Elements at the 5′ end ofXistharbor SPEN-independent transcriptional antiterminator activity

• DOI: 10.1101/2020.05.13.090506
• Publication Date: 2020-05-14T14:45:51Z
• Authors: Jackson B. Trotman; David M. Lee; Rachel E. Cherney; Sue O. Kim; Kaoru Inoue; Megan D. Schertzer; Steven R. Bischoff; Dale O. Cowley; J. Mauro Calabrese
• Keywords: 0301 basic medicine; X Chromosome; Transcription, Genetic; Gene Expression Regulation, Developmental; RNA-Binding Proteins; Mouse Embryonic Stem Cells; Polyadenylation; Chromatin; DNA-Binding Proteins; Mice; 03 medical and health sciences; X Chromosome Inactivation; RNA and RNA-protein complexes; Animals; RNA, Long Noncoding; Gene Silencing; Repetitive Sequences, Nucleic Acid

AbstractTheXistlncRNA requires Repeat A, a conserved RNA element located in its 5′ end, to induce gene silencing during X-chromosome inactivation. Intriguingly, Repeat A is also required for the production ofXist. While silencing by Repeat A requires the protein SPEN, how Repeat A promotesXistproduction remains unclear. We report that in mouse embryonic stem cells, expression of a transgene comprising the first two kilobases ofXist(Xist-2kb) causes transcriptional readthrough of multiple downstream polyadenylation sequences. Readthrough required Repeat A and the ~750 nucleotides downstream but did not require SPEN. Despite associating with SPEN and chromatin,Xist-2kb did not robustly silence transcription, whereas a transgene comprisingXist’s first 5.5 kilobases robustly silenced transcription and read through its polyadenylation sequence. Longer, splicedXisttransgenes also induced robust silencing yet terminated efficiently. Thus, in contexts examined here,Xistrequires sequence elements beyond its first two kilobases to robustly silence transcription, and the 5′ end ofXistharbors SPEN-independent transcriptional antiterminator activity that can repress proximal cleavage and polyadenylation. In endogenous contexts, this antiterminator activity may help produce full-lengthXistRNA while rendering theXistlocus resistant to silencing by the same repressive complexes that the lncRNA recruits to other genes.