Invasion of the CAG Triplet Repeats by a Complementary Peptide Nucleic Acid Inhibits Transcription of the Androgen Receptor and TATA-binding Protein Genes and Correlates with Refolding of an Active Nucleosome Containing a Unique AR Gene Sequence

Lidia C Boffa,Patricia L Morris,Elisabetta M Carpaneto,Marjorie Louissaint,Vincent G Allfrey

doi:10.1074/jbc.271.22.13228

Abstract

The DNA sequence of the genes for the androgen receptor (AR) and TATA-binding protein (TBP), like many other genes encoding transcription factors, contains a series of tandem CAG repeats. Here we explore the capacity of complementary peptide nucleic acids (PNAs) to invade the CAG triplets of the AR and TBP genes in human prostatic cancer cells and show that the PNAs readily entered the nuclei of lysolecithin-permeabilized cells and effectively inhibited sense transcription of unique AR and TBP DNA sequences downstream of the site of PNA.DNA hybridization, but not upstream of that site. These PNAs had little or no effect on transcription of the c-myc gene, which lacks a CAG triplet domain. Conversely, a PNA complementary to a unique sequence of the c-myc gene did not inhibit transcription of the AR or TBP genes but did inhibit c-myc transcription. Comparisons of PNA effects on sense and antisense transcription of the AR, TBP, and c-myc genes confirm that progression of the RNA polymerase complex beyond the site of PNA.DNA hybridization is impaired in both directions. Suppression of the AR gene results in refolding of a transcriptionally active nucleosome containing a unique 17-mer AR DNA sequence.

Highlights

Peptide nucleic acids (PNAs)1 are synthetic structural homologues of DNA and RNA in which the entire phosphate-sugar backbone of the polynucleotide has been replaced by a flexible polyamide backbone consisting of 2-aminoethyl glycine units
We have tested the effects of CAG-specific and c-mycspecific PNAs on androgen receptor (AR), TATA-binding protein (TBP), and c-myc transcription in human prostatic cancer cell lines, and we show that each PNA can selectively target its complementary DNA sequence in the intact chromatin of permeabilized cells
Over 33 transcription factors are characterized by domains of tandem glutamine residues encoded by CAG triplet repeats

Summary

Introduction

Peptide nucleic acids (PNAs) are synthetic structural homologues of DNA and RNA in which the entire phosphate-sugar backbone of the polynucleotide has been replaced by a flexible polyamide backbone consisting of 2-aminoethyl glycine units. We have shown previously that a biotinylated PNA targeted to CAG repeats will strand invade those DNA sequences in their transcriptionally active states in intact chromatin [21]. By combining the use of the biotinylated CAG-specific PNA with techniques that permit a clear separation of transcriptionally active and inactive chromatin restriction fragments [22], it became possible to capture the active chromatin fragments containing the stable PNA1⁄7DNA hybrids on streptavidin-agarose magnetic beads. Those chromatin fragments were shown to contain the transcriptionally active DNA for the TBP of human colonic cancer cells. Detailed comparisons of PNA inhibitions of sense and antisense transcription of the AR, TBP, and c-myc genes confirm that progression of the RNA polymerase complex beyond the site of PNA1⁄7DNA hybrid formation is impaired in both directions

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