Purine– and pyrimidine–triple-helix-forming oligonucleotides recognize qualitatively different target sites at the ribosomal DNA locus

Abstract

Triplexes are noncanonical DNA structures, which are functionally associated with regulation of gene expression through ncRNA targeting to chromatin. Based on the rules of Hoogsteen base-pairing, polypurine sequences of a duplex can potentially form triplex structures with single-stranded oligonucleotides. Prediction of triplex-forming sequences by bioinformatics analyses have revealed enrichment of potential triplex targeting sites (TTS) at regulatory elements, mainly in promoters and enhancers, suggesting a potential function of RNA-DNA triplexes in transcriptional regulation. Here, we have quantitatively evaluated the potential of different sequences of human and mouse ribosomal RNA genes (rDNA) to form triplexes at different salt and pH conditions. We show by biochemical and biophysical approaches that some of these predicted sequences form triplexes with high affinity, following the canonical rules for triplex formation. We further show that RNA triplex-forming oligos (TFOs) are more stable than their DNA counterpart, and point mutations strongly affect triplex formation. We further show differential sequence requirements of pyrimidine and purine TFO sequences for efficient binding, depending on the G-C content of the TTS. The unexpected sequence specificity, revealing distinct sequence requirements for purine and pyrimidine TFOs, shows that in addition to the Hoogsteen pairing rules, a sequence code and mutations have to be taken into account to predict genomic TTS.

Nucleosomes Stabilize ssRNA-dsDNA Triple Helices in Human Cells

“…To test this hypothesis, we used the En3 TTS that we applied to the nucleosome binding studies and analyzed its functional role in rDNA transcriptional regulation ( Figure 4G). The En3 TFO binds to the regulatory region of the rDNA transcription unit, and we recently showed that only the pyrimidine (Y-RNA_TFO) and not the purine (R-RNA_TFO) En3 TFO forms a stable triplex in vitro (Maldonado et al, 2018). Transfecting the TFO revealed a significant increase in the 47S pre-rRNA expression level with the pyrimidine TFO, whereas the purine TFO that does not bind to this site shows no effect ( Figure 4G).…”

“…To study the functional role of triplexes in chromatin, we used the triplex-forming motif En3 located in the enhancer of mouse rDNA that was previously characterized (Maldonado et al, 2018). Fluorescently labeled DNA templates of 200 bp harboring or lacking the En3 TTS were generated by PCR.…”

“…Genome-wide analyses have predicted millions of potential triplex-forming motifs in mammalian genomes, which are highly enriched at regulatory regions and especially at gene promoters (Buske et al, 2012;Goñ i et al, 2004;Wu et al, 2007). We recently showed that the sequence specificity and binding mode of TFOs is highly dependent on the GC content and perfect complementarity to the TTS, increasing the specificity of triplex binding sites and therefore increasing their regulatory potential by targeting specific ncRNAs (Maldonado et al, 2018).…”

lncRNA PAPAS tethered to the rDNA enhancer recruits hypophosphorylated CHD4/NuRD to repress rRNA synthesis at elevated temperatures

“…Our results show that PAPAS interacts directly with the rDNA enhancer. The murine rDNA enhancer repeats are separated by T stretches of different lengths (Kuhn et al 1990;Maldonado et al 2018). Intriguingly, at active rRNA genes, these repeats wrap around UBF, which allows PAPAS to bind to the T stretches that separate individual enhancer repeats.…”

“…To increase the resolution of the capture assay, we separated the rDNA promoter from the enhancer by digestion of genomic DNA with AccI and SalI, which cleave within the upstream terminator T 0 between the rDNA promoter and the enhancer elements (Grummt et al 1986). This experimental approach revealed that PAPAS comprising nucleotides −275/−336 captured the corresponding region in the rDNA enhancer that contains the TSS-proximal T stretch, termed En3-TFR (Maldonado et al 2018). RNA comprising PAPAS sequences from +5 to −55 did not capture significant amounts of rDNA (Fig.…”