for each were defined in the PCE. After modification of nuclear extract and gel shift analysis procedures, Nkx2.8 was identified in six AFP-positive cell lines. Transient-transfection analysis did not show transcriptional stimulation by Nkx2.8 or other active NK2 factors, which only interfered with gene expression. However, two sets of analysis demonstrated the relationship of Nkx2.8 to AFP expression: chromatin immunoprecipitation demonstrated that Nkx2.8 bound to the active AFP promoter, and antisense inhibition of Nkx2.8 mRNA translation selectively reduced expression of both the endogenous human AFP gene and transfected reporters containing the rat AFP promoter.

Modulation of the far-upstream enhancer of the rat alpha-fetoprotein gene by members of the ROR alpha, Rev-erb alpha, and Rev-erb beta groups of monomeric orphan nuclear receptors.

Expression of the oncodevelopmental alpha-fetoprotein (AFP) gene is tightly regulated and occurs in the yolk sac, fetal liver and intestine, and cancerous liver cells. Transcription of the AFP gene is under the control of three enhancers that are very tissue specific. We have shown that the most upstream of these enhancers, located at -6 kb, works through the combined action of liver-enriched factors and nuclear receptors that bind to three regions of this DNA regulatory element. This study showed that orphan nuclear receptors of the ROR alpha, Re-verb alpha, and Rev-erb beta groups can bind as monomers with high affinity and specificity to an evolutionarily conserved AGGTCA motif in the functionally important region 1 of this AFP enhancer. Transient transfection experiments performed with human HepG2 hepatoma cells showed that overproduction of ROR alpha 4 stimulated the activity of the AFP enhancer in a dose-dependent manner, while that of Rev-erb alpha and Rev-erb beta had the opposite effect. These effects were highly specific and required the integrity of the AGGTCA motif. The action of these nuclear receptors also occurred in the context of the entire 7-kb regulatory region of the rat AFP gene. These results suggest that altering the amounts or activities of these orphan receptors in cells of hepatic or endodermal origin could modulate AFP gene expression in response to a variety of developmental or carcinogenic stimuli.

Identification of a cis-acting element in the rat alpha-fetoprotein gene and its specific binding proteins in F9 cells during retinoic acid-induced differentiation.

Mouse F9 embryonic teratocarcinoma stem cells can be induced to differentiate into visceral endoderm. Following retinoic acid (RA) treatment, alpha-fetoprotein (AFP), a differentiation marker, is expressed and secreted. The mechanism by which RA regulates AFP expression during differentiation is not clear. The relatively late induction of AFP indicates that the AFP gene may not be a primary target of RA activity during F9 cell differentiation. In this study, a CAT reporter plasmid containing the rat AFP 5'-regulatory region (-7040 to +7) adjacent to the CAT gene (pAFPCAT) was stably transfected into F9 cells and used to delineate a cis-acting element which associates with AFP gene activation. Similar spatial and temporal expression patterns between the transcriptional activity of the recombinant AFP gene and the endogenous AFP gene demonstrate that this stably transfected F9 system can be used to dissect both cis-elements and trans-acting factors responsible for RA-induced AFP expression. Using a series of deletion mutants of the pAFPCAT, the region between -2611 to -1855 was found to be important in AFP-induction. Subsequent analysis identified a functional sequence (-1905 to -1891, 5'-ACTAAAATGGAGACT-3') that differentially binds nuclear proteins from undifferentiated and differentiated F9 cells. This sequence, designed as differentiation-associated sequence (DAS) for its unique binding of a nuclear protein (DAP-II) that appears during RA-induced F9 differentiation, acts as a regulatory protein factor in AFP gene activation.

Competitive Binding of Xenobiotic Oestrogens to Rat Alpha-Fetoprotein and to Sex Steroid Binding Proteins in Human and Rainbow Trout (Oncorhynchus mykiss) Plasma

