Interaction of noncoding RNA with the rDNA promoter mediates recruitment of DNMT3b and silencing of rRNA genes

Abstract

Noncoding RNAs are important components of regulatory networks controlling the epigenetic state of chromatin. We analyzed the role of pRNA (promoterassociated RNA), a noncoding RNA that is complementary to the rDNA promoter, in mediating de novo CpG methylation of rRNA genes (rDNA). We show that pRNA interacts with the target site of the transcription factor TTF-I, forming a DNA:RNA triplex that is specifically recognized by the DNA methyltransferase DNMT3b. The results reveal a compelling new mechanism of RNA-dependent DNA methylation, suggesting that recruitment of DNMT3b by DNA:RNA triplexes may be a common and generally used pathway in epigenetic regulation.Supplemental material is available at http://www.genesdev.org.Received April 28, 2010; revised version accepted August 26, 2010. Noncoding RNAs (ncRNAs) play a profound and complex role in regulating gene expression (Goodrich and Kugel 2009). While the mechanistic details of how RNA and chromatin are connected remain unclear, there is increasing evidence that epigenetic regulation likely represents a balanced interplay of both RNA and chromatin fields (Bernstein and Allis 2005;Mattick 2009). Previous studies have established that ncRNA plays a key role in epigenetic silencing of rRNA genes. Mammalian genomes contain several clusters of tandemly arrayed rRNA genes (rDNA), of which only a subset is transcribed. Silent rDNA repeats are marked by heterochromatic histone modifications and CpG methylation of the rDNA promoter . The establishment and maintenance of the heterochromatic state is mediated by NoRC (Strohner et al. 2001), a chromatin remodeling complex comprising the ATPase SNF2h and a large subunit, termed TIP5 (TTF-Iinteracting protein #5). NoRC silences rRNA genes by recruiting enzymes that mediate heterochromatin formation and silencing (Zhou et al. 2002;Zhou and Grummt 2005). Transcriptional silencing involves DNA methylation at a critical CpG residue (CpG-133) within the upstream control element (UCE) of the rDNA promoter, thereby impairing binding of the transcription factor UBF and abrogating transcription complex formation . Importantly, NoRC function requires the association of TIP5 with RNA that originates from an RNA polymerase I (Pol I) promoter located in the intergenic spacer ;2 kb upstream of the pre-rRNA transcription start site (Mayer et al. 2006). These intergenic transcripts are of low abundance and usually do not accumulate in vivo because they are rapidly degraded or processed into 150-to 250-nucleotide (nt) RNAs that are shielded from degradation by binding to NoRC. These NoRC-associated transcripts are termed pRNA (for promoter-associated RNA), as their sequence matches the rDNA promoter. TIP5 recognizes the secondary structure of pRNA, and the interaction of TIP5 with pRNA changes the structure of both pRNA and NoRC in an induced fit mechanism (Mayer et al. 2008). Antisensemediated depletion of pRNA leads to nucleoplasmic distribution of NoRC, hypomethylation of rDNA, and enhanced Pol I transcription. ''RNA refeeding'' a...

Subnuclear relocalization and silencing of a chromosomal region by an ectopic ribosomal DNA repeat

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Transcription control by long non-coding RNAs

“…This mechanism is similar to the formation of a DNA:RNA triplex by pRNA (promoterassociated RNA) at the rDNA promoter which signals the recruitment of the DNA methyltransferase DNMT3b to silence the transcription of rRNA genes. 59 The pervasive transcription observed near annotated genes 7 might act similarly to generate lncRNAs locally that regulate the assembly of nearby transcription complexes.…”