The mechanisms and biological roles of Polycomb repressive complex (PRC) recruitment by long noncoding RNAs (lncRNAs) remain unclear. To gain insight, we expressed two lncRNAs that recruit PRCs to multi-megabase domains, Airn and Xist, from an ectopic locus in mouse stem cells and compared effects. Unexpectedly, ectopic Airn recruited PRC1 and PRC2 to chromatin with a potency resembling Xist yet did not repress genes. Compared with PRC2, PRC1 was more proximal to Airn and Xist, where its enrichment over C-rich elements required the RNA-binding protein HNRNPK. Fusing Airn to Repeat A, the domain required for gene silencing by Xist, enabled gene silencing and altered local patterns but not relative levels of PRC-directed modifications. Our data suggest that, endogenously, Airn recruits PRCs to maintain rather than initiate gene silencing, that PRC recruitment occurs independently of Repeat A, and that protein-bridged interactions, not direct RNA contacts, underlie PRC recruitment by Airn, Xist, and other lncRNAs.

We report that when expressed at similar levels from an isogenic locus, the Airn lncRNA induces Polycomb deposition with a potency that rivals Xist . However, when subject to the same degree of promoter activation, Xist is more abundant and more potent than Airn . Our data definitively demonstrate that the Airn lncRNA is functional and suggest that Xist achieved extreme potency in part by evolving mechanisms to promote its own abundance.

AbstractWe report that when expressed at similar levels from an isogenic locus, theAirnlncRNA induces Polycomb deposition with a potency that rivalsXist. However, when subject to the same degree of promoter activation,Xistis more abundant and more potent thanAirn. Our data definitively demonstrate that theAirnlncRNA is functional and suggest thatXistachieved extreme potency in part by evolving mechanisms to promote its own abundance.

Vibert et al. (2022) demonstrate that oncogenic transcription factor fusion proteins bind otherwise silent genomic regions, producing RNAs that can be spliced, exported, and translated. These "neogenes'' represent possible targets for immunotherapy and may even be universal byproducts of altered transcription in cancer.

Abstract Dysregulation of DNA methylation and mRNA alternative cleavage and polyadenylation (APA) are both prevalent in cancer, but they have been studied as independent processes. We discovered a DNA methylation regulated APA mechanism when we compared genome-wide DNA methylation and polyadenylation site usage between DNA methylation-competent and DNA methylation-deficient cells. Here we show that removal of DNA methylation enables CTCF binding and recruitment of the cohesin complex, which in turn, promotes proximal polyadenylation site usage. In this DNA de-methylated context, depletion of the RAD21 cohesin complex component can recover distal polyadenylation site usage. We also confirmed, using chromosomal conformation capture (3C), that CTCF and the cohesin complex mediate chromatin loops in the absence of DNA methylation to modulate polyadenylation site usage. Leveraging data from The Cancer Genome Atlas, we authenticated the relationship between DNA methylation and mRNA polyadenylation isoform expression in vivo. This DNA methylation regulated APA mechanism demonstrates how aberrant DNA methylation impacts transcriptome diversity and highlights the potential sequelae of global DNA methylation inhibition as a cancer treatment. Citation Format: Angela H. Ting, Vishal Nanavaty, Elizabeth Abrash, Byron Lee, Emily Fink, Tae Hyun Hwang, Changjin Hong. DNA methylation at the intersect of chromatin structure and transcriptome diversity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1081.

Dysregulation of DNA methylation and mRNA alternative cleavage and polyadenylation (APA) are both prevalent in cancer and have been studied as independent processes. We discovered a DNA methylation-regulated APA mechanism when we compared genome-wide DNA methylation and polyadenylation site usage between DNA methylation-competent and DNA methylation-deficient cells. Here, we show that removal of DNA methylation enables CTCF binding and recruitment of the cohesin complex, which, in turn, form chromatin loops that promote proximal polyadenylation site usage. In this DNA demethylated context, either deletion of the CTCF binding site or depletion of RAD21 cohesin complex protein can recover distal polyadenylation site usage. Using data from The Cancer Genome Atlas, we authenticated the relationship between DNA methylation and mRNA polyadenylation isoform expression in vivo. This DNA methylation-regulated APA mechanism demonstrates how aberrant DNA methylation impacts transcriptome diversity and highlights the potential sequelae of global DNA methylation inhibition as a cancer treatment.