Structural and functional analyses of DNA bending induced by Sp1 family transcription factors

DNA bending induced by eukaryotic transcription factors may play a direct role in the activation of transcription by bringing together factors bound at non-adjacent sites or facilitate binding of factors involved in the formation of an initiation complex. The ubiquitously expressed zinc-finger transcription factor Sp1 is involved in the regulation of a variety of viral and cellular genes. During the past few years proteins homologous to Sp1 have been described constituting a family of Sp1 transcription factors. We have used gel electrophoretic methods to analyse the extent, location and direction of the DNA bend induced by four different Sp1 family proteins upon binding to a consensus GC box. We found that the Sp1 family proteins induce an asymmetric bend in DNA directed towards the major groove, with a bend centre displaced towards the 3' end of the GC box. The zinc-finger domain was alone responsible for introducing this distortion. The magnitude of the induced bend varied between the different proteins. Construction of a hybrid protein and mutation of the 3' end of the GC box indicated that zinc finger 1 is important both for the magnitude of the bend angle, location of the bend centre and the binding affinity. Transactivation studies of a Sp1-dependent promoter revealed that a 5 bp insertion between the TATA box and the GC box, or inversion of the GC box significantly reduced the promoter activity, indicating that protein-induced bending could be important for promoter activity. However, no stimulatory effect could be observed in cotransfections with the DNA binding domain of Sp1 in Drosophila SL-2 cells, suggesting that the bending activity alone is not sufficient for transactivation.