DNA looping between sites for transcriptional activation: self-association of DNA-bound Sp1.

Abstract

The Spl protein activates transcription from many eukaryotic promoters. Spl can act in vivo from enhancer sites that are distal to the promoter and exhibit synergistic interaction with promoter-proximal binding sites. To investigate possible protein-protein interactions between DNA-bound Spl molecules, we have used electron microscopy to visualize the DNA-protein complexes. At the SV40 promoter, we observed the expected localized interaction at the Spl sites; in addition, we found that DNA-bound Spl served to associate two or more DNA molecules. At a modified thymidine kinase promoter, we observed a localized interaction at each of two binding locations that were separated by 1.8 kbp; in addition, we noted a substantial fraction of DNA molecules in which the distant binding regions were joined by a DNA loop. As judged by studies with mutant Spl proteins, the distant interactions depended on the glutamine-rich regions of Spl required for transcriptional activation. We conclude that DNA-bound Spl can self-associate, bringing together distant DNA segments. From the correlation between DNA looping in vitro and synergistic activation of the modified thymidine kinase promoter shown previously in vivo, we suggest that Spl exerts its transcriptional synergism by a direct protein-protein association that loops the intervening DNA. Our experiments support the DNA-looping model for the function of transcriptional enhancers.[Key Words: Spl protein; DNA looping; DNA binding; transcription]Received December 26, 1990; revised version accepted January 23, 1991.The controlled initiation of transcription in prokaryotes and eukaryotes depends on the action of regulatory proteins from sites that are too distant for a direct interaction with promoter-bound proteins on linear DNA. Three principal models have been proposed for positive regulation from distant (enhancer) sites (Dynan and Tjian 1985;Echols 1986;Ptashne 1986). In the first, the regulatory protein (or RNA polymerase) associates with the DNA at the enhancer site and then traverses the DNA to the promoter site (scanning model). In the second, the enhancer-binding protein initiates a change in DNA structure that is propagated from the enhancer to the promoter, thereby activating transcription (structural transmission model). In the third, the enhancerbound regulatory protein activates transcription by a direct protein-protein interaction with RNA polymerase at the promoter (or other proteins that contact polymerase) (DNA-looping or nucleoprotein model).DNA looping is currently favored as the most likely mechanism for distant regulatory interactions. The interaction between DNA-bound proteins has been firmly established as the central structural feature for regulated 3Present address:

DNA looping by Ku and the DNA-dependent protein kinase

“…DNA looping is a well established phenomenon that may play a role in the regulation of transcription, DNA replication, and recombination (62,63). A common example is loop formation by the self-association of transcription factors bound at two distant sites (56)(57)(58). The looping mediated by Ku, however, does not appear to be localized to specific binding sites because the two unrelated DNA sequences examined here both display Ku-associated loops.…”

“…DNA loops were formed in the presence of Ku at a frequency of Ϸ10-15%, a relatively high percentage for in vitro DNA looping (e.g., compare with refs. [56][57][58]. No obvious sequence specificity for the looping effect was detected; loops formed in an apparently random distribution along the length of pUC19 DNA as well as a short, unrelated sequence.…”

Mutations in the promoter reveal a cause for the reduced expression of the human manganese superoxide dismutase gene in cancer cells

“…SP-1 activation depends on three Zn-®nger structures responsible for DNA binding and at least one of two glutamine-rich regions that are required for transcription activation (Kadonaga et al, 1988;Courey and Tjian, 1988). Contact between SP-1 proteins at nonadjacent sites on a short DNA fragment causes the formation of extrastructures, such as DNA-looping (Su et al, 1991) or DNA-bending (Ikeda et al, 1993). The human MnSOD promoter is composed of multiple SP-1 and AP-2 binding sites on a 200 bp DNA fragment.…”

Biological consequences of tightly bent DNA: The other life of a macromolecular celebrity

“…Though there are a host of interesting examples of transcriptional regulation that involve DNA looping, we focus almost exclusively on the dissection of the role of looping in 85 Repression and activation 210 Gal repressor 85 Repression 115 Deo repressor 85 Repression 270, 599, 869 Nag repressor 86 Repression 93 NtrC 87 Activation 110-140 l repressor 84,88 Repression and activation $2400 XylR 89 Activation $150 PapI 87,90 Activation $100 -globin locus 91,92 Activation 40,000-60,000 RXR 93 Activation 30-500 SpGCF1 94 Activation, domain intercommunication 50-2500 HSTF 95 Activation 23 p53 96 Repression and activation 50-3000 Sp1 [97][98][99] Activation $1,800 c-Myb and C/EBP 100 Activation $80…”