Silent pericentromeric repeats speak out.

Pericentromeres are essential structural components of the chromosome that function to ensure proper sister chromatid segregation during cell division. These chromosomal domains are composed of major satellite DNA repeats whose constitutively heterochromatic structure facilitates kinetochore formation (1). Characteristically demarcated by the silencing histone mark trimethyl-histone-3lysine-9, pericentromeres had long been thought to be transcriptionally silent. However, recent reports have demonstrated that transcription from pericentromeric satellite repeats is required for proper chromosome formation during differentiation (2, 3). Aberrant transcription from these regions has also been observed in certain cancers (4, 5). Thus far, the biological relevance of the resulting RNA products has remained a mystery. In PNAS, Bersani et al. (6) and Tanne et al. (7) have now ascribed distinct functional properties to a specific class of noncoding RNAs (ncRNAs) that are produced from pericentromeric satellite repeats in cancer cells. The findings from these complementary studies have broad implications for the role of repeat-derived ncRNAs in cancer development and prognosis. In Bersani et al. (6), the authors build on their previous observations that specific subclasses of satellite repeats are overexpressed in a variety of cancers (5). In the process of establishing model systems to study this phenomenon they discovered that one particular subclass of pericentromeric repeat, HSATII, is highly expressed when cancer cell lines are cultured as 3D tumor spheres. Intriguingly, the authors found that HSATII transcripts are partially susceptible to DNase I digestion and propose that HSATII RNA exists in the cell as a DNase-sensitive DNA:RNA heteroduplex (Fig. 1A). Bersani et al. (6) explore the possibility that HSATII transcripts are reverse-transcribed into DNA:RNA intermediates using DNA: RNA hybrid immunoprecipitation coupled with quantitative PCR. This method takes advantage of the S9.6 monoclonal antibody that has specificity toward DNA:RNA hybrids. The authors observed that introduction of in vitro-transcribed HSATII RNA into 293T cells, which do not express these transcripts endogenously, resulted in the detection of significant levels of HSATII DNA: RNA hybrids. Moreover, HSATII-expressing 3D tumor spheres treated with general reverse transcriptase inhibitors display a significant reduction in endogenous HSATII DNA: RNA hybrids. Altogether, these results provide compelling evidence that HSATII transcripts are indeed reverse-transcribed. The authors provide suggestive evidence that telomerase reverse transcriptase (hTERT) may be involved in the reverse transcription of HSATII RNAs. This is surprising because hTERT typically complexes with a wellcharacterized RNA subunit that serves as a short RNA template and the ability of the enzyme to reverse-transcribe long sequences of nonspecific RNA has not been previously described. Future studies will be required to confirm the exact role of hTERT and/or alternative reverse transcriptases in this process. Reverse transcriptase activity is a hallmark property of retroelements that amplify in the genome through RNA intermediates. Using a combination of quantitative and qualitative assays, Bersani et al. (6) find that HSATII RNA expression is correlated with HSATII DNA copy number gains. These gains are restricted to pericentromeric domains and result in the expansion of existing HSATII loci (Fig. 1A). Blocking reverse transcription of HSATII RNA with reverse transcriptase inhibitors in an in vivo tumor xenograft model resulted in decreased HSATII DNA copy number gains, further supporting the role of HSATII RNA in HSATII repeat expansions. These findings raise a number of important questions. How are HSATII DNA: RNA hybrids converted into double-stranded DNA for integration? What is the enzyme that mediates HSATII integration? Although the authors implicate hTERT in the reverse transcription of HSATII RNA, this enzyme does not have DNA-dependent DNA-polymerase or integrase functionality. The mechanism for RNA-mediated HSATII expansion is likely complex and will be an exciting area for future research. To evaluate potential physiological consequences of these HSATII expansions Bersani et al. (6) used data from The Cancer Genome Atlas (TCGA) project (8). TCGA is a publically Pericentromeric Heterochromatin HSATII HSATII HSATII HSATII HSATII Nucleus Cytoplasm HSATII ncRNA Innate pattern recognition receptor?