# Unleashing XIST from X-chromosome inactivation

Céline Morey, Claire Rougeulle, Jean-François Ouimette

► To cite this version:

Céline Morey, Claire Rougeulle, Jean-François Ouimette. Unleashing XIST from X-chromosome inactivation. Current Opinion in Cell Biology, 2024, 92, pp.102446. 10.1016/j.ceb.2024.102446. hal-05406874

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# Unleashing XIST from X-chromosome inactivation
Céline Morey, Claire Rougeulle and Jean-François Ouimette

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Recognition that the most abundant class of genes present in the human genome are those producing long noncoding RNA (lncRNA) has hyped research on this category of transcripts. One such prototypical RNA, Xist, has particularly fueled interest. Initially characterized for its specific expression from the inactive X (Xi), recent studies have uncovered the molecular mechanisms underlying its essential role in the initiation of X-chromosome inactivation, from its exquisitely precise transcriptional regulation to the plethora of protein interactors forming the Xist ribonucleoprotein (RNP) that mediate its gene silencing activity. Here, we will discuss the recent advances that have broadened our knowledge of Xist functions, challenging classical models and revealing unsuspected, unconventional actions of its RNP.

## Addresses

Epigenetics and Cell Fate, CNRS, Université Paris Cité, Paris, France

Corresponding author: Rougeulle, Claire (claire.rougeulle@u-paris.fr)

**Current Opinion in Cell Biology 2025, 92:102446**

This review comes from a themed issue on Cell Nucleus (2024)

Edited by **Evi Soutoglou** and **Noriko Saitoh**

For complete overview of the section, please refer the article collection - Cell Nucleus (2024)

Available online 27 November 2024

https://doi.org/10.1016/j.ceb.2024.102446

0955-0674/© 2024 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

## Introduction

LncRNAs play crucial roles in regulating gene expression at various levels, from nuclear organization, chromatin modification, transcription, and posttranscriptional processing. Since its discovery in 1991, the Xist lncRNA has been well characterized for its ability to regulate, chromosome-wide, X-linked gene expression via the process of X-chromosome inactivation (XCI), which contributes to dosage compensation in mammals by silencing one of the two X chromosomes in females. Xist is a eutherian restricted gene that produces a ~17 kb lncRNA that coats *in cis* the chromosome from which it is expressed. Functional modules of the Xist RNA, which overlap with several conserved tandem repeat sequences (repeat A, B, C, D, E, F), have been shown to recruit various enzymes and protein complexes that form the

functional Xist RNP [1–5]. While Xist has been mainly studied for its action in the initiation of XCI in the mouse, recent studies in human and mouse have revealed noncanonical and non-XCI-related properties of XIST/Xist. Capitalized and noncapitalized letters will refer to human and mouse lncRNA, respectively.

## Xist and the RNP chronicles: decoding chromosome silencing

The production and accumulation of mature Xist RNA strictly on the future inactive X chromosome (Xi) marks the onset of XCI in the mouse. This key event involves monoallelic transcriptional upregulation (reviewed in Ref. [6]), efficient splicing and RNA folding, as well as posttranscriptional modifications. Indeed, among the Xist RNA interactors are RBM15/RBM15B that allow the recruitment of the METTTL3/14 complex, triggering the deposition of m6A at several specific sites on Xist RNA [7] (Figure 1a). Xist m6A RNA methylation has been proposed to participate in X chromosome transcriptional repression, although this is still debated [8,9]. In addition, whether through direct RNA-protein or *via* downstream protein-protein interactions, XIST RNA molecules serve as concentrating agents [10–12**] for:

1. The HDAC activator SPEN, a key player in histone deacetylation and RNA Polymerase II eviction, and thus in triggering transcriptional repression [2,13,14]. Its levels seem to correlate to the efficiency of gene silencing [11];

2. Members of the Polycomb Repressive Complex 1, RYBP, and PCGF5, involved in the deposition of H2AK119Ub. This mark appears to be upstream of PRC2 recruitment [15];

3. CELF1, MATR3, PTBP1, and TDP43, which are RNA binding proteins that might be essential for tethering Xist RNA to the Xi and for Xist-independent maintenance of XCI [10];

4. CIZ1 and hnRNPU, both of which participate in nuclear spatial organization processes and Xist anchoring [16,17].

Along with other cofactors, this supramolecular complex forms the Xist RNP, which initially assembles in a limited number of nucleation foci to eventually concentrate and spread over the entire X chromosome [11], in a manner that may be directed by the high-order conformation of the Xist-expressing locus in relation to the rest of the
<figure><img src="image_3.png" /><figcaption>**Figure 1**</figcaption></figure>