The ability of a variety of “environmental oestrogens” to compete with radiolabelled steroids to rat α-fetoprotein (AFP) and to sex steroid binding proteins was investigated in human and rainbow trout (Oncorhynchus mykiss) plasma. For [3H]oestradiol binding to AFP, diethylstilbestrol and 4-nonylphenoxyacetic acid showed significant competition at concentrations about 100-fold greater than oestradiol (relative binding affinities ≈ 1% c.f. oestradiol). All other compounds (phytooestrogens: coumestrol, daidzein, genistein; others: 4-nonylphenol, 4-tert-octylphenol, 4-nonylphenoldiethoxylate, 4-tert-butylphenol, bisphenol-A (Bis-A), bis(2- ethylhexl)phthalate, dioctylphthalate, dibutyl phthalate, 2,4′DDT (op' enantiomer), 2,4′-DDE (mixed enantiomers), kepone) showed only very weak or no competition (relative binding affinities <<0.1% c.f. oestradiol). The situation for both human and fish plasma was very similar, with only very high concentrations (>>1000 fold more than the natural ligand) of a few of the compounds showing any ability to displace the natural ligand. These results suggest that environmental oestrogenic agents are unlikely to produce biological effects by displacing endogenous steroids from plasma steroid binding proteins unless they are present in very high concentrations.

An immunoaffinity-purified Trypanosoma cruzi antigen suppresses cellular proliferation through a TGF-beta-mediated mechanism.

Two subfractions with opposite immunological properties were obtained from the flagellar antigens (FF) of Trypanosoma cruzi epimastigotes by immunoaffinity chromatography. The ligand-bound material (Ag 123) contained four polypeptide bands of 97, 55, 38 and 14 kDa. The nonretained flow-through (FT), induced a potent proliferation of murine naive splenocytes. In contrast, Ag 123 inhibited the proliferative capacity of the FT as well as the proliferation mediated by the mitogen Concanavalin A (Con A). The suppressive effect of Ag 123 on the Con A-mediated proliferation was neutralized by an anti-TGF-beta monoclonal antibody. Both Ag 123 and FF stimulated high serum levels of TGF-beta in injected mice. Ag 123 also induced in vitro secretion of TGF-beta by murine splenocytes. These results demonstrate that Ag 123 is a potent stimulator of TGF-beta both in vivo and in vitro. Oligopeptides derived from the 38 kDa protein present in Ag 123 showed homology with human and rat alpha-fetoproteins (AFP). Ag 123 seems to have a key role in the immunosuppression that develops during early stages in the infection with T. cruzi.

Flavonoids inhibit estrogen binding to rat alpha-fetoprotein.

There is considerable interest in the role(s) of plant-derived compounds such as bioflavonoids in regulating steroid hormone action in mammals, and in particular, the possible effects of the bioflavonoids on the growth of steroid-dependent breast and prostate tumors and on possible abnormal development of steroid-sensitive tissues. Studies of the hormone-like actions of bioflavonoids often use fetal or neonatal rats, which contain high levels of serum alpha-fetoprotein (AFP), a protein that binds estradiol with a Kd approximately 5 x 10(-9) M. Interaction of bioflavonoids with AFP could affect the availability of estrogens to estrogen-responsive cells, as well as the actions of bioflavonoids. These considerations motivated us to study the effect of several flavonoids (quercetin, rutin, naringenin, chrysin, apigenin, kaempferol, myricetin, morin, fisetin) and isoflavonoids (daidzein, genistein) on estrogen binding to rat AFP. We found that naringenin, a flavanone, and quercetin and kaempferol, flavonols, inhibit estrogen binding to AFP with apparent Kds of about 5 x 10(-7) M. To our surprise, the two isoflavonoids, daidzein and genistein, have Kds of about 5 x 10(-6) M for AFP. This 10-fold [correction of 1Q-fold] difference in affinity for AFP between flavonoids and isoflavonoids suggests that AFP has a specificity for the flavonoid structure. Moreover, the affinities of bioflavonoids for rat AFP are sufficiently high to suggest that flavonoids and isoflavonoids could modulate estradiol and estrone binding to rat AFP in vivo, when present at dietary levels. Additionally, the potency of the plant estrogens may be altered by binding to AFP. The flavonoids that we tested have different hydroxyl and glucoside substituents on the A, B, and C rings, which allows us to define some of the spatial requirements for binding to AFP. We find that 5,7-hydroxyl groups in ring A and a 4'-hydroxyl group in ring B are important for binding to AFP. This information, combined with molecular modeling studies, may elucidate the molecular basis for recognition of flavonoids and estrogens by AFP. Also, these findings indicate that the flavonoid levels in the diet need to be considered in studies of the effects of various xenobiotics and endocrine manipulations on experimental animals, particularly during development when serum estrogen binding protein concentrations are often elevated. Finally, bioflavonoids should be useful tools for understanding the variety of estrogen actions initiated by different structural classes of estrogens.

