Browsing by Author "Currie, R. Alexander"

Now showing 1 - 2 of 2
  • Thumbnail Image
    Item
    Interaction of Oct-1 with TFIIB. Implications for a novel response elicited through the proximal octamer site of the lipoprotein lipase promoter
    (American Society for Biochemistry and Molecular Biology, 1995-08-18) Nakshatri, Harikrishna; Nakshatri, Poornima; Currie, R. Alexander
    The ubiquitous human POU domain protein, Oct-1, and the related B-cell protein, Oct-2, regulate transcription from a variety of eukaryotic genes by binding to a common cis-acting octamer element, 5′-ATTTGCAT-3′. The binding of Oct-1 and Oct-2 to the functionally important lipoprotein lipase (LPL) promoter octamer site was stimulated by the general transcription factor, TFIIB. Comparative analysis of the LPL, histone H2B (H2B), and herpes simplex virus ICPO gene promoter octamer sites revealed that nucleotide sequences within and flanking the octamer sequence determined the degree of TFIIB-mediated stimulation of Oct-1 DNA binding. TFIIB was found to decrease the rate of dissociation of Oct-1 from the LPL octamer site, whereas it increased the rate of association, as well as decreased the rate of dissociation, of Oct-1 from the H2B octamer site. A monoclonal antibody against TFIIB immunoprecipitated a ternary complex containing TFIIB, Oct-1, and the LPL and H2B octamer binding sites. TFIIB did not alter the DNase I footprints generated by Oct-1 on the LPL and H2B promoters. However, Oct-1 prevented TATA-binding protein and TFIIB from footprinting the perfect TATA box sequence located 5′ of the LPL NF-Y binding site. In transfection experiments, transcription from reporters containing the LPL octamer, and either the SV40 or the yeast transcription factor GAL4-dependent enhancers, initiated at a precise position within the octamer sequence. Transcription from reporters containing the H2B octamer and the SV40 enhancer initiated at several positions within and flanking the octamer site, whereas transcription initiated at a precise position within the octamer from reporters with both the H2B octamer and the GAL4-dependent enhancer. These results suggest that octamers and their flanking sequences play an important role in positioning the site of transcription initiation, and that this could be a function of the interaction of Oct-1 with TFIIB.
  • Thumbnail Image
    Item
    Subunit Association and DNA Binding Activity of the Heterotrimeric Transcription Factor NF-Y Is Regulated by Cellular Redox
    (American Society for Biochemistry and Molecular Biology, 1996-11-15) Nakshatri, Harikrishna; Bhat-Nakshatri, Poornima; Currie, R. Alexander
    NF-Y is a heterotrimeric transcription factor that specifically recognizes a CCAAT box motif found in a variety of eukaryotic promoter and enhancer elements. The subunit association and DNA binding properties of the NF-Y complex were examined as a function of redox state using recombinant NF-YA, NF-YB, and NF-YC subunits. Reduction of NF-YB by dithiothreitol (DTT) was essential for reconstitution of specific NF-Y CCAAT box DNA binding activity in vitro. Approximately 30% of the Escherichia coli-derived NF-YB subunit existed as intermolecular disulfide-linked dimers. NF-YB mutants in which the highly conserved cysteine residues at positions 85 and 89 had been converted to serines existed only as monomers and did not require DTT for functional NF-Y DNA binding activity. DTT was required, however, for the functional association of NF-YC with wild-type NF-YB but not with the NF-YB cysteine mutants. The cellular redox factors Ref-1 and adult T-cell leukemia-derived factor stimulated the DNA binding activity of recombinant NF-Y in the absence of DTT. Cells treated with 1-chloro-2,4-dinitrobenzene, an irreversible inhibitor of thioredoxin reductase, exhibited reduced endogenous NF-Y DNA binding activity. Together these results suggest that the cellular redox environment of mammalian cells is an important posttranscriptional regulator of NF-Y subunit association and DNA binding activities.