**The multiple functions of the XIST lncRNA.** (a). In female mammalian cells, the *Xist* lncRNA initially accumulates to approximately 50 sites along the inactive X chromosome (Xi), exploiting the 3D conformation of the *Xist* locus in relation to the rest of the chromosome. *XIST/Xist* serves as a concentrating agent for various interacting factors with diverse enzymatic activities leading to gene repression, 3D organization of the Xi, and long-term maintenance of silencing. The modules of the *Xist/XIST* RNP together with *Xist/XIST* repeats A, B, C, and E hosting the interactions are depicted on the right. The composition of the RNP has been defined using both mouse and human models. (b). X-inactivation independent functions of *XIST*. *XIST* RNP induces the transcriptional dampening of X-linked genes on both X-chromosomes and of loci on neighboring autosomes in naïve preimplantation-like human cells. *XIST* RNP may trigger immunogenic reactions when released from the cell during the cell cycle or upon cell injury. This may result in female-biased autoimmune diseases. *XIST* may serve as a competing endogenous RNA through the binding of microRNAs. This function, when perturbed, has been associated with tumorigenesis.

chromosome [18] (Figure 1a). In the context of XCI, this
induces changes to the chromatin structure and triggers
the reconfiguration of the coated chromosome to a het-
erochromatin Xi compartment. For this transition,
several pieces of evidence suggest that the *Xist* RNP
relies on nuclear compartmentalization (elegantly
reviewed in Ref. [19]), although this might not affect
directly the ability of the transcription machinery to
access the *Xist*-coated chromosome [20*]. Importantly,
*Xist* RNPs are also functional in triggering long-distance
transcriptional silencing in a transgenic context [21,22].
This striking ability of *Xist* lncRNA to induce gene
silencing may have overshadowed other functions related
to intrinsic properties of *Xist* RNP, independent of XCI.

Dampening and delocalizing XIST activity

Although XCI occurs in females of all mammalian species
studied, significant differences in the underlying strat-
egy and kinetics have been observed among species,
particularly between rodents and primates. The initial
observations, in preimplantation human female embryos,
that both X chromosomes remain active despite being
coated by XIST RNA, suggested noncanonical functions
of XIST [23,24]. Intriguingly, XIST RNA in preXCI
human early embryonic cells exhibits a more dispersed
configuration than in human postXCI cells, which may
suggest that the XIST RNP does not locally concentrate
as efficiently on the coated chromosomes in preXCI cells
[23,25]. Whether this is a consequence of an inefficient/
incomplete *XIST* RNP assembly that prevents chromatin remodeling or an alternative chromosome conformation has not yet been elucidated (for discussion see Ref. [26]). Despite the lack of XCI induction at this stage, cells of preimplantation female embryos display accumulation of H3K27me3 over their X chromosomes [27**] and reduced expression of X-linked genes through a process termed X-chromosome dampening [24].

Dispersed *XIST* RNA distribution has also been observed in naïve human pluripotent stem cells [25,28], which recapitulate features of preimplantation embryos, where it has been associated with the X-chromosomes as well as with autosomal loci (Figure 1b). In these cells, *XIST* dampens the expression of X-linked and autosomal contacted loci in a manner that depends, at least partially, on SPEN [27,29]. These surprising observations might be explained by nonexclusive hypotheses. Context-dependent composition of the *XIST* RNP could result in qualitative or quantitative differences in its ability to silence target loci; this is related to recent findings that the continuous requirement of *XIST* to maintain the repression of a subset of X-linked genes in human B cells relies on B-cell specific *XIST* interactors [30]. Interestingly, *Xist* RNA can be delocalized from the Xi in specific immune cells, reinforcing the hypothesis that its activity can be modulated in a cell-type-specific manner [31]. Alternatively, *XIST* RNA levels might influence the extent of silencing observed on the targeted loci. It has indeed been recently reported that increasing *XIST* levels modulate escape from XCI [32*] and also results in *XIST* RNPs targeting autosomal loci [12**].