Chicken ovalbumin upstream promoter-transcription factor, hepatocyte nuclear factor 3, and CCAAT/enhancer binding protein control the far-upstream enhancer of the rat alpha-fetoprotein gene.

We have further characterized the most distal of the three alpha-fetoprotein (AFP) enhancers required for expression of the AFP gene in fetal hepatocytes and yolk sac endodermal cells. Almost total rat AFP enhancer 3 (E3) activity is driven by a 160-bp fragment at -6 kb containing three target regions for nuclear proteins that cooperate to stimulate transcription from the AFP and the thymidine kinase promoters in HepG2 hepatoma cells. Region 1, recently shown to be crucial for correct function of the enhancer in liver of transgenic mice, is recognized by two sets of transcription factors that bind to partly overlapping sites, 1a and 1b, in a noncooperative and nonexclusive manner. Site 1a contains a motif, AGGTCA, which is recognized by chicken ovalbumin upstream promoter transcription factors (COUP-TFs), but not by hepatocyte nuclear factor 4. Hepatocyte nuclear factor 3 (HNF3) and CCAAT/enhancer binding protein (C/EBP), which bind to regions 2 and 3, respectively, are likely responsible for the liver-specific E3 action. They play a key role by acting in synergy. The participation of nuclear receptors such as COUP-TFs, with C/EBP and HNF3, in the tight control of the distal AFP enhancer is a new, and perhaps key, step toward understanding the regulation and function of this enhancer, which may remain active throughout development.

RXR-mediated regulation of the alpha-fetoprotein gene through an upstream element.

Retinoic acid (RA) is known to have potent effects on development and differentiation. RA exerts its effects on transcription through two distinct classes of nuclear receptors, the retinoic acid receptor (RAR) and the retinoid X receptor (RXR), that bind to specific RA-responsive elements (RARE) in target genes. alpha-Fetoprotein (AFP), a hepatocyte differentiation, maturation, and carcinogenesis marker, is transcriptionally upregulated by RA in McA-RH8994 hepatoma cells. Using deletion mapping analysis, we have identified a RARE-like sequence that is located between -2406 and -2378 of the transcription initiation site of the rat AFP gene. Sequence analysis demonstrated that this cis-acting element consists of three direct repeats and one inverted repeat of a GGGTCA-like half-site. The putative RARE can specifically bind to both RXR homodimers and RAR/RXR heterodimers as determined by gel mobility shift assays. A DR1 direct repeat was more efficient than a DR5 direct repeat oligonucleotide in competition for binding of the putative RARE to RXR and RAR/RXR. A mutagenesis study indicated that to have a full-strength induction, all the repeats were required. To further analyze the function of this element in vivo, a reporter gene construct of the putative RARE combined with the thymidine kinase promoter was cotransfected with RAR and RXR expression plasmids in CV1 cells. CAT assays demonstrated that overexpression of RXRalpha conferred the best RA response, consistent with our previous observation that 9-cis-RA is more potent than all-trans-RA for inducing the expression of the AFP gene. In addition, the RXR selective ligand LG100153 alone can stimulate the expression of the AFP gene. Our data suggest that an RXR-mediated pathway exists for modulation of AFP gene expression through a specific element.

In vitro and in vivo hepatoma cell-specific expression of a gene transferred with an adenoviral vector.

Recombinant adenoviruses are widely used for the transfer of foreign genes into various mammalian cells. However, the utilization of these vectors for cancer gene therapy requires the specific and efficient expression of the transferred gene in tumor cells. To obtain targeted expression in hepatoma cells, we constructed recombinant adenoviral vectors containing transcriptional elements from either the rat alpha-fetoprotein (AFP) or the human insulin-like growth factor II (IGFII) genes driving expression of the nuclear beta-galactosidase gene (nls lacZ). In vitro infection revealed that the AFP but not the IGFII transcriptional regulatory sequence controlled nls lacZ expression specifically in hepatoma cells. The same specificity was obtained in vivo in subcutaneous human hepatic tumors generated by engraftment of Huh7 hepatoma cells in nude mice as well as in primary liver tumors developed in rats and mice. No marker gene expression was detectable after AFP-nls lacZ gene transfer to normal rat liver parenchyma despite evidence for the presence of DNA encoding the nls lacZ gene. However, in vivo experiments with primary liver tumors in rats and mice also revealed that primary hepatoma cells were poorly infected by adenoviral vectors. Peritumoral and normal tissues were infected efficiently by adenoviral vectors. We conclude that hepatoma cell-specific expression of a transgene can be achieved with AFP regulatory sequences but that adenoviral vectors may not be the preferable vector for transferring genes in vivo in primary liver tumors.

