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Features RNA Illuminating Xist Features RNA Illuminating Xist Heather Coker, Benoit Moindrot, Greta Pintacuda and Neil Brockdorff (University of Oxford, UK) The Central Dogma proposed that RNA, encoded by DNA in the genome, acts as the template used by cells for protein production. The simplicity of RNA as a discrete mediator of information has subsequently been challenged by the discovery of non-protein-coding RNAs. Understanding Downloaded from http://portlandpress.com/biochemist/article-pdf/37/2/24/5434/bio037020024.pdf by guest on 30 September 2021 of this intricate new field has been fuelled by the development of new research techniques. In this article, we consider how recent advances in microscopy have added to our current understanding of the non-coding RNA Xist (X-inactive specific transcript). In female placental mammals, the process RNA cloud that can be visualized using fluorescent in of X-inactivation results in the permanent situ hybridization (FISH). This technique, in which silencing of one of the two X-chromosomes. The fluorescent probes are directed against Xist RNA inactive X-chromosome (Xi) is embedded into a (RNA-FISH), has been of particular use in visualizing repressive heterochromatic region known as the the effect of genetic changes upon the structure and Barr body, a DNA-dense region in the nucleus formation of the Xist domain (Figure 1). of XX somatic cells visible by light microscopy. Xist has been identified in all placental mammals, Xist is regarded as the ‘master regulator’ of this and shown to be composed of six broadly conserved6 complex process, which corrects the genetic segments of short repeats, named A to F (Figure imbalance of close to a thousand X-linked genes 2). Whereas the 3ʹ end of the transcript appears between XX females and XY males (see Figure 1 for to function redundantly, the conserved A-repeat an overview). region at the 5ʹ end of the transcript, is of particular importance3. This region contains 7.5 copies of a Xist transcript 26-nt GC-rich core sequence, separated by AT-rich spacers of variable length7. It has been predicted Xist encodes a long non-coding RNA (15–17 that each 26-nt monomer might fold into a structure kb) mono-allelically expressed from the inactive comprising two stem–loops8. Interestingly, the 5ʹ X-chromosome, which is responsible for both the end of the recently discovered marsupial orthologue initiation and maintenance in cis of its silencing 1,2. of Xist (Rsx) has the potential to form similar Indeed, the ectopic expression of a Xist transgene stem–loop structures9. Critically, in the absence is sufficient to induce silencing of the transgene- of these conserved A-repeats, the ability to silence carrying chromosome, mimicking endogenous genes is lost, despite the fact that Xist still coats the silencing of the Xi3,4. Recently, the remarkable chromosome3. The ability to coat the chromosome silencing capability of Xist was successfully exploited also appears to rely, at least in part, on the C-repeats for potential therapeutic benefit, when a human since Xist RNA domains seem to disappear shortly XIST transgene was targeted to, and silenced, the after the injection of locked nucleic acids targeting additional chromosome 21 in Down’s syndrome the C-repeat10. induced pluripotent stem cells5. The Xist transcript is spliced, capped and Formation of a repressive compartment harbours a poly(A) tail as is the case for conventional protein-coding messenger RNAs (mRNAs). However, Xist is thought to initiate the formation of a Xist is not exported, but remains in the nucleus and repressive compartment by the recruitment of coats the inactive chromosome, forming a stable chromatin-modifying complexes to the soon-to-be Keywords: Barr body, Abbreviations: 3D-SIM, 3D structured illumination microscopy; FISH, fluorescent in situ hybridization; PRC, Polycomb repressive chromatin, non-coding complex; SAF-A, scaffold attachment factor A; Xi, inactive X-chromosome; Xist, X-inactive specific transcript. RNA, X-chromosome inactivation, Xist 24 April 2015 © Biochemical Society RNA Features Downloaded from http://portlandpress.com/biochemist/article-pdf/37/2/24/5434/bio037020024.pdf by guest on 30 September 2021 Figure 1. The heterochromatic nature of the inactive X-chromosome. Xist RNA coats in cis the inactive X (top panel) and triggers its downstream heterochromatinization. Exclusion of RNA polymerase II (PolII) and active histone modifications (H3K4me3, H4ac and H3K9ac), is accompanied by recruitment of the Polycomb repressive complexes (PRC1 and PRC2) depositing the repressive modifications (H3K27me3 and H2AK119ub). inactive X-chromosome. One of the earliest events promoter. It has been suggested that this would in occurring after the coating of the chromosome by turn enhance the expression of Xist RNA through Xist RNA is the exclusion of both RNA polymerase an as yet uncharacterized positive-feedback loop. II and euchromatic histone modifications such However, biochemical analysis has been impeded by as H3K9ac (acetylated Lys9 of histone H3) and the fact that the long Xist RNA transcript