Identification of cellular differentiation-dependent nuclear factors that bind to a human gene for thymidylate synthase.

Three nuclear factors were identified that interact with sequences in the 5'-upstream region of the human thymidylate synthase gene. Two of these factors interact with a sequence around the initiation codon of the thymidylate synthase gene. The amounts of these two factors changed dramatically as human promyelocytic leukemia HL-60 cells differentiated into macrophage-like cells by the treatment with 1,25-dihydroxyvitamin D3. The change was closely correlated with the decrease in the amount of thymidylate synthase mRNA during the differentiation. These findings suggest that the specific nuclear factors are involved in the regulation of the expression of human thymidylate synthase gene during the differentiation of HL-60 cells.     

Identification of DU 145 prostate cancer cell proteins that bind to the carboxy-terminal peptide of human PTHrP in vitro

Peptides spanning the range of human parathyroid hormone-related protein (PTHrP) have been shown to bind heat shock protein-70 expressed on the surface of cancer cells with cytoprotective consequences in vitro. The present study focused on identification of intracellular proteins that interact with the carboxy-terminal peptide of human PTHrP. Using affinity chromatography, we applied extracts of DU 145 prostate cancer cells over PTHrP (140-173)-Sepharose and eluted with 8 M urea. After concentration and electrophoresis, protein bands were excised and subjected to mass spectroscopy analyses. Proteins identified included those associated with protection from oxidative stress, DNA repair, protection from apoptosis, and proteins involved in membrane trafficking and cytoskeletal rearrangement. These novel protein-protein interactions further support the hypothesis that the carboxy-terminus of PTHrP plays a role in cell survival.     

Identification of cell membrane proteins that bind visna virus.

Visna virus infects cells of ovine origin by attaching to a cell surface receptor via its envelope glycoprotein. The identity of the visna virus receptor is not known. To identify the molecule responsible for binding the virus to target cells, virus overlay protein blot assays were used to examine the molecular weights of cell surface molecules which bind purified virus. Molecules on the surface of goat synovial membrane (GSM) cells and sheep choroid plexus (SCP) cells of approximately 15, 30, and 50 kDa bound to visna virus. The binding of visna virus to these proteins was reduced by preincubating virus with neutralizing antibodies. 125I-labeled cell membrane preparations of GSM and SCP cells were used to affinity purify these virus-binding proteins. These proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and had molecular masses of 15, 30, and 50 kDa. Antibodies to the 50-kDa protein bound to the surface of both live SCP and GSM cells in immunofluorescence assays. In addition, antibodies to the 50-kDa protein blocked the binding of [35S]methionine-labeled visna virus to SCP cells in culture. Antibodies raised against the 15- and 30-kDa proteins did not block virus binding to cells. The blocking activity of antibody of the 50-kDa protein provided data that this protein is the molecule which visna virus recognizes and binds to on the surface of target cells.     

Identification of cellular factors that bind specifically to the Epstein-Barr virus origin of DNA replication.

The specific binding of HeLa cell factors to DNA sequences at the Epstein-Barr virus (EBV) latent origin of DNA replication was detected by gel shift experiments and DNase I footprinting analysis. These cellular proteins protected at least five discrete regions of the DNA replication origin. The viral protein required for EBV plasmid replication, EBV nuclear antigen 1 (EBNA-1), binds to specific sequences within the origin region. The HeLa cell proteins competed with EBNA-1 for binding to EBV origin DNA in vitro, leading to the possibility that these cellular proteins regulate EBV DNA replication by displacing EBNA-1 at the origin sites.     

Purification of multiple erythroid cell proteins that bind the promoter of the alpha-globin gene.

Three erythroid cell factors that bind the murine alpha-globin promoter were enriched more than 1,000-fold by conventional and DNA sequence affinity chromatography. Visualization of enriched polypeptides revealed simple patterns suggesting that each binding activity was purified. Two of the purified proteins, alpha-CP1 and alpha-CP2, have been shown previously to interact with distinct binding sites that overlap in the alpha-globin CCAAT box. Affinity purification of alpha-CP1 revealed seven polypeptides with Mrs raging from 27,000 to 38,000. In contrast, purified alpha-CP2 was made up of a polypeptide doublet with Mrs of 64,000 and 66,000. The third purified binding activity, alpha-IRP, interacted with sequences that formed an inverted repeat (IR) between the alpha-globin CCAAT and TATAA boxes. Affinity-purified alpha-IRP was made up of a single polypeptide with an Mr of 85,000. We confirmed that the purified polypeptides corresponded to alpha-CP1-, alpha-CP2-, and alpha-IRP-binding activities by UV cross-linking experiments (alpha-CP2 and alpha-IRP) or by renaturation of binding activity after elution of polypeptides from sodium dodecyl sulfate-polyacrylamide gels (alpha-CP1 and alpha-CP2). The apparent complexity of the polypeptides accounting for alpha-CP1 binding activity prompted a further physical characterization of this factor. Sedimentation of affinity-purified alpha-CP1 in glycerol gradients containing 100 mM KCl showed that all seven polypeptides migrated as a complex that cosedimented with alpha-CP1-binding activity. In contrast, when sedimented in glycerol gradients containing 500 mM KCl, alpha-CP1 dissociated into at least two components. Under these conditions, alpha-CP1-binding activity was reduced or lost. Activity was reconstituted, however, by combining fractions that were enriched in the two components. These results were confirmed by experiments in which we showed that alpha-CP1-binding activity can be recovered only by combining distinct sets of polypeptides that were isolated and renatured from sodium dodecyl sulfate-polyacrylamide gels. Our results suggest that the seven polypeptides visualized after affinity purification of alpha-CP1 interact to form a heterotypic complex (or set of complexes) required for alpha-CP1-binding activity.     

Two tissue-specific factors bind the erythroid promoter of the human porphobilinogen deaminase gene.

We have studied the erythroid-specific promoter of the human gene coding for Porphobilinogen Deaminase (PBGD) by DNaseI footprinting, gel retardation and methylation interference assays. We show that this promoter, which is inducible during MEL cell differentiation, contains three binding sites for the erythroid-specific factor NF-E1 and one site for a second erythroid-specific factor, which we name NF-E2. NF-E1 is a factor that also binds the promoter and the enhancer (present in the 3' flanking region) of the human beta-globin gene. NF-E2 has not yet been described and although it binds to a sequence containing the Ap1 consensus, it appears to be different from Ap1.