High affinity binding site for nuclear factor I next to the hepatitis B virus S gene promoter.

The hepatitis B virus (HBV) surface antigen (HBsAG) is encoded by the S gene under the regulation of a promoter in the pre-S1 region. The S gene promoter does not contain a 'TATA' box-like sequence, but there is a sequence resembling, in part, the late promoter of Simian virus 40 (SV40). In an attempt to study the regulation of the S gene promoter we looked for cellular proteins which bind to this region. We report here that a nuclear protein is tightly bound to the HBV genome at a position approximately 190 bases upstream from the S gene promoter. Evidence is provided to show that (a) this nuclear protein is the nuclear factor I (NF-I) that was previously found to be bound to the inverted terminal repeat of the adenovirus (Ad) DNA and to enhance Ad DNA replication in vitro and (b) this NF-I binding site is required for optimal activity of the S gene promoter.     

Control of the adipsin gene in adipocyte differentiation. Identification of distinct nuclear factors binding to single- and double-stranded DNA

The mouse adipsin gene encodes a serine protease with complement factor D activity that is expressed during adipocyte differentiation and is deficient in several animal models of obesity. We have investigated the regulation of adipsin expression by transfecting preadipocytes and adipocytes with plasmids containing the 5'-flanking region of the adipsin gene linked to a reporter gene. Constructions containing a -950 to +35 segment of the adipsin promoter were preferentially expressed in adipose cells. Deletion experiments identified a region from -114 to -38 which contains a large inverted repeat sequence and negatively regulated gene expression in preadipocytes and positively regulated expression in fat cells. Exonuclease III protection and gel retardation assays indicated that this region of duplex DNA had multiple binding sites for nuclear factors, several of which were preadipose specific. In addition, we also identified two distinct factors that bound symmetrically and sequence specifically to the inverted repeat sequences only when they were in single-stranded form; one of these factors was induced during adipocyte differentiation. These results suggest that the control of the adipsin promoter in differentiation may involve an interplay of multiple regulated DNA-binding proteins, including two that have preferential affinity for single-stranded DNA.     

Characterization of nuclear factors for elicitor-mediated activation of the promoter of the pea phenylalanine ammonia-lyase gene 1.

The nuclear factors presumably associated with the activation of the gene encoding phenylalanine ammonia-lyase by a fungal elicitor were characterized in pea (Pisum sativum L.) epicotyls. The TATA-proximal region was dissected and putative cis-regulatory elements in the promoter of pea phenylalanine ammonia-lyase gene 1 were examined by gel-mobility shift and in vitro footprinting analyses. Specific binding of the nuclear factors to the promoter-proximal regions of pea phenylalanine ammonia-lyase gene 1 associated with elicitor-mediated activation was detected at a region containing consensus sequence motifs of boxes 2 and 4 and other AT-rich sequences. The analyses of DNA fragments containing the deleted promoter regions suggested that a residue from -183 to -173 (ATTAGTAAGTGAT) was essential for a maximal activity of forming low-mobility complex (LMC) in the gel-mobility shift assay, and synthetic oligonucleotides confirmed the presence of at least one nuclear component associated with the formation of an active LMC. Competition experiments and treatment with Hoechst 33258 provided direct evidence that the formation of LMC with the promoter fragments from genes encoding phenylalanine ammonia-lyase and chalcone synthase in pea contained one or more of the same proteins that recognize AT-rich sequence motifs for binding. It also suggests that common high-mobility group-like proteins might be involved in the regulation of elicitor-inducible genes in pea.     

Characterization of cis-acting elements in light regulation of the nuclear gene encoding the A subunit of chloroplast isozymes of glyceraldehyde-3-phosphate dehydrogenase from Arabidopsis thaliana.

