SummaryWe double-tagged Xist (inactivated X chromosome-specific transcript), a prototype long non-coding RNA pivotal for X chromosome inactivation (XCI), using the programmable RNA sequence binding domain of Pumilio protein, one tag for live-cell imaging and the other replacing A-repeat (a critical domain of Xist) to generate “ΔA mutant” and to tether effector proteins for dissecting Xist functionality. Based on the observation in live cells that the induced XCI in undifferentiated embryonic stem (ES) cells is counteracted by the intrinsic X chromosome reactivation (XCR), we identified Kat8 and Msl2, homologs of Drosophila dosage compensation proteins, as players involved in mammalian XCR. Furthermore, live-cell imaging revealed the obviously undersized ΔA Xist cloud signals, clarifying an issue regarding the previous RNA fluorescence in situ hybridization results. Tethering candidate proteins onto the ΔA mutant reveals the significant roles of Ythdc1, Ezh2, and SPOC (Spen) in Xist-mediated gene silencing and the significant role of Ezh2 in Xist RNA spreading.
We carried out padlock capture, a high-resolution RNA allelotyping method, to study X chromosome inactivation (XCI). We examined the gene reactivation pattern along the inactive X (Xi), after Xist (X-inactive specific transcript), a prototype long non-coding RNA essential for establishing X chromosome inactivation (XCI) in early embryos, is conditionally deleted from Xi in somatic cells (Xi∆Xist). We also monitored the behaviors of X-linked non-coding transcripts before and after XCI. In each mutant cell line, gene reactivation occurs to ~6% genes along Xi∆Xist in a recognizable pattern. Genes with upstream regions enriched for SINEs are prone to be reactivated. SINE is a class of retrotransposon transcribed by RNA polymerase III (Pol III). Intriguingly, a significant fraction of Pol III transcription from non-coding regions is not subjected to Xist-mediated transcriptional silencing. Pol III inhibition affects gene reactivation status along Xi∆Xist, alters chromatin configuration and interferes with the establishment XCI during in vitro differentiation of ES cells. These results suggest that Pol III transcription is involved in chromatin structure re-organization during the onset of XCI and functions as a general mechanism regulating chromatin configuration in mammalian cells.
Using amino-labeled oligonucleotide probes, we established a simple, robust and low-noise method for simultaneous detection of RNA and DNA by fluorescence in situ hybridization, a highly useful tool to study the large pool of long non-coding RNAs being identified in the current research. With probes either chemically or biologically synthesized, we demonstrate that the method can be applied to study a wide range of RNA and DNA targets at the single-cell and single-molecule level in cellular contexts.
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