Hepatoma cell-specific expression of a retrovirally transferred gene is achieved by α-fetoprotein but not insulinlike growth factor II regulatory sequences

To target gene expression to malignant hepatic cells, we have constructed recombinant retroviral vectors containing a reporter gene encoding nuclear beta-galactosidase (nls-LacZ) under transcriptional control of regulatory sequences from the rat alpha-fetoprotein (AFP) or human insulinlike growth factor II (IGFII) genes. The AFP and IGFII P3 promoters activate transcription during fetal development and are often reactivated in hepatocellular carcinoma (HCC). Infection of several cultured cell types with the retroviral vector containing the IGFII P3 sequence resulted in expression of the reporter gene in all cell lines tested, including those that do not produce IGFII. In contrast, selective expression was achieved by vectors containing the AFP transcriptional regulatory sequence. Nuclear beta-galactosidase activity was detectable in cells from lines that produce AFP, and not in cells that do not express the AFP gene. In most infected cell lines, retroviral RNA synthesis from the 5' LTR was inhibited, and deletion of the retroviral LTR enhancer did not change expression from either the IGFII P3-nls-LacZ or the AFP-nls-LacZ cassettes. After treatment of cells with 12-O-tetradecanoylphorbol-13-acetate and epidermal growth factor (EGF), the decrease in concentrations of endogenous AFP messenger RNA (mRNA) and nls-LacZ mRNA transcribed from the transferred AFP regulatory sequence were similar. In the context of an integrated provirus, the AFP transcriptional regulatory sequence is therefore subject to similar regulatory control as that of the endogenous gene. These data show that the AFP sequence, and not the IGFII P3 promoter we used, is suitable for targeting gene expression to malignant hepatic cells.

The c- jun Proto-oncogene Down-regulates the Rat α-Fetoprotein Promoter in HepG2 Hepatoma Cells without Binding to DNA

The effects of a phorbol ester (TPA) and of members of the Jun and Fos oncoprotein family on the activity of the rat alpha-fetoprotein (AFP) promoter were checked by using transient expression experiments in HepG2 hepatoma cells. TPA blocked the activity of the rat AFP promoter in a dose-dependent manner. Overexpression of c-Jun specifically repressed the rat AFP promoter but not the albumin promoter. JunB and JunD were poorer inhibitors. c-Fos expression did not potentiate the negative effect of Jun. The Jun-induced repression does not require binding of c-Jun to the AFP promoter. DNase 1 footprinting experiments did not display any high affinity binding site for Jun on the AFP promoter. Integrity of the c-Jun DNA binding domain is not required for the c-Jun protein to block the AFP promoter. The N-terminal part of Jun, which contains the activating domain, is responsible for the repression as shown by using Jun-Gal4 chimera. Jun likely exerts its negative control on the AFP promoter via protein-protein interactions with a not yet identified trans-activating factor within the -134 to +6 region or with a component of the general machinery of transcription. Jun proteins can thus be key intermediates in regulatory cascades which result in the differential modulation of the AFP and albumin gene expression in the course of liver development and carcinogenesis.