## XIST RNP, a potential immunogenic complex

Female individuals have a higher risk of developing autoimmunity compared to males [33,34]. Enhanced escape from XCI of some X-linked genes with immune functions that may occur upon perturbation of XCI has been recently associated with this bias [35,36]. In parallel, several observations suggest that *XIST* RNP itself could trigger innate immune responses [37,38**]. *XIST* RNA sequence is enriched in UU dinucleotides [37] — a motif known to be a preferential single-strand RNA (ssRNA) ligand for TLR7 activation, a Toll-like receptor involved in the development of Systemic Lupus Erythematosus (SLE) [39]. This motif is predominant in the A repeat of *XIST/Xist*, which folds into a stem-loop structure harboring m6A [7]. m6A-dependent binding of YTHDC1 unfolds the stem-loop structure, making it accessible to RNA binding proteins or to ssRNA receptors such as TLR7 [40]. In addition, several known interactors of *Xist* RNA were reported to serve as autoantigens in human autoimmune diseases [38**,41], while DNA components from the Xi chromatin associated with *XIST* RNPs could also trigger immune response [38**]. Hence, under certain circumstances, i.e., during the cell cycle, upon cell death — apoptosis or

senescence — or upon tissue injury, *XIST* may be released from the cell nucleus and exposed in the extracellular space where *XIST* RNPs and associated Xi DNA may cluster and activate immune-receptors (Figure 1b). Indeed, DNA-autoantibody and RNA-autoantigen immune complexes activate the TLR7, TLR8, and TLR9 pathways of the innate immune system in patients with SLE and in SLE mouse models [42].

To test the immunogenic properties of mouse *Xist* RNP, Dou et al. have induced, in male mice, the expression of a transgenic form of *Xist*, devoid of A repeat, from chromosome 11 [38**]. In these animals, pristane administration promotes an SLE-like phenotype, with a response at the levels of gene expression, chromatin accessibility profiles, and CD4+ T cell repertoire, which closely resemble that observed in female counterparts, while it has no effect in control males. If *Xist* induction elicits overt autoimmunity in SJL/J male mice, it is however not sufficient to do so in the C57BL/6 refractory background, suggesting an influence of the genetic background in modulating *Xist*-induced SLE phenotypes. Importantly, in this system, the use of a mutated form of *Xist* lacking repeat A allows to discard artifactual effects that may have resulted from the ectopic silencing of chromosome 11. While this suggests that *Xist* repeat A and associated silencing activity are not required for *Xist* RNP immunogenicity, it also questions the physiological relevance of the findings. Indeed, it is not clear whether this truncated version of *Xist* RNA promotes the formation of *Xist* RNP aggregates and displays immunogenic properties similar to full-length *Xist*. In addition, inducing *Xist* expression in adult males, a naïve context that has never been exposed to *Xist* RNP, may not be strictly comparable to a female background which is likely immunized against endogenous *Xist* RNP. Finally, the extent to which this response is specific to *Xist* with respect to other nuclear lncRNAs such as *Neat1* or *Malat1* that recruit similar constitutive RNA binding proteins and also bind chromatin is unclear. These observations nevertheless open new perspectives for understanding the molecular basis of autoimmunity and for early diagnosis of autoimmune conditions based on measurements of *XIST* RNP levels in the serum.

Hence *Xist* RNA association with Xi chromatin has two opposite outcomes: 1) it prevents spontaneous autoimmunity through *Xist*-mediated maintenance of XCI and control of escapees; 2) it may activate the immune system upon release of *Xist* RNP/DNA complex from the cell. These involve two independent mechanisms of action. The first is cell autonomous, and takes place in the nucleus of specific immune cells where it controls the levels of X-linked products with immune function. The second relies on a cell-extrinsic activation of immune cells present in the extracellular space (Figure 1b). These antagonist actions indicate that tight mechanisms of *XIST* RNP expression and localization
may have been selected during evolution to buffer the immune response in females.

### XIST, a molecular sponge for many microRNAs associated with various pathologies

In recent years, reports on *XIST* acting as a miR sponge, with widespread involvement in various cancers and other conditions, have been accumulating. This modus operandi has notably been implicated in the regulation of cell proliferation, migration, invasion, angiogenesis, chemo-resistance, and evasion from apoptosis of cancer cells. Because of its large size, *XIST* potentially interacts with a plethora of miRs, which in turn may modulate the expression of several downstream genes (Figure 1b). Target genes have been reported to include members of key regulatory pathways such as WNT [43,44] or NOTCH [45] signaling, NF-kB and TLR pathways [46,47], and many others. *XIST* multibinding capacity may impact various miR regulatory networks at the same time. Hence, abnormal *XIST* levels have been associated with contrasting phenotypes depending on the cell type. For example, *XIST* overexpression is associated either with oncogenic or tumor suppressor effects in colon cancer [43] and hepatocellular carcinoma [44], respectively.