associates H3K4me3 (trimethylated Lys4 of histone H3). with the insoluble fraction of the nucleus, meaning This is subsequently followed by the recruitment of Polycomb repressive complex 1 (PRC1) and Polycomb repressive complex 2 (PRC2), respectively ubiquitinating H2AK119 (Lys119 of histone H2A) and trimethylating H3K27 (Lys27 of histone H3). Further repressive chromatin marks, such as H3K9me2/3 (di/tri-methylated Lys9 of histone H3) and H4K20me3 (trimethylated Lys20 of histone H4) begin to accumulate, along with other chromatin modifiers, together contributing to the formation of a repressive compartment (Figure 1). So how do we know that Xist orchestrates this succession of events? Much of our knowledge regarding the mechanism of Xist has been obtained from the combination of biochemical, genetic and imaging data. Several lines of evidence have suggested that PRC2 might bind to the A-repeat Figure 2. Conservation of Xist in mammals. The Xist gene shows a high degree of homology of Xist RNA via its EZH2 or SUZ12 subunits11–13. between species, particularly evident in the six repeat regions (A–F). Genetic and biochemical In particular, a transcript that encompasses the studies have suggested that A-repeats are important for gene silencing, B- and F-repeats for A-repeats has been proposed to bind directly Polycomb recruitment, and C-repeats for the correct localization of Xist RNA. Adapted from6 to PRC2, leading to its recruitment at the Xist under Creative Commons License. April 2015 © Biochemical Society 25 Features RNA that experiments have been mainly carried out in vitro. This may explain inconsistencies in the data, for example, while biochemical studies suggest that Xist lacking A-repeats should not lead to the deposition of H3K27me3 (trimethylated Lys27 of histone H3) and H2AK119ub (ubiquitinated Lys119 of histone H2A), the A-repeats are in fact dispensable for the recruitment of PRC2 in vivo14,15. Biochemical experiments have also proposed the nuclear scaffold attachment factor A [SAF-A or heteronuclear ribonucleoprotein U (hnRNP-U)] as a candidate protein that may interact with Xist16. Downloaded from http://portlandpress.com/biochemist/article-pdf/37/2/24/5434/bio037020024.pdf by guest on 30 September 2021 Recruitment of SAF-A to the Xi requires Xist RNA and appears to be dependent on the SAF-A RNA-binding domain recognizing the C-repeats of Xist16–18. Depletion of SAF-A disperses the Xist cloud that coats the chromosome, and impairs both the gene silencing and the establishment of H3K27me3 domains. It has been proposed that because SAF-A also contains a matrix attachment region, it might be responsible for the trapping of Xist in the nuclear matrix. New perspectives Work published recently by Cerase et al.19 and Smeets et al.20 has circumvented some of the limitations of biochemical analysis of Xist RNA function by exploiting advanced super-resolution microscopy to reappraise Xi organization relative to Xist RNA and other markers. Super-resolution 3D structured illumination microscopy (3D-SIM) achieves an overall 8-fold increase in image resolution compared with conventional light microscopy. This increased resolution, in combination with the use of milder sample denaturation conditions, enabled Smeets et al.20 to assess the distribution of Xist in relation to the underlying chromatin structure of the Barr body (visualized with the fluorescent DNA stain DAPI). Contrary to previous supposition, 3D-SIM illustrated that the Xi domain was not impenetrably heterochromatic, but rather exhibited a ‘sponge-like’ organization with DAPI- poor channels indicating the potential for movement of factors between the interior of the Barr body and the nuclear pores (Figure 3). The concept of an accessible network of channels within the Xi, albeit narrowed and restricted compared with that associated with transcriptionally competent autosomes, immediately challenges Figure 3. Super-resolution (3D-SIM) imaging of the Barr body. The Barr domain, defined our ideas of the Xi as a tightly packaged inert by dense DAPI-stained chromatin and H3K27me3, is permeated by a network of narrow chromosome. Indeed, when Smeets et al.20 and channels that contact nuclear pores (bottom panel, in green). Xist and SAF-A reside in Cerase et al.19 proceeded to investigate the Xi further, these channels, which may also allow limited access of RNA polymerase II (PolII). using the combined power of 3D-SIM, RNA-FISH 26 April 2015 © Biochemical Society RNA Features and immunodetection, they discovered that, whereas Heather Coker gained her PhD in Biochemistry from King’s College, University of epigenetic marks placed by PRC2 were co-localized London. She then spent time at Clare Hall Laboratories (Cancer Research UK), as expected with the DAPI-dense chromatin, Xist before joining Neil Brockdorff in the Department of Biochemistry, University of was found within the DAPI-poor interchromatin Oxford, as a Wellcome Trust postdoctoral researcher interested in understanding channels. SAF-A was also present within these the processes and dynamics of X-chromosome inactivation.
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