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      Live-Cell Imaging and Functional Dissection of Xist RNA Reveal Mechanisms of X Chromosome Inactivation and Reactivation

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          Summary

          We 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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          Highlights

          • •

            A Pumilio-based system allows efficient double-tagging of Xist RNA in live cells

          • •

            Induced XCI in undifferentiated ES cells reveals the roles of Kat8 and Msl2 in XCR

          • •

            Live-cell imaging reveals the undersized “ΔA mutant” Xist signals

          • •

            Tethering proteins onto “ΔA mutant” reveals their role in Xist-mediated silencing

          Abstract

          Genetics; Molecular Biology; Cell Biology; Developmental Biology

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          Most cited references16

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          The Xist lncRNA exploits three-dimensional genome architecture to spread across the X chromosome.

          Jesse Engreitz, Amy Pandya-Jones, Patrick McDonel … (2013)
          Many large noncoding RNAs (lncRNAs) regulate chromatin, but the mechanisms by which they localize to genomic targets remain unexplored. We investigated the localization mechanisms of the Xist lncRNA during X-chromosome inactivation (XCI), a paradigm of lncRNA-mediated chromatin regulation. During the maintenance of XCI, Xist binds broadly across the X chromosome. During initiation of XCI, Xist initially transfers to distal regions across the X chromosome that are not defined by specific sequences. Instead, Xist identifies these regions by exploiting the three-dimensional conformation of the X chromosome. Xist requires its silencing domain to spread across actively transcribed regions and thereby access the entire chromosome. These findings suggest a model in which Xist coats the X chromosome by searching in three dimensions, modifying chromosome structure, and spreading to newly accessible locations.
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            XIST RNA paints the inactive X chromosome at interphase: evidence for a novel RNA involved in nuclear/chromosome structure

            HF Willard, JA McNeil, JB Lawrence (1996)
            The XIST gene is implicated in X chromosome inactivation, yet the RNA contains no apparent open reading frame. An accumulation of XIST RNA is observed near its site of transcription, the inactive X chromosome (Xi). A series of molecular cytogenetic studies comparing properties of XIST RNA to other protein coding RNAs, support a critical distinction for XIST RNA; XIST does not concentrate at Xi simply because it is transcribed and processed there. Most notably, morphometric and 3-D analysis reveals that XIST RNA and Xi are coincident in 2- and 3-D space; hence, the XIST RNA essentially paints Xi. Several results indicate that the XIST RNA accumulation has two components, a minor one associated with transcription and processing, and a spliced major component, which stably associates with Xi. Upon transcriptional inhibition the major spliced component remains in the nucleus and often encircles the extra-prominent heterochromatic Barr body. The continually transcribed XIST gene and its polyadenylated RNA consistently localize to a nuclear region devoid of splicing factor/poly A RNA rich domains. XIST RNA remains with the nuclear matrix fraction after removal of chromosomal DNA. XIST RNA is released from its association with Xi during mitosis, but shows a unique highly particulate distribution. Collective results indicate that XIST RNA may be an architectural element of the interphase chromosome territory, possibly a component of nonchromatin nuclear structure that specifically associates with Xi. XIST RNA is a novel nuclear RNA which potentially provides a specific precedent for RNA involvement in nuclear structure and cis-limited gene regulation via higher-order chromatin packaging.
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              A novel role for Xist RNA in the formation of a repressive nuclear compartment into which genes are recruited when silenced.

              Edith Heard, Anton Wutz, Julie Chaumeil … (2006)
              During early mammalian female development, one of the two X chromosomes becomes inactivated. Although X-chromosome coating by Xist RNA is essential for the initiation of X inactivation, little is known about how this signal is transformed into transcriptional silencing. Here we show that exclusion of RNA Polymerase II and transcription factors from the Xist RNA-coated X chromosome represents the earliest event following Xist RNA accumulation described so far in differentiating embryonic stem (ES) cells. Paradoxically, exclusion of the transcription machinery occurs before gene silencing is complete. However, examination of the three-dimensional organization of X-linked genes reveals that, when transcribed, they are always located at the periphery of, or outside, the Xist RNA domain, in contact with the transcription machinery. Upon silencing, genes shift to a more internal location, within the Xist RNA compartment devoid of transcription factors. Surprisingly, the appearance of this compartment is not dependent on the A-repeats of the Xist transcript, which are essential for gene silencing. However, the A-repeats are required for the relocation of genes into the Xist RNA silent domain. We propose that Xist RNA has multiple functions: A-repeat-independent creation of a transcriptionally silent nuclear compartment; and A-repeat-dependent induction of gene repression, which is associated with their translocation into this silent domain.
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                Author and article information

                Contributors
                Lingyi Chen
                Li-Feng Zhang
                Journal
                Journal ID (nlm-ta): iScience
                Journal ID (iso-abbrev): iScience
                Title: iScience
                Publisher: Elsevier
                ISSN (Electronic): 2589-0042
                Publication date PMC-release: 13 September 2018
                Publication date Collection: 26 October 2018
                Publication date (Electronic): 13 September 2018
                Volume: 8
                Pages: 1-14
                Affiliations
                [1 ]School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Singapore
                [2 ]State Key Laboratory of Medicinal Chemical Biology, Key Laboratory of Bioactive Materials, Ministry of Education, Tianjin Key Laboratory of Protein Sciences and College of Life Sciences, Nankai University, Tianjin 300071, China
                Author notes
                [∗ ]Corresponding author lingyichen@ 123456nankai.edu.cn
                [∗∗ ]Corresponding author zhanglf@ 123456ntu.edu.sg
                [3]

                Present address: Beijing Leadman Biochemistry Co., Ltd., No.5 Xinghai Road, BDA, Beijing, China

                [4]

                Lead Contact

                Article
                Publisher ID: S2589-0042(18)30142-1
                DOI: 10.1016/j.isci.2018.09.007
                PMC ID: 6159346
                PubMed ID: 30266032
                SO-VID: ca4b890f-d9de-48fd-8a22-d0c6fab65b7c
                Copyright © © 2018 The Author(s)
                License:

                This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

                History
                Date received : 4 July 2018
                Date revision received : 4 September 2018
                Date accepted : 7 September 2018
                Categories
                Subject: Article

                Keywords: genetics,molecular biology,cell biology,developmental biology
                Data availability:
                Keywords: genetics, molecular biology, cell biology, developmental biology

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