The XCI research community has raised valid concerns regarding *XIST*'s function as a competing endogenous RNA. Indeed, *XIST* lncRNA is preferentially expressed in female cells and localizes to the nucleus, two characteristics that seem incompatible with canonical miR function in the cytosol and with involvement in pathologies affecting both female and male individuals. In addition, the folding of *XIST* lncRNA into an RNP complex may mask miR target sequences, which would either require the presence of free *XIST* lncRNA molecules in the nuclear space or high local concentrations of miRs in order to destabilize *XIST* RNP and access to miR complementary sequences. These considerations are rarely taken into account or discussed in the studies describing *XIST* lncRNA acting as a miR sponge. In particular, in *situ* analyses of *XIST* lncRNA localisation hardly show any convincing signal (i.e., an *XIST* RNA domain in the nucleus in female cells), the sex of the cell lines/animal models under investigation is often overlooked and shRNAs that are not supposed to enter the nucleus are generally used as *XIST* lncRNA interfering reagent.

Some associations between *XIST* lncRNA and specific miRs have however been confirmed in independent studies yet sometimes in different cell types/pathogenic contexts [48–53]. Some biochemical analyses report the presence of *XIST* lncRNA in the cytoplasmic fraction [48] or in the exosomes [50]. *XIST* may also be briefly

accessible to nonnuclear miRs for sponging during cell division, although this has not been shown as yet [54]. In-depth sequence analyses have moreover revealed that some miRs are translocated to the nucleus upon maturation thanks to a specific hexanucleotide stretch included within the miR sequence [54,55]. In addition, sex-specific pathogenic effects associated with *XIST*-mediated sponging of miR-92b have been observed [44] and a case of transient de-repression of *XIST* in patients and in male mice upon ischemic stroke has been described. In the latter, the increase of *Xist* lncRNA levels is thought to be accompanied by the binding of miR-92a to *Xist* and to result in alleviation of the ischemic phenotype [56]. *XIST* may also be abnormally expressed in male cancer cells that harbor supernumerary X chromosomes [57]. Hence, the ability of *XIST* lncRNA to regulate genes *via* miR sponging requires critical yet open-minded assessment using approaches that integrate XCI-related knowledge on *XIST* function.

### Conclusions

From its discovery in the early 1990s, the *XIST* lncRNA has served as a paragon for function and modus operandi of ncRNAs at large. Long considered as a nuclear RNA produced from a single X chromosome in XX female cells, which reconfigures the chromatin of the chromosome allowing for chromosome-wide transcriptional silencing, recent observations prompted us to revisit this somewhat reductive view. Indeed, novel functionalities have emerged.

1. *XIST* may coat both X chromosomes without promoting full gene silencing, which provides a way to finely tune X-chromosome expression depending on the developmental stage, cell type, or species.

2. *XIST* RNP associates with autosomal genes within the X chromosome nuclear vicinity. This implies that chromosome territories are spatially organized to allow specific *trans*-interactions with *XIST* RNPs.

3. *XIST* RNP may have bystander functions either inside or outside of the cell that are linked to its ability to concentrate proteins, RNA, and DNA.

4. *XIST* may sequester microRNAs whether in the frame of *XIST* RNP, at the Xi, within the nucleoplasm, or in the cytoplasm; this needs to be clarified and may depend on the cellular context.

These recent findings spur constant broadening of our perspectives on the functions and molecular mechanisms of *XIST*. Whether others remain to be discovered and how these regulatory functions are integrated in XX individuals remains to be fully understood.
**Declaration of competing interest**

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

**Acknowledgments**

Research in the Rougeulle lab is funded by the Agence Nationale de la Recherche (ANR- 14-CE10-0017), European Research Council under the European Union's Horizon 2020 research and innovation program (101020423), Labex 'Who Am I?' (ANR-11-LABX-0071) and Université Paris Cité IdEx (ANR-18-IDEX-0001) funded by the French Government through its 'Investments for the Future' program.

**Data availability**

No data was used for the research described in the article.

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This study uses an inducible Xist transgene introduced on chromosome 11 of male mice to show that ectopic expression of Xist RNP in male mice converts chromatin landscape and gene expression into a female-like profile. In addition, Xist RNP expression induces lupus upon administration of pristane in males of the SJL/J permissive genetic background.

39. Brown GJ, Cañete PF, Wang H, Medavy A, Bones J, Roco JA, He Y, Qin Y, Cappello J, Ellyard JI, et al.: TLR7 gain-of-function genetic variation causes human lupus. *Nature* 2022, **605**: 349–356.

40. Jones AN, Tikhaia E, Mourão A, Sattler M: Structural effects of m6A modification of the Xist A-repeat AUCG tetraloop and its recognition by YTHD1C. *Nucleic Acids Res* 2022, **60**: 2350–2362.

41. Lu Z, Guo JK, Wei Y, Dou DR, Zarnegar B, Ma Q, Li R, Zhao Y, Liu F, Choudhry H, et al.: Structural modularity of the XIST ribonucleoprotein complex. *Nat Commun* 2020, **11**:6163.

