XIST dampens X chromosome activity in a SPEN-dependent manner during early human development.

XIST (X-inactive specific transcript) long noncoding RNA (lncRNA) is responsible for X chromosome inactivation (XCI) in placental mammals, yet it accumulates on both X chromosomes in human female preimplantation embryos without triggering X chromosome silencing. The XACT (X-active coating transcript) lncRNA coaccumulates with XIST on active X chromosomes and may antagonize XIST function. Here, we used human embryonic stem cells in a naive state of pluripotency to assess the function of XIST and XACT in shaping the X chromosome chromatin and transcriptional landscapes during preimplantation development. We show that XIST triggers the deposition of polycomb-mediated repressive histone modifications and dampens the transcription of most X-linked genes in a SPEN-dependent manner, while XACT deficiency does not significantly affect XIST activity or X-linked gene expression. Our study demonstrates that XIST is functional before XCI, confirms the existence of a transient process of X chromosome dosage compensation and reveals that XCI and dampening rely on the same set of factors.

Non-Coding RNA Silencing in Mammalian Cells by Antisense LNA GapmeRs Transfection.

The assessment of non-coding RNAs (ncRNAs) functions highly relies on loss of function studies. However, due to their exclusive or partial nuclear localization, many small and long ncRNAs are not efficiently silenced by RNA interference. Antisense LNA GapmeRs constitute a good alternative to RNAi. They allow an effective knockdown of ncRNAs with sizes greater than 80 nucleotides, regardless of their cellular localization. This chapter focuses on the silencing of two different nuclear ncRNAs (ANRIL and SATIII RNAs) in mammalian cells using antisense LNA GapmeRs with two different transfection methods: calcium phosphate-mediated transfection and LipofectamineTM 2000.

Study of Genome-Wide Occupancy of Long Non-Coding RNAs Using Chromatin Isolation by RNA Purification (ChIRP).

Long noncoding RNAs (lncRNAs) have recently emerged as masters of gene expression regulation by exerting their functions in all cell compartments through a wide repertoire of mechanisms. A high portion of lncRNAs are robustly enriched in the chromatin fraction suggesting a broad regulatory role in the nuclear compartment. Despite the advances in this field, the interaction between lncRNAs and the chromatin is still poorly understood. This led to the emergence of numerous hybridization capture assays such as the Chromatin Isolation by RNA Purification (ChIRP) which revealed at high resolution the genomic binding sites of several nuclear lncRNAs. In this chapter, we describe the ChIRP protocol that was successfully applied to the lncRNA ANRIL. We also provide a user-friendly bioinformatic pipeline for ChIRP-seq data analysis.

Implication of repeat insertion domains in the trans-activity of the long non-coding RNA ANRIL.

Long non-coding RNAs have emerged as critical regulators of cell homeostasis by modulating gene expression at chromatin level for instance. Here, we report that the lncRNA ANRIL, associated with several pathologies, binds to thousands of loci dispersed throughout the mammalian genome sharing a 21-bp motif enriched in G/A residues. By combining ANRIL genomic occupancy with transcriptomic analysis, we established a list of 65 and 123 genes potentially directly activated and silenced by ANRIL in trans, respectively. We also found that Exon8 of ANRIL, mainly made of transposable elements, contributes to ANRIL genomic association and consequently to its trans-activity. Furthermore, we showed that Exon8 favors ANRIL's association with the FIRRE, TPD52L1 and IGFBP3 loci to modulate their expression through H3K27me3 deposition. We also investigated the mechanisms engaged by Exon8 to favor ANRIL's association with the genome. Our data refine ANRIL's trans-activity and highlight the functional importance of TEs on ANRIL's activity.