We have characterized cis-acting elements involved in light regulation of the nuclear gene (GapA) encoding the A subunit of chloroplast glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in Arabidopsis thaliana. Our results show that a 1.1-kb promoter fragment of the GapA gene is sufficient to confer light inducibility and organ specificity in transgenic Nicotiana tabacum (tobacco) plants, using the beta-glucuronidase gene of Escherichia coli as the reporter gene. Deletion analysis indicates that the -359 to -110 bp region of the GapA gene is necessary for light responsiveness. Within this region there are three copies of a decamer repeat (termed the Gap box) having the consensus sequence 5'-CAAATGAA(A/G)A-3', which has not been characterized in the promoter regions of other light-regulated genes. A deletion (to -247) producing loss of one copy of these elements from the GapA promoter reduces light induction by two- to threefold compared with a promoter deletion (to -359) with all three Gap boxes present, while deletion of all three Gap boxes (to -110) abolishes light induction completely. Gel mobility shift experiments using tobacco nuclei as the source of nuclear proteins show that GapA promoter fragments that contain these repeats bind strongly to a factor in the nuclear extract and that binding can be abolished by synthetic competitors consisting only of a monomer or dimer of the Gap box. Furthermore, a trimer, dimer, and monomer of the Gap box show binding activity and, like the authentic GapA promoter-derived probes, show binding activities that are correlated with Gap box copy number. These results strongly suggest that these repeats play important roles in light regulation of the GapA gene of A. thaliana.     

Interaction of a nuclear factor-1-like protein with the regulatory region of the human polyomavirus JC virus

We have initiated a study to identify host proteins which interact with the regulatory region of the human polyomavirus JC (JCV), which is associated with the demyelinating disease, progressive multifocal leukoencephalopathy. We examined the interaction of nuclear proteins prepared from different cell lines with the JCV regulatory region by DNA binding gel retardation assays. Binding was detected with nuclear extracts prepared from human fetal glial cells, glioma cells, and HeLa cells. Little or no binding was detected with nuclear extracts prepared from human embryonic kidney cells. Competitive binding assays suggest that the nuclear factor(s) which interacted with the JCV regulatory region was different from those which interacted with the regulatory region of the closely related polyomavirus SV40. We found three areas in the JCV regulatory region protected from DNase I digestion: site A, located just upstream from the TATA sequence in the first 98-base pair (bp) repeat; site B, located upstream from the TATA sequence in the second 98-bp repeat; and site C, located just following the second 98-bp repeat. There were some differences in the ability of the nuclear factor(s) from the two brain cell lines and HeLa cells to completely protect the nucleotides within the footprint region. The results from the DNase I protective studies and competitive DNA binding studies with specific oligonucleotides, suggest that nuclear factor-1 or a nuclear factor-1-like factor is interacting with all three sites in the JCV regulatory region. In addition, the results suggest that the nuclear factor which interacts with the JCV regulatory region from human brain cell lines is different from the factor found in HeLa cells.     

Multiple interactions between nuclear proteins of Zea mays and the promoter of the Shrunken gene

Summary Nuclear proteins were extracted from isolated nuclei of immature maize kernels. The promoter region (1.5 kb) of the Shrunken gene, which is highly transcribed in the developing endosperm of the kernel, was scanned for protein-DNA interactions. Several promoter fragments showed protein-DNA complex formation in gel retardation experiments. Two different nucleo-protein complexes (MNP1 and MNP2) have been distinguished in competition and DNase I footprinting experiments. Both nuclear DNA-binding activities are able to recognize multiple sites distributed over a 1.5 kb upstream region of the Shrunken gene. Some of the binding sites established in the in vitro reconstitution experiments are located near to DNase I hypersensitive sites found in the promoter of the Shrunken gene (Frommer and Starlinger 1988).     

Characterization of nuclear factors binding to AT-rich element in the rat p53 promoter

In this study, we identified AT-rich element located at positions -504 to -516 in the rat p53 promoter by DNase I foot printing assay. This region was previously identified as a positive regulatory element in the murine p53 promoter and designated as PBF1 (p53 binding factor 1) binding site. However, the proteins binding to this AT-rich element have not been identified yet. Therefore, we characterized the binding protein by various biochemical methods. First, we confirmed that by the oligonucleotide competition assay, nuclear factors bound to the AT-rich element in a sequence-specific manner. Two binding proteins were identified in southwestern blotting analysis and the molecular masses of the proteins were 60 and 40 kDa, respectively. The proteins were stable to denaturants or ionic strength. Treatment of chelators showed that the binding proteins did not require divalent cation for DNA-binding activity. In addition, the binding proteins were labile to protease treatment. This study showed that 60 and 40 kDa proteins bound to AT-rich element and the physico-chemical properties provided new insights into the binding proteins.