Previous work identified four upstream cis-acting elements required for tissue-specific expression of the a-fetoprotein (AFP) gene: three distal enhancers and a promoter. To further define the role of the promoter in regulating AFP gene expression, segments of the region were tested for the ability to direct transcription of a reporter gene in transient expression assay. Experiments showed that the region within 250 base pairs of the start of transcription was sufficient to confer liver-specific transcription. DNase I footprinting and band shift assays indicated that the region between -130 and -100 was recognized by two factors, one of which was highly sequence specific and found only in hepatoma cells. Competition assays suggested that the liver-specific binding activity was HNF-1, previously identified by its binding to other liver-specific promoters. Mutation of the HNF-1 recognition site at -120 resulted in a significant reduction in transcription in transfection assays, suggesting a biological role for HNF-1 in the regulation of AFP expression.The restriction of gene expression to specific tissues in a multicellular organism has been attributed to the interaction between cis-acting DNA elements and tissue-specific transcription factors (16,22,24,26,31,45). cis-Acting elements have been identified close to genes as part of promoters and at varying distances from the gene as part of enhancers (2,11,32). In several instances, the same tissue-specific cisacting element has been found in both positions, as is the case with the B-cell-specific octamer motif found in the promoter of the immunoglobulin light-chain gene and in the enhancer of the heavy-chain gene (26,38,40,46). On the other hand, there are several promoter elements that have yet to be identified in enhancers. For example, recognition sites for the ubiquitous transcription factors Spl and NF-1 appear to be restricted to the region near the start of transcription (3, 21).The regulatory sequences of genes expressed in liver have been intensively studied as a model for tissue-specific gene expression. These studies have resulted in the identification of several trans-acting factors that appear to be present at higher levels in liver than in other cell types (1,16,22,39). The protein C/EBP, which was originally identified in rat liver extracts on the basis of its ability to bind the promoter of the herpes simplex virus (HSV) thymidine kinase (TK) gene and the enhancer of the Moloney leukemia virus (15), has since been shown to trans-activate the liver-specific albumin gene by interactions with both its promoter and enhancer (19,25). Darnell and colleagues have identified two liver-enriched trans-acting proteins, HNF-3 and HNF-4, that bind to both enhancers and promoters of the transthyretin and al-antitrypsin genes (5). In addition, the binding sites for another liver-specific trans-acting protein, HNF-1, appear to be restricted to the promoter regions of a large number of liver-specific genes, including the rat at-fetoprotein (AFP) gene (6, 7).We wishe...

Retroviruses contain three genes for replication: gag, pol, and env, which encode polyprotein precursors for the internal capsid proteins, reverse transcriptase, and envelope glycoprotein, respectively (1). Murine leukemia virus (MuLV) differs from most other retroviruses in that it encodes two different pathways for gag gene expression (2). These two pathways begin with two independent translation products, gPr80gag and Pr65ag. Pr65gag is processed by proteolytic cleavage to yield the internal capsid proteins of the virus particle and is analogous to the gag polyprotein precursors of other retroviruses (3-5). gPr8Ogg contains the amino acid sequences of Pr65gag as well as 4-6 kilodaltons (kDa) of amino-terminal protein (6, 7). The additional amino-terminal peptides result in glycosylation of gPr8O'ag during translation (8, 9). gPr8 gag is processed by the further addition of carbohydrate and exported to the cell surface where it appears as a glycoprotein of 95 kDa (8-11). It may be also be released into the extracellular medium as cleavage products of 55 and 40 kDa (8,12). Glycosylated gag products are not incorporated into MuLV virions, but they associate with the extracellular matrix (13).While the function of Pr65gag is known, the role of gPr809ae in MuLV infection is not known. Glycosylated gag might provide some function required for viral replication, or it might play an accessory role. We previously isolated mutants of Moloney MuLV (M-MuLV)-infected mouse fibroblasts that did not express gag proteins