Martin, M.; Jalife, C.; Mendez, C.; Montecinos, C.; Brown-Brown, D.; Diaz-Tejeda, F.; Caffi, V.; Zavala, K.; Kugler, F.; Molina-Gonzalez, L.; Valenzuela-Nieto, G.; Laengst, G.; Mardones, G. A.; Munita, R.; Opazo, J. C.; Maldonado, R.

MicroRNAs (miRNAs) are known for their roles in post-transcriptional gene regulation in the cytoplasm. However, increasing evidence indicates that a subset of miRNAs localizes to the nucleus and associates with chromatin. Here, we systematically characterize the chromatin-associated small non-coding RNA landscape of the human pancreatic cancer cell line PANC-1. Using chromatin RNA immunoprecipitation followed by small RNA sequencing, we show that chromatin-associated small RNAs are distinct from the bulk nuclear population and are predominantly composed of miRNAs. Among these, miR-21 emerges as the most abundant chromatin-associated type. We identify a defined subset of chromatin-associated miRNAs that fulfill sequence and genomic context requirements for RNA-DNA triplex formation and preferentially localize to regulatory genomic elements. Biochemical analyses demonstrate that Argonaute 2 (Ago2) directly interacts with RNA-DNA triple helical structures in vitro, with contributions from both its N-terminal and PIWI domains, suggesting a mechanistic link between miRNAs, Ago2, and chromatin engagement. Evolutionary sequence conservation and phyletic distribution analyses reveal that triplex-forming chromatin-associated miRNAs are largely restricted to anthropoid primates, in contrast to non-triplex-forming miRNAs, which display broad conservation across jawed vertebrates. These findings indicate that triplex-mediated miRNA-chromatin interactions represent an evolutionarily recent regulatory innovation. Together, our results uncover a previously unappreciated layer of miRNA function at the chromatin level, in which a subset of miRNAs engages Ago2 via RNA-DNA triplex formation, potentially modulating transcriptional programs in a lineage-specific manner.