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
Journal of Biological Chemistry
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
... NA sequences downstream of the site of PNA⅐DNA 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 PNA⅐DNA 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. 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synthetic structural homologues of DNA and RNA in which the entire phosphate-sugar backbone of the polynucleotide has been replaced by a flexible polyamide backbone consisting of 2-aminoethyl glycine units. Each unit is linked to an appropriate purine or pyrimidine base to create the sequence required for hybridization to the targeted nucleic acid (1-3). The absence of phosphate groups in the PNA molecule facilitates its invasion of negatively charged DNA duplexes containing the complementary base sequences (4 -6). Under appropriate conditions, PNAs show greater discrimination and form more stable hybrids with DNA than the corresponding DNA⅐DNA duplexes (1, 4 -8). When a PNA mol-ecule invades a targeted DNA sequence, one strand of the DNA is displaced (1, 5, 6, 9) , whereas the PNA binds quickly to its complementary DNA sequence by Watson-Crick base pairing (7). Studies of homopyrimidine and homopurine PNAs show that this rapid and highly specific association with the complementary DNA strand is followed by the addition of a second PNA molecule to the PNA⅐DNA duplex to form a very stable (PNA) 2 -DNA triplex, while the noncomplementary DNA strand is left in the single-stranded state (8 -10). PNA invasion of DNA duplex strands to form specific and stable PNA⅐DNA hybrids has profound implications for both positive and negative control of transcription. For example, it has been shown that DNA loops displaced as a consequence of PNA binding act as artificial transcription promoters (11), but PNA binding to the transcribed strand of a targeted DNA sequence blocks transcript elongation beyond the site of PNA⅐DNA hybridization (5, (12) (13) (14) . We have tested the effects of PNAs complementary to the CAG triplet repeats that occur in many genes involved in transcriptional control. More than 33 transcription factors, including the human TATA-binding protein (TBP) (15-17) and the androgen receptor (18 -20), are characterized by the presence of polyglutamine tracts containing more than 20 residues encoded by tandem CAG triplet repeats. 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 PNA⅐DNA 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. Moreover, the selectivity of the PNA probe for the CAG triplet repeats was confirmed by the fact that the streptavidinbound chromatin fragments did not contain DNA for the c-myc protooncogene, which is amplified and actively transcribed in the same cells, but lacks a domain of tandem CAG triplets (21). We have now tested the effects of CAG-specific and c-mycspecific PNAs on AR, TBP, and c-myc transcription in human prostatic cancer cell lines, and we now show that each PNA can selectively target its complementary DNA sequence in the intact chromatin of permeabilized 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 PNA⅐DNA hybrid formation is impaired in both directions.