リコンビナント分子を用いたＡＦＰの機能部位の同定

We would like to focus on two topics which were recently made in our laboratoty on structure/function relationship of alpha-fetoprotein (AFP) by recombinant DNA technology: identification of estrogen binding site of rat AFP, and evaluation of glycosylation of two possible N-glycosylation sites in rat AFP. In order to identify estrogen binding site of rat AFP, a series of rat-human chimeric AFPs was designed, since rat AFP binds to estrogen while human AFP does not. Out of 7 rat-human chimeric AFPs, only those which carry a rat segment 426-467 in human back ground sequence conserved the estrogen binding activity, indicating the segment 426-467, where 15 amino acid substitutions were observed, contained the residues responsible for estrogen binding. A variety of human mutant AFPs in which some human-specific amino acid residues were substituted with those of rat AFP was obtained by site-directed mutagenesis employing polymerase chain reaction. Among 17 human mutant AFPs carrying different combinations of substitutions, those carrying 436 A→V, 437A→S, 438 T→I, 450 L→R, 451 L→S, 457 A→L, 461 I→Y conserved sufficient binding activities, strongly suggesting that these amino acid residues were responsible for the estrogen binding activity in rat AFP. The rat AFP resolves on polyacrylamide gel electrophoreses (PAGE) into two discrete bands, “Slow”and“Fast”, and this electrophoretic pattern was shown by us to depend on the heterogeneity of N-glycosylation. For evaluation of glycosylation at two possible N-glycosylation sites in rat AFP, we designed three mutant rat AFPs, which had either substitution of 93Asn→Gln, 229Asn→Gln, or 93Asn→Gln+229Asn→Gln, as well as recombinant rat AFP without mutation. SDS-PAGE before and after glycopeptidase F treatment of the mutant AFPs indicated that the“Fast”had one carbohydrate moiety at 229Asn and the“Slow”had two carbohydrate moieties at 93Asn and 229Asn. It was therefore indicated that 229Asn was fully glycosylated while 93Asn was partially.

A novel hepatocytic transcription factor that binds the alpha-fetoprotein promoter-linked coupling element.

We recently characterized a promoter-linked coupling element (PCE) in the rat alpha-fetoprotein (AFP) gene required for strong transcriptional stimulation by distant enhancers (P. Wen, N. Crawford, and J. Locker, Nucleic Acids Res. 21:1911-1918, 1993). In this study, oligonucleotide gel retardation and competition experiments defined the PCE as a 12-bp binding site, TGTCCTTGAACA, an imperfect inverted repeat from -166 to -155 near the AFP promoter. A factor that bound this site (PCF) was abundant in HepG2 nuclear extracts and detectable in extracts from several other AFP-producing hepatocarcinoma cell lines and fetal liver. Hepatocytic cell lines that did not express AFP, nonhepatocytic cell lines, adult liver, and fetal brain did not show the factor. Experiments excluded the possibility that PCF activity was due to binding of glucocorticoid receptor or an AP1-like factor that bound overlapping sites. Competition experiments with several mutant oligonucleotides determined that the optimum PCF binding site was TGTCCTTGAAC(A/T). Mutations decreased binding or totally abolished binding activity. In expression plasmids, PCE mutations strongly reduced gene expression. UV cross-linking to a PCE probe identified peptide bands near 34 kDa. PCF was purified by heparin-Sepharose chromatography followed by affinity binding to oligomerized PCE DNA. The product resolved as a complex of three peptides (PCF alpha 1, PCF alpha 2, and PCF beta, 32 to 34 kDa) on sodium dodecyl sulfate-acrylamide gels. The peptide sizes and gel patterns are unlike those of any of the well-described hepatic transcription factors, and the binding site has not been previously reported. PCF thus appears to be a novel transcription factor.

A novel hepatocytic transcription factor that binds the alpha-fetoprotein promoter-linked coupling element.

We recently characterized a promoter-linked coupling element (PCE) in the rat alpha-fetoprotein (AFP) gene required for strong transcriptional stimulation by distant enhancers (P. Wen, N. Crawford, and J. Locker, Nucleic Acids Res. 21:1911-1918, 1993). In this study, oligonucleotide gel retardation and competition experiments defined the PCE as a 12-bp binding site, TGTCCTTGAACA, an imperfect inverted repeat from -166 to -155 near the AFP promoter. A factor that bound this site (PCF) was abundant in HepG2 nuclear extracts and detectable in extracts from several other AFP-producing hepatocarcinoma cell lines and fetal liver. Hepatocytic cell lines that did not express AFP, nonhepatocytic cell lines, adult liver, and fetal brain did not show the factor. Experiments excluded the possibility that PCF activity was due to binding of glucocorticoid receptor or an AP1-like factor that bound overlapping sites. Competition experiments with several mutant oligonucleotides determined that the optimum PCF binding site was TGTCCTTGAAC(A/T). Mutations decreased binding or totally abolished binding activity. In expression plasmids, PCE mutations strongly reduced gene expression. UV cross-linking to a PCE probe identified peptide bands near 34 kDa. PCF was purified by heparin-Sepharose chromatography followed by affinity binding to oligomerized PCE DNA. The product resolved as a complex of three peptides (PCF alpha 1, PCF alpha 2, and PCF beta, 32 to 34 kDa) on sodium dodecyl sulfate-acrylamide gels. The peptide sizes and gel patterns are unlike those of any of the well-described hepatic transcription factors, and the binding site has not been previously reported. PCF thus appears to be a novel transcription factor.

