Triple Helix-forming Oligonucleotide Corresponding to the Polypyrimidine Sequence in the Rat α1(I) Collagen Promoter Specifically Inhibits Factor Binding and Transcription

Abstract

Type I and III fibrillar collagens are the major structural proteins of the extracellular matrix found in various organs including the myocardium. Abnormal and progressive accumulation of fibrillar type I collagen in the interstitial spaces compromises organ function and therefore, the study of transcriptional regulation of this gene and specific targeting of its expression is of major interest. Transient transfection of adult cardiac fibroblasts indicate that the polypurine-polypyrimidine sequence of ␣1(I) collagen promoter between nucleotides ؊200 and ؊140 represents an overall positive regulatory element. DNase I footprinting and electrophoretic mobility shift assays suggest that multiple factors bind to different elements of this promoter region. We further demonstrate that the unique polypyrimidine sequence between ؊172 and ؊138 of the promoter represents a suitable target for a single-stranded polypurine oligonucleotide (TFO) to form a triple helix DNA structure. Modified electrophoretic mobility shift assays show that this TFO specifically inhibits the protein-DNA interaction within the target region. In vitro transcription assays and transient transfection experiments demonstrate that the transcriptional activity of the promoter is inhibited by this oligonucleotide. We propose that TFOs represent a therapeutic potential to specifically influence the expression of ␣1(I) collagen gene in various disease states where abnormal type I collagen accumulation is known to occur.In response to tissue injury or invasion, a healing response is invoked that ultimately leads to an accumulation of fibrillar type I collagen. This is true for many systemic organs and the heart. Such a healing response, when unabated and invoked in the absence of injury, leads to a progressive interstitial fibrosis that proves pathologic. Parenchymal cell function is compromised by a disproportionate concentration of type I collagen, a characteristic feature of interstitial fibrosis in different organs (1-9). Various stages of organ dysfunction are marked by the activation and repression of type I collagen gene, thereby allowing for the design of specific agents to promote the necessary or adaptive phenotype or to repress the onset of pathologic interstitial fibrosis.A wide array of hormones, cytokines, and growth factors have been implicated in the mediation of fibrous tissue formation (10 -20). Many of these factors mediate their action through transcriptional mechanisms. Therefore, the study of transcriptional regulatory elements within the ␣1(I) and ␣2(I) collagen gene promoters and their trans-acting protein factors is of major interest. Effector cells that bring about fibrosis include interstitial fibroblasts and phenotypically transformed fibroblast-like cells termed myofibroblasts (21).Several cis-acting elements in the ␣1(I) and ␣2(I) collagen genes located on both sides of the transcription start site as well as their trans-acting factors have been identified (for recent reviews, see . Very little is known about the factor(s) bi...

Activation of Fibroblast Procollagen α1(I) Transcription by Mechanical Strain Is Transforming Growth Factor-β-dependent and Involves Increased Binding of CCAAT-binding Factor (CBF/NF-Y) at the Proximal Promoter

“…A repressor element, known to bind cKrox (60) or BFCOL-1 (61) under basal conditions, has been mapped to the sequence spanning the junction between regions in the mouse gene equivalent to Regions B and C. However, the role of this element in regulating COL1A1 transcription during cellular stress and in response to TGF-␤ has not been evaluated. Furthermore, in rat cardiac fibroblasts a strong positive element has been mapped to Region B under basal conditions, and an upstream repressor has been suggested to counteract the effect of this positive element (62). Our data agree with this because the reported repressor element would be disrupted in the Ϫ197 construct and give rise to high basal levels.…”

Accessibility of nuclear DNA to triplex-forming oligonucleotides: The integrated HIV-1 provirus as a target

“…The next step was to extend these data to experiments with chromosomally located genes in mammalian cells. There have been several reports describing inhibition of gene expression in eukaryotic cells using triplex-forming oligonucleotides to target endogenous genes (12)(13)(14)(15)(16)(17). However no direct evidence was provided that this inhibition was due to triplex formation rather than any other oligonucleotide-directed mechanism.…”

“…There have been several reports describing the use of triplex-forming oligonucleotides to inhibit the expression of endogenous genes (12)(13)(14)(15)(16)(17). However, none of these reports provided direct evidence for the implication of triplex formation at the target site in the inhibitory activity.…”

Sticky DNA, a Self-associated Complex Formed at Long GAA·TTC Repeats in Intron 1 of the Frataxin Gene, Inhibits Transcription

“…Two mechanisms have been proposed to explain the transcriptional inhibition by triplexes. Formation of a triplex may block the transcription elongation by RNA polymerase (29 -31) or may interfere with the binding of transacting factors to sequences that control transcription (32)(33)(34). Recently, Grabczyk and Usdin proposed another model, in which an RNA polymerase advancing within a long GAA⅐TTC repeat causes the transient formation of an R⅐R⅐Y type of triplex, which then creates a pause site (35).…”