The equilibrium constant was determined for the binding of the transcription factor Sp1 to a single consensus GC box DNA recognition site, (5'-GGGGCGGGGC-3'). For these experiments, single copies of the recognition site were synthesized and cloned in a standard plasmid background. Binding was measured either by a footprinting assay modified so that the binding reaction was at equilibrium, or by a gel mobility shift assay. The concentration of active Sp1 in the reactions and the dissociation constant were determined by computer-assisted fitting to theoretical curves. Values for the dissociation constant obtained in different experiments ranged from 4.1 X 10(-10) M to 5.3 X 10(-10) M. Several variants of the consensus recognition site were also tested. An A-substituted variant (5'-GGGGAGGGGC-3') and a T-substituted variant (5'-GGGGTGGGGC-3') were bound 3-fold and 6-fold more weakly than the consensus site, respectively. A G-substituted variant (5'-GGGGGGGGGC-3') was bound at least 30-fold more weakly than the consensus site. These findings help distinguish between alternative models for Sp1-DNA recognition. They are consistent with the presence of specific hydrogen-bond contacts between Sp1 and the central C-G base pair, but provide no particular evidence to support a model where local DNA structure is the dominant factor in the interaction.

The human hepatitis B virus (HBV) is a hepatotropic virus that replicates through an RNA intermediate referred to as the pregenome. The promoter that directs the synthesis of the pregenome and several other transcripts with heterogeneous 5' ends is of particular interest because ofits role in regulating key functions during the viral life cycle. We have examined the liver-specific characteristics of this promoter by DNA-protein interactions and by demonstrating the in vivo function of the promoter using the luciferase reporter gene expression system. The DNA-protein interactions in this region appear to be almost entirely liver-specific. Among these, a liver-specific nuclear factor, CCAAT/enhancer binding protein, binds to at least five sites on this promoter. Transient cotransfection experiments using CCAAT/enhancer binding protein expression vectors and the core promoter in the context of either the native hepatitis B virus genome or the luciferase reporter gene demonstrate that CCAAT/enhancer binding protein at low concentration modestly activates expression from the core promoter but represses at high concentration.The human hepatitis B virus (HBV) causes acute and chronic hepatitis, and the infection has been associated with hepatocellular carcinoma (1). There are four genes encoded by the viral genome: SipreS, Cle, Pol, and X (1). The transcription of these genes is controlled by at least four promoters (1-7). Additionally, an enhancer element has been identified in the HBV genome (8, 9). The core or pregenomic promoter regulates synthesis ofthe 3.6-kilobase (kb) RNAs that include mRNAs for several viral proteins (core, e, and pol) and the pregenomic RNA. Transcriptional regulation of these viral macromolecules makes this promoter a key element of the viral life cycle. The core promoter exhibits enhancerdependence for efficient liver-specific activity (6,(9)(10)(11)(12).In this study, we have used a series of restriction fragments and deletion mutations to define the boundaries of the HBV core/pregenomic promoter by using the firefly luciferase reporter gene (13). Furthermore, DNase I protection analysis was employed to identify nucleotide sequences that are binding sites for trans-acting cellular factors. This analysis revealed at least seven regions of DNA-protein interactions, most of which appear to be liver-specific. We show here that the CCAAT/enhancer binding protein (C/EBP), a liverspecific factor, binds to at least five sites in the promoter region. C/EBP was first purified from rat liver and shown to bind to several viral and cellular promoters/enhancers (14)(15)(16)(17)(18).To assess the role of C/EBP in the core promoter function, cotransfection experiments were carried out with a C/EBP expression vector and the core promoter linked to a reporter gene or in the context ofHBV genome. These studies indicate that C/EBP, at low concentrations, can function as a modest trans-activator of the core promoter. Increasing concentrations of C/EBP, however, resulted in trans-repression of the core ...

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The liver-specific enhancer I of the human hepatitis B virus contains several regions of DNA-protein interaction. Located within this element are also the domains of a promoter controlling the synthesis of the X open reading frame. Functional domains of the enhancer I and the X gene promoter were identified using DNase I protection analysis, deletion mutagenesis, and cell transfections. A unique liverspecific interaction was identified within this element whose binding site includes a direct sequence repeat, 5'-AGTAAA-CA(GTA-3'. The factor(s) binding to this sequence motif was purified by oligonucleotide-affinity chromatography. Binding of this factor appears to play a key role in determining the overall enhancer function. Additionally, the interaction of several purified factors is presented. Cotransfection of liver cells with expression vectors encoding transcriptional factors resulted in trans-activation of the promoter/enhancer function. Based on the results of genetic analysis a model outlining the functional domains of the enhancer/promoter region is presented.The human hepatitis B virus (HBV) infects hepatocytes and causes acute and chronic hepatitis. Expression of the HBV genes appears to be modulated by transcriptional control elements that display liver specificity. This is facilitated by the interaction of trans-acting cellular factors with upstream regulatory sequences. There are four genes encoded by the viral genome (Fig. 1A): S/preS, C/e, Pol, and X (1), whose transcription is controlled by four promoters (see review, ref. 1). Two enhancer elements have been identified in the HBV genome by the use of reporter genes in heterologous systems (3-5), both of which display liver specificity (3-7). The enhAncer element, termed enhancer I, is located between 966 and 1308 nt (3, 4, 7) and also has been speculated to contain the functional domains of the X gene promoter. This enhancer has been previously shown to direct its influence on the promoters of C, X, and, to a modest extent, the S ORF (8-12). Deletion of this enhancer from the genome results in a general decline of transcription from vi-al promoters (13).Analysis of DNA-protein interactions of both enhancers has revealed binding regions for several cellular transcriptional factors (6, 14-17). These protein binding sites display homology to known sequence motifs for which factors have been identified and purified. Some of these interactions have been shown to be liver specific (6,(14)(15)(16)(17). A second element, designated as enhancer II, was identified relatively recently (5) and maps approximately within the core/pregenomic promoter at nt 1645-1803 in the HBV adw2 genome.In this report, using deletion mutagenesis studies of the enhancer/X promoter complex, we have identified key elements crucial for enhancer function and those required for X promoter activity. Two regions of DNA-protein interaction were shown to be essential for enhancer activity. The transcription factor EF-C binds to one of these sites (18), and the other site binds a...