Members of the CAAT/enhancer-binding protein, hepatocyte nuclear factor-1 and nuclear factor-1 families can differentially modulate the activities of the rat α-fetoprotein promoter and enhancer

The promoter of the rat alpha-fetoprotein (AFP) gene, which makes the expression of the developmentally regulated AFP gene specific to the liver, is a putative target for transcription factors of the CAAT/enhancer-binding protein (C/EBP), hepatocyte nuclear factor-1 (HNF-1) and nuclear factor-1 (NF-1) families. We have evaluated the influence of these factors on the activity of the AFP promoter by transfection of HepG2 hepatoma cells with the appropriate expression vector plus a CAT plasmid under the control of the AFP promoter. A similar plasmid bearing the rat albumin promoter was used as a control. C/EBP alpha, C/EBP beta and D-binding protein (DBP) acted as trans-activators on the AFP promoter, whereas liver inhibitory protein (LIP), a truncated form of C/EBP beta, was a potent negative regulator of the promoter. C/EBP alpha also bound to and stimulated the activity of the AFP enhancer at -2.5 kb. Interestingly, HNF-1 beta was found to be more potent than HNF-1 alpha in activating the AFP promoter. This effect was specific, as it did not occur with the rat albumin promoter. HNF-1 beta, which is produced earlier than HNF-1 alpha during liver development, would thus have the greater influence on the AFP promoter in early development. Both HNF-1s allowed expression of the AFP promoter in cells of nonhepatic origin. Overexpression of NF-1 induced a specific decrease in the activity of the AFP promoter. This strongly suggests that competition between NF-1 and HNF-1 for binding to their overlapping binding sites on the AFP promoter is critical for modulating its activity. Thus changing combinations of these trans-acting factors may tightly modulate the AFP promoter activity in the course of liver development and carcinogenesis.

Enhancement of reporter gene de novo methylation by DNA fragments from the alpha-fetoprotein control region.

The 5'-upstream region of the rat alpha-fetoprotein (AFP) gene strongly increased de novo methylation of an adjacent chloramphenicol acetyltransferase (CAT) gene upon transfection into F9 mouse embryonal carcinoma cells. The same effect was exerted by a distal 775-base pair (bp) fragment and by 300- and 1-kb fragments preceding the transcriptional start site, but not by other parts of the control region. Further division of the larger, strongly active fragments resulted in a gradual decrease of methylation and clonal variation in the methylation patterns. The effect of the 775-bp fragment did not depend on its orientation. It was ablated by insertion of the mouse metallothionein I promoter between the AFP gene fragment and the CAT gene, but not by its insertion upstream of the AFP gene fragment. Two fragments from the AFP control region increasing methylation contained B1 and B2 small interspersed repetitive elements, respectively. B1 and B2 sequences of different origin also acted strongly to increase methylation. These findings support the idea that mammalian genes contain specific sequences involved in regulating their methylation. The effects of these sequences appear to be exerted in cis, to be dependent on proximity, but not on orientation, and to require an optimal size of 500-700 bp. Small retrotransposon sequences within such elements may be particularly effective in attracting de novo methylation.

A promoter-linked coupling region required for stimulation of alpha-fetoprotein transcription by distant enhancers.

We previously demonstrated that the rat alpha-fetoprotein (AFP) gene has three upstream enhancers that stimulate the AFP promoter additively in HepG2 cells (1). In this paper, deletion analysis demonstrated that a promoter-linked segment from -178 to -155 was required for full activity when the enhancers were distant from the promoter, even at less than their normal genomic distances, but dispensable when the enhancers were moved close to the promoter. This 'promoter-coupling element' appears to interact simultaneously with all three enhancers. Deletion analysis also localized a transcription stimulatory and a negative region in the promoter. Though these latter regions controlled the strength of the isolated promoter, they did not affect 'coupling' to the distant enhancers, and transcription stimulation by these distal promoter elements was small compared to the distant enhancers. Overall, the distant enhancers, acting through the promoter-coupling element, accounted for 70% of the activity of the transfected AFP gene. Footprint analysis with HepG2 nuclear extracts demonstrated protein binding at two sites near the promoter-coupling element. The data indicate a positive transcription control mechanism by which distant enhancers stimulate the AFP promoter through a specific promoter-linked element.