Unusual Sp1-GC box interaction in a parvovirus promoter

Abstract

The P4 promoter of the parvovirus minute virus of mice contains a single degenerate GC box sequence which binds the transcription factor Spl with high affinity. The two protomers of murine Spl were affinity purified, and their interactions with the P4 promoter were examined. Several unusual features were observed. Methylation interference experiments demonstrated that Spl makes contacts with both DNA strands, including the central guanine as well as an adenine residue on the cytidine-rich strand of the GC box. UV photocrosslinking revealed that the 95and the 105-kDa protomers cross-link exclusively to opposite strands of the GC box. These results suggest that the phosphorylation of the 95-kDa Spl protomer results in a change in the way Spl is positioned on the P4 GC box and identifies a high-affinity GC box motif.

ras oncogene-dependent activation of the P4 promoter of minute virus of mice through a proximal P4 element interacting with the Ets family of transcription factors

“…Cooperation of the EBS and GC box in promoter P4 activity. In agreement with previous reports (1,22,49), disruption of the GC box of promoter P4 led to a reduction of transcriptional activity, arguing for the involvement of Sp1-like factors in P4 up-regulation. This possibility was confirmed by showing that exogenous Sp1 supplied to Sp1-free D. melanogaster SL2 cells increased their capacity for sustaining P4-driven gene expression.…”

“…3 (lane 6), a monoclonal antibody directed against Sp1 (IC6) specifically enhanced retardation of complex C2, indicating the involvement of Sp1 in this complex. In agreement with a previous report (49), purified human Sp1 protein was found to associate with the GC-P4 probe, giving rise to a complex that comigrated with C2 (Fig. 3, lane 9) and was supershifted by the monoclonal anti-Sp1 antibody IC6 (Fig.…”

“…The activity of the P4 promoter of MVMp is regulated by a number of DNA elements located upstream from the transcription start site (1,22,27,47). In particular, a proximal GC box that constitutes a high-affinity binding site for Sp1-like proteins proved to be essential for P4 promoter activity (1,49). In the present work, an EBS was identified immediately upstream from this GC box.…”

“…The MVMp GC box interacts with Sp1 and Sp3 proteins. The GC box of the MVMp P4 promoter has high sequence homology to the consensus binding site for the Sp1 family of transcription factors (30,35,36) and has been found to constitute a high-affinity binding site for Sp1-like proteins (1,49). To identify the Sp1 family members that interact with the GC box of promoter P4 in FREJ4 cells, EMSAs were performed ( Fig.…”

“…At least a fraction of these elements is typically found within a 50-to 200-nt-long region located upstream from the transcription start site (43), as was shown for the P4 promoter (1,22). Various regulatory motifs, in particular proximal GC and TATA boxes (1,49), upstream E and Y boxes (27), and cyclic AMP (cAMP) response elements (47), have been reported to contribute to P4 promoter activity. The present study aimed at analyzing another putative P4 promoter element that is located immediately upstream from the GC box and consists of the sequence 5Ј-AAGGAAG-3Ј.…”