Tsix transcription- versus RNA-based mechanisms in Xist repression and epigenetic choice

Recent inquiries have revealed a surprisingly large number (>2500) of naturally occurring antisense transcripts [1-4], but their function remains largely undiscovered. A better understanding of antisense mechanisms is clearly needed because of their potentially diverse roles in gene regulation and disease [5-8]. A well-documented case occurs in X inactivation, the mechanism by which X-linked gene expression is equalized between XX females and XY males [9]. The antisense gene Tsix [6] determines X chromosome choice and represses the noncoding silencer, Xist [10-12]. In principle, Tsix action may involve RNA, the act of transcription, or local chromatin. Here, we create novel Tsix alleles to distinguish transcription-versus RNA-based mechanisms. When Tsix transcription is terminated before Xist (Tsix(TRAP)), Tsix cannot block Xist upregulation, suggesting the importance of overlapping antisense transcription. To separate the act of transcription from RNA, we knocked in Tsix cDNA in the reverse orientation (Tsix(cDNA)) to restore RNA levels in cis without concurrent transcription across Xist. However, Tsix(cDNA) cannot complement Tsix(TRAP). Surprisingly, both mutations disrupt choice, indicating that this epigenetic step requires transcription. We conclude that the processed antisense RNA does not act alone and that Tsix function specifically requires antiparallel transcription through Xist. A mechanism of transcription-based feedback regulation is proposed.