Enhancement of transcription factor, USF, binding to the adenovirus major late promoter: effect of dithiothreitol and high mobility group protein-1

• Published in: Biochimica et biophysica acta Vol. 1395; no. 2; pp. 228 - 236
• Main Authors: Marmillot, Philippe, Scovell, William
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 21-01-1998
• Subjects: Adenoviridae - genetics; Bivalent homotetramer USF protein; Dithiothreitol - pharmacology; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; E-box/Inr; High Mobility Group Proteins - metabolism; High Mobility Group Proteins - pharmacology; Humans; Oxidation-Reduction; Promoter Regions, Genetic; Recombinant Fusion Proteins - metabolism; Transcription Factors - genetics; Transcription Factors - metabolism; Upstream Stimulatory Factors; Viral Proteins; Bivalent homotetramer USF protein; Ad MLP, adenovirus-2 major late promoter; Inr, initiator sequence; E-box/Inr; PIC, preinitiation complex; USF, up-stream stimulatory factor; HMG-1, high mobility group protein-1; PMSF, phenylmethylsulfonylfluoride; TBP, TATA-binding protein; EMSA, electrophoretic mobility shift assay; b/HLH/Z protein, basic helix-loop-helix leucine zipper protein; DTT, dithiothreitol
• ISSN: 0167-4781; 0006-3002; 1879-2634
• DOI: 10.1016/S0167-4781(97)00153-X

Up-stream stimulatory factor (USF)
1 is a human transcription factor which binds specifically to the E-box in the Ad MLP located at −58 from the start site. The nature of USF binding on a Ad MLP DNA fragment was investigated in the presence of DTT and also in the presence of purified HMG-1 using electrophoretic mobility shift assay. We show that the binding capacity of USF for the E-box increases significantly with increasing DTT concentrations. At the higher DTT levels, a second USF–DNA complex is formed in which there is co-occupation of both the E-box and the initiator sequence. The stability of the second complex is largely refractory to an excess of unlabeled oligonucleotide which contains the initiator sequence. These findings indicate a cooperative binding interaction between USF ligands bound simultaneously at the E-box and the Inr sequence. Two models are proposed which are consistent with these data. Furthermore, experiments indicate that the presence of HMG-1, a nuclear protein known to influence transcriptional activity, increases USF binding activity at the E-box by as much as 100%. These findings indicate that both reducing conditions and HMG-1 may act as modulators of USF-regulated transcription.