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.

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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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A highly useful tool for studying lncRNAs is simultaneous RNA-DNA FISH, which reveals the localization and quantitative information of RNA and DNA in cellular contexts. However, a simple combination of RNA FISH and DNA FISH often generates disappointing results because the fragile RNA signals are often damaged by the harsh conditions used in DNA FISH for denaturing the DNA. Here, we describe a robust and simple RNA-DNA FISH protocol, in which amino-labeled nucleic acid probes are used for RNA FISH. The method is suitable to detect single-RNA molecules simultaneously with DNA.

Xist (inactivated X chromosome specific transcript) is a prototype long noncoding RNA in charge of epigenetic silencing of one X chromosome in each female cell in mammals. In a genetic screen, we identify Mageb3 and its homologs Mageb1 and Mageb2 as genes functionally required for Xist-mediated gene silencing. Mageb1-3 are previously uncharacterized genes belonging to the MAGE (melanoma-associated antigen) gene family. Mageb1-3 are expressed in undifferentiated ES cells and early stages of in vitro differentiation, a critical time window of X chromosome inactivation. Mageb3 showed both cytoplasmic and nuclear localization without enrichment on the inactive X (Xi). Mageb3 interacted with Polycomb group ring finger 3 (Pcgf3), a RING finger protein involved in recruiting Polycomb activities onto Xi. Mageb3 overexpression stabilized Pcgf3 protein. Mageb1-3 gene knockout affected H3K27me3 enrichment and the spreading of gene silencing along Xi. These data suggested that Mageb3 might regulate the recruitment of the Polycomb complex onto Xi and subsequent H3K27me3 modification through Pcgf3. Moreover, the nucleolar enrichment of Mageb3 was diminished when nuclear matrix factor hnRNP U is overexpressed, implying the interaction between Mageb3 and nuclear matrix, which is another possible mechanism for Mageb3 to regulate X chromosome inactivation.

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