c‐fos Protooncogene Transcription can be Modulated by Oligonucleotide‐Mediated Formation of Triplex Structures in vitro

Abstract

A homopurine . homopyrimidine sequence of the c:fos promoter was chosen as a target for a triple helix oligonucleotide. Eight DNA oligonucleotides that ranged from 14 to 31 bp were shown to form a triple helix with three sequences within the c-fos promoter region. Reactive derivatives of homopyrimidine oligonucleotides bearing the 5'-or 3'-terminal DNA alkylation aromatic 2-chloroethylamino group were also synthesized. It was concluded, based on the physical properties of the DNA oligonucleotide complex, that the oligonucleotide forms a colinear triplex with the duplex binding sites. We investigated in detail, using electrophoretic mobility and footprinting protection, whether such oligonucleotide . DNA complexes are of benefit in designing high-affinity probes for a natural DNA sequence in the mouse c-fos gene. Our results demonstrate that four different DNA targets within the c-fos promoter region can form triplex structures with synthetic oligonucleotides in a sequence-specific manner. Moreover, in vitro modifications of the retinoblastoma-gene-product-binding site of the cTfos promoter at position -83 in front of the cAMP/cAMP-responsive element binding site and fos-binding site 3/activator-protein-2-like (FBS3/AP-2-like) site at position -431 by triple helix forming oligonucleotides cause dramatic suppression of ,fox-chloramphenicol acetyltransferase activity in endothelial cells. These results provide a basis for the development of a specific oligonucleotide target forming triplex-DNA complex, and emphasize the importance of a target forming triplex as a basis for control of gene expression and cell proliferation.Keywords: cTfos ; triplex ; promoter ; transcription factor ; gene regulation, Intermolecular DNA triple helix formation offers the opportunity to achieve precise sequence recognition, and has a potential use in gene therapy [I, 21. The formation of a specific complex between two strands of polyuridylic acid and one strand of polyadenylic acid in the presence of divalent cations was first reported in 1957 131. Recent studies have revealed the site-specific formation of short intermolecular triplexes by a wide variety of techniques, which include different kinds of footprintings, affinity cleavage, and NMR [4-1 I]. Chemical and physical evidence indicates that the nucleotide bases of the third strand occupy the major groove of the Watson-Crick target duplex [12, 131. A triple helix in nucleic acid results from specific Hoogsteen-type or reverse Hoogsteen-type hydrogen-bonding interactions between bases in a homopurine strand of a Watson-Crick duplex and an additional third strand. Several DNA triplets have been characterized resulting in two distinct classes of structural motifs in DNA triplexes. In the case of pyrimidine . purine . pyrimidine (Y*RY) triplexes (so-called pyrimidine motif), the third pyrimidine-rich strand runs parallel to the purine strand of the target duplex resulting in the formation of T*AT and C+*GC base triple combinations [4, 6, 111. The requirement for protonation of the th...

The Human c-Src Proto-oncogene Promoter Contains Multiple Targets for Triplex-Forming Oligonucleotides

“…During our initial characterization, we noticed that the promoter contained a total of four closely linked homopolypurine/homopolypyrimidine (Pu:Py) tracts ranging in length from 11 to 31 bp. Similar Pu:Py tracts have been reported in the promoter region of a number of genes (Thomas et al, 1995;Olivas and Maher, 1996;Svinarchuk et al, 1996b;Durland et al, 1991;Lavrovsky et al, 1996) and are often required for optimal transcriptional activity (Johnson et al, 1988;Mavrothalassitis et al, 1990;Firulli et al, 1994). Indeed, one of these c-Src Pu:Py tracts spans a critical Sp binding site in the promoter, and two of the other Pu:Py tracts contain the sequence CTTCCT or CTTCCC, which are known to bind certain Ets family members (Yu et al, 1997).…”

“…Such structures formed within the promoter of a target gene could, theoretically, inhibit transcription by several means, such as the inhibition or displacement of critical rrani-acting factors (Reddoch and Miller, 1995) or the inhibition of RNA polymerase elongation (Krasilnikov et al, 1997). A number of groups have now employed such TFOs to target these sequences in a variety of genes, and several have demonstrated significant inhibition of transcription in vitro and in vivo (Giovannangeli et al, 1996;Kim and Miller, 1995;Lavrovsky et al, 1996). This body of work suggests that triplex-mediated gene therapy may have genuine clinical promise.…”

Gene transfer of human heme oxygenase into coronary endothelial cells potentially promotes angiogenesis

“…Several transcriptional motives, such as NF-B, STAT, AP-1, and AP-2, have been shown to activate the HO-1 promoter and upregulate HO-1 gene expression Lavrovsky et al, 1996]. Similar transcriptional factor binding sites have been identified on the promoter region of bFGF and VEGF [Ryuto et al, 1996;Gille et al, 1997], which suggests that physical interaction or competition among these factors may be involved in the activation of several genes, such as HO-1 , c-fos [Lavrovsky et al, 1996], and bFGF. Hypoxia and hypoxia ischemia causes elevation of HO-1 mRNA [Lee et al, 1997;Matz et al, 1996] and several other glycolytic enzyme genes that are hypoxia inducible [Semenza et al, 1994].…”

Triplex-Forming Oligonucleotides Can Modulate Aquaporin-5 Gene Expression in Epithelial Cells

“…The efficient delivery of TFOs to intact cells, and their subsequent translocation to the nucleus, is a practical concern. In the past, TFOs primarily have been most successfully used to selectively inhibit gene transcription in permeabilized cell systems (Frank-Kamenetskii and Mirkin, 1995;Grigoriev et al, 1993;Ing et al, 1993;Lavrovsky et al, 1996;Okada et al, 1994;Orson et al, 1991;Postel et al, 1991;Tu et al, 1995).…”