Locked nucleic acids (LNAs) reveal sequence requirements and kinetics of Xist RNA localization to the X chromosome

Abstract

A large fraction of the mammalian genome is transcribed into long noncoding RNAs. The RNAs remain largely uncharacterized as the field awaits new technologies to aid functional analysis. Here, we describe a unique use of locked nucleic acids (LNAs) for studying nuclear long noncoding RNA, an RNA subclass that has been less amenable to traditional knockdown techniques. We target LNAs at Xist RNA and show displacement from the X chromosome with fast kinetics. Xist transcript stability is not affected. By targeting different Xist regions, we identify a localization domain and show that polycomb repressive complex 2 (PRC2) is displaced together with Xist. Thus, PRC2 depends on RNA for both initial targeting to and stable association with chromatin. H3K27-trimethyl marks and gene silencing remain stable. Time-course analysis of RNA relocalization suggests that Xist and PRC2 bind to different regions of the X at the same time but do not reach saturating levels immediately. Thus, LNAs provide a tool for studying an emerging class of regulatory RNA and offer a window of opportunity to target epigenetic modifications with possible therapeutic applications.

Fig. 1.

LNAs targeting Xist repeat C abolish Xist RNA localization to Xi. (A) Alignment of 14 tandem mouse repeat C. Mismatches are shown in red. The regions targeted by LNAs are indicated by a red line. (B) Xist RNA FISH at indicated time points after LNA nucleofection. Results shown for LNA-C1, but LNA-C2 gives similar results. Xist RNA, green. Asterisk, diffuse Xist cluster; arrows, pinpoint Xist signals. (C) Quantitation of the results in B. (D) Alignment of mouse and human repeat C regions targeted by the LNAs (Left). (E) XIST RNA FISH performed in human 293 cells nucleofected with LNAs. XIST RNA, red.

Fig. 2.

Xist RNA displacement is accompanied by loss of PRC2 localization and recovery occurs initially near Xist. (A) Real-time qRT-PCR analysis of Xist levels, normalized to Gapdh RNA. (B) Xist RNA FISH in cells treated with or without ActD after LNA nucleofection. Xist RNA, green. (C) Xist RNA FISH on metaphase chromosomes after LNA nucleofection. Xist RNA, red. (D) Xist RNA FISH (green) followed by Xist DNA FISH (red) in metaphase spreads from cells at 3 h post-LNA treatment. Arrows, Xist locus. (E) Immunostaining for Ezh2 (green). (F) Immunostaining for H3K27me3 (green). Xist RNA FISH (red) was performed to confirm loss of Xist from Xi at 1 h. (G) Two-color RNA FISH detecting Hprt and Xist RNA simultaneously at 3 h after nucleofection.

Fig. 3.

A broader domain around repeat C is required for Xist localization. (A) Map of Xist exon/intron structure and locations of LNAs used. (B) Time-course Xist RNA FISH analysis in cells treated with indicated LNAs. Xist, green. (C) qRT-PCR of Xist levels, normalized to Gapdh quantities. (D) Immunostaining for H3K27me3 (green) and RNA FISH for Xist (red). (E) Western blot with Ezh2 antibodies. Actin is used as a loading control. (F) Xist RNA FISH (green) after nucleofection with indicated LNAs. (G) Ezh2 immunostaining after nucleofection with indicated LNAs.

Fig. 4.

Ezh2 recovery after LNA nucleofection is uniform along Xi but slow in kinetics. (A) Schematic representation of X genes. (B) Ezh2 ChIP analysis at X genes. (C) ChIP analysis of Ezh2 after LNA nucleofection. *P < 0.05 by the Student's t test. (D) ChIP analysis of Ezh2 enrichment on the autosomal En1 promoter.