42. Busconi L, Lau CM, Tabor AS, Uccellini MB, Ruhe Z, Akira S, Viglianti GA, Rifkin IR: Marshak-Rothstein A: DNA and RNA autoantigenic as autoadjuvants. *J Endotoxin Res* 2006, **12**: 379–384.

43. Sun N, Zhang G, Liu Y: Long non-coding RNA XIST sponges miR-34a to promotes colon cancer progression via Wnt/β-catenin signaling pathway. *Gene* 2018, **665**:141–148.

44. Zhuang LK, Yang YT, Ma X, Han B, Wang ZS, Zhao QY, Wu LQ, Qu ZQ: MicroRNA-92b promotes hepatocellular carcinoma progression by targeting Smad7 and is mediated by long non-coding RNA XIST. *Cell Death Dis* 2016, **7**:e2203. e2203.

45. Wang X, Zhang G, Cheng Z, Dai L, Jia L, Jing X, Wang H, Zhang R, Liu M, Jiang T, et al.: Knockdown of LncRNA-XIST suppresses proliferation and TGF-β1-induced EMT in NSCLC through the notch-1 pathway by regulation of miR-137. *Genet Test Mol Biomarkers* 2018, **22**:333–342.

46. Zhang Y, Zhu Y, Gao G, Zhou Z: Knockdown XIST alleviates LPS-induced WI-38 cell apoptosis and inflammation injury via targeting miR-370-3p/TLR4 in acute pneumonia. *Cell Biochem Funct* 2019, **37**:348–358.

47. Wei M, Li L, Zhang Y, Zhang Z, Liu H, Bao H: LncRNA X inactive specific transcript contributes to neuropathic pain development by sponging miR-154-5p via inducing toll-like receptor 5 in CCI rat models. *J Cell Biochem* 2019, **120**:1271–1281.

48. Yu H, Xue Y, Wang P, Liu X, Ma J, Zheng J, Li Z, Li Z, Cai H, Liu Y: Knockdown of long non-coding RNA XIST increases blood–tumor barrier permeability and inhibits glioma angiogenesis by targeting miR-137. *Oncogenesis* 2017, **6**:e303. e303.

49. Zhao Y, Li S, Xia N, Shi Y, Zhao C: Effects of XIST/miR-137 axis on neuropathic pain by targeting TNFAIP1 in a rat model. *J Cell Physiol* 2018, **233**:4307–4316.

50. Cheng K, Pan J, Liu Q, Ji Y, Liu L, Guo X, Wang Q, Li S, Sun J, Gong M, et al.: Exosomal lncRNA XIST promotes perineural invasion of pancreatic cancer cells via miR-211-5p/GDNF. *Oncogene* 2024, **43**:1341–1352.

51. Li L, Lv G, Wang B, Kuang L: The role of lncRNA XIST/miR-211 axis in modulating the proliferation and apoptosis of osteo-arthritis chondrocytes through CXCR4 and MAPK signaling. *Biochem Biophys Res Commun* 2018, **503**:2555–2562.
52. Li Z, Zhang Y, Ding N, Zhao Y, Ye Z, Shen L, Yi H, Zhu Y: Inhibition of lncRNA XIST improves myocardial I/R injury by targeting miR-133a through inhibition of autophagy and regulation of SOCS2. *Mol Ther Nucleic Acids* 2019, **18**: 764–773.

53. Wei W, Liu Y, Lu Y, Yang B, Tang L: LncRNA XIST promotes pancreatic cancer proliferation through miR-133a/EGFR. *J Cell Biochem* 2017, **118**:3349–3358.

54. Marshall EA, Stewart GL, Sage AP, Lam WL, Brown CJ: Beyond sequence homology: cellular biology limits the potential of XIST to act as a miRNA sponge. *PLoS One* 2019, **14**, e0221371.

55. Hwang H-W, Wentzel EA, Mendell JT: A hexanucleotide element directs microRNA nuclear import. *Science* 2007, **315**:97–100.

56. Wang C, Dong J, Sun J, Huang S, Wu F, Zhang X, Pang D, Fu Y, Li L: Silencing of lncRNA XIST impairs angiogenesis and exacerbates cerebral vascular injury after ischemic stroke. *Mol Ther Nucleic Acids* 2021, **26**: 148–160.

57. Sadagopan A, Nasim IT, Li J, Achom M, Zhang C-Z, Viswanathan SR: Somatic XIST activation and features of X chromosome inactivation in male human cancers. *Cell Systems* 2022, **13**:932–944. e5.