at the cell surface, and they were deficient for virus production. However, these mutants were cellular, not viral, in nature and they produced normal amounts of gPr8g'ag intracellularly (14). To obtain a more definitive answer, we constructed two mutants of M-MuLV at the recombinant DNA level and recovered virus by transfection. The constructions. and characterizations of the mutant viruses are described here.MATERIALS AND METHODS Cells and Viruses. All cells were grown in Dulbecco-modified Eagle's medium/10% calf serum; Mouse NIH-3T3 cells were described previously (15), as were M-MuLV-infected NIH-3T3 cells (clone A9) (16).The UV-XC assay for MuLV was carried out as described (17). Assay of viral reverse transcriptase by the addition of exogenous poly(rA):oligo(dT) template-primer (16) and banding of virus in sucrose density gradients (18) was carried out as described.Recombinant DNA Cloning. A phage recombinant DNA clones (A-MLV clones 48, 61, and 63) carrying integrated copies of M-MuLV provirus were described previously (19). pMSV-1 is a plasmid clone of unintegrated Moloney murine sarcoma virus (M-MSV) DNA permuted about the unique HindIII site (20). pSLT is a subclone of pMSV-1 deleted from the Sal I site in M-MSV DNA to the Sal I site in the pBR322.vector (see Fig. 2) and was kindly provided by Inder Verma. P90(Abl), a plasmid clone of unintegrated Abelson MuLV (Ab-MuLV) (P90 strain) DNA, was kindly provided by Owen Witte. Plasmid vector pBR328 has been described by Soberon et al. (21).Restriction ...

Aberrant expression of the ␣-fetoprotein (AFP) gene is a diagnostic tumor marker of hepatocellular carcinoma. We find that AFP gene expression is repressed by the TP53 family member p73 during normal hepatic development and when p73␣ or p73␤ is introduced into cultured hepatoma cells that express AFP. Transient co-transfection of p53 family members showed that p53 and transactivating (TA)-p73, but not TA-p63, repress endogenous AFP transcription additively or independently. p53-independent functions of p73 are further supported by delayed, p73-associated compensation of AFP repression during development of the p53-null mouse. Chromatin immunoprecipitation assays of normal and p53-null mouse liver tissue showed that TA-p73 binds at a previously identified p53 repressor site (؊860/؊830) within the distal promoter of AFP at a level equivalent to p53 in wild type liver, with increased binding of TA-p73 to chromatin in the absence of p53. Sequential chromatin immunoprecipitation analyses revealed that TA-p73 and p53 bind simultaneously to their shared regulatory site in wild type liver. Like the founding family member p53, TA-p73 represses AFP expression by chromatin structure alteration, targeting reduction of acetylated histone H3 lysine 9 and increased dimethylated histone H3 lysine 9 levels. However, chromatin-bound TA-p73 is associated with elevated di-and tri-methylated histone H3 lysine 4 levels in p53-null liver and hepatoma cells, concomitant with a reduced ability to repress transcription compared with p53.The p53 superfamily encompasses a number of p53-, p63-, and p73-specific isoforms, defined by their conserved domains and structural homology (1-3). Alternative splicing and/or divergent promoter usage can lead to considerable diversity in the composition of functional domains and regulatory motifs among specific family members. The most striking structural differences between p53 and p63/p73 include the extended C termini of full-length, transactivating (TA) 4 isoforms, and the truncated N termini of the ⌬N isoforms, of p63 and p73 (1). The TA isoforms of p63/p73 activate the transcription of specific, p53-regulated genes with functions in cell cycle arrest and apoptosis, as well as regulate other genes in a p53-independent manner. Dominant negative forms of p53, which lack DNA binding ability, may form heteromeric complexes with p73 or p63 (4) and interfere with p63/p73 function (5, 6). Although mutations of p63/p73 are not frequently associated with tumorigenesis (7,8), correlations between aberrantly high levels of ⌬N isoform expression and specific tumor formation have been reported (8, 9). Developmentally, ⌬N-p73 plays an essential role in protection of embryonic neurons from p53-mediated cell death (10). These studies and the observed requirement for p63/p73 in p53-induced apoptosis in E1A-expressing mouse embryo fibroblasts suggest an intricate network of influence among the p53 family members (11). The phenotypes of mice genetically engineered for the loss of the individual genes encoding p53, p6...