TERRA RNA antagonizes ATRX and protects telomeres

Through an integration of genomic and proteomic approaches to advance understanding of long noncoding RNAs, we investigate the function of the telomeric transcript, TERRA. By identifying thousands of TERRA target sites in the mouse genome, we demonstrate that TERRA can bind both in cis to telomeres and in trans to genic targets. We then define a large network of interacting proteins, including epigenetic factors, telomeric proteins, and the RNA helicase, ATRX. TERRA and ATRX share hundreds of target genes and are functionally antagonistic at these loci: whereas TERRA activates, ATRX represses gene expression. At telomeres, TERRA competes with telomeric DNA for ATRX binding, suppresses ATRX localization, and ensures telomeric stability. Depleting TERRA increases telomerase activity and induces telomeric pathologies, including formation of telomere-induced DNA damage foci and loss or duplication of telomeric sequences. We conclude that TERRA functions as an epigenomic modulator in trans and as an essential regulator of telomeres in cis. We used CHIRT-seq to identify of TERRA interacting chromatin. We used “CHIRT,” a hybrid ChIRP and CHART method optimized for TERRA capture. Several capture probe sets were designed: (i) TERRA antisense (TERRA-AS), to capture transcripts containing UUAGGG, and (ii) TERRA sense (TERRA-S, the reverse complement), to control for strand-specificity and further rule out DNA capture artifacts due to probe hybridization to genomic DNA rather than the intended RNA target. We sequenced two critical controls: (i) an RNaseH- control in which RNaseH was omitted in the elution step, which should preclude elution of RNA-dependent interactions; and (ii) a TERRA-S control, which would not hybridize to TERRA RNA but could pull down genomic DNA. We analyzed RNA-seq after TERRA depletion using single-stranded antisense oligonucleotides (ASO) with locked nucleic acids (LNA) chemistry.