Xist condensates: perspectives for therapeutic intervention

Abstract

X-chromosome inactivation (XCI) is a crucial mechanism of dosage compensation in female mammals ensuring that genes from only one X chromosome are expressed, initiated through expression of the long noncoding RNA Xist. Recent evidence underscores the significance of molecular crowding-most likely via liquid-liquid phase separation (LLPS)-in forming Xist RNA-driven condensates critical for establishing and sustaining the silenced state. By integrating existing knowledge and emerging ideas, we provide a comprehensive perspective on the molecular underpinnings of XCI and outline how manipulation of LLPS-based mechanisms offers new avenues for novel therapeutic approaches.

Keywords: Condensate-modifying therapeutics; Condensates; Liquid-liquid phase separation; RNA-binding proteins; X-chromosome inactivation; X-linked disorders; X-reactivation; Xist.

Fig. 1

Comparative overview of Xist RNA repeat regions in humans and mice. A The Xist repeats exhibit evolutionary conservation between humans and mice, although most repeat regions vary considerably in copy number. B Xist (shown in red) comprises five functionally distinct repeat regions whose coordinated activities drive X‑chromosome inactivation (XCI): the A‑repeat at the 5′ end is highly conserved in both sequence and copy number and nucleates XCI initiation by recruiting SPEN to block RNA polymerase II (yellow) and promote removal of histone acetylation marks (green dots); the F‑repeat anchors the inactive X (Xi) to the nuclear lamina via the Lamin B Receptor (LBR), preserving higher‑order chromatin organization and sustained silencing; the B/C‑repeats engage Polycomb repressive complexes PRC1 and PRC2 to deposit H2AK119ub and H3K27me3, reinforcing chromatin compaction and maintenance of gene repression; the D‑repeat directs Xist RNA in cis along the Xi chromosome to ensure thorough coating of its target; and the E‑repeat drives phase separation to form Xist granules that concentrate silencing factors essential for stable gene silencing

Fig. 2

Spatial organization of the active and inactive X chromosome in the nucleus. Chromosomes are organized into distinct territories within the nucleus and are further divided into compartments (right), A compartment (green), which contains highly transcribed regions and B compartment which comprises heterochromatin and is divided into B1 (in blue, characterized by an abundance of Polycomb mark H3K27me3) and B4 subcompartment (in pink, tethered to the nuclear lamina). Top. The active X chromosome exhibits a chromatin arrangement dominated by A compartments (euchromatin, green), which allow RNA polymerase II to transcribe genes efficiently. Additionally, B compartments (heterochromatin, pink and blue) are present, although less prominent. Bottom. The spatial configuration of the inactive X chromosome (Xi) differs significantly. During differentiation, certain domains from the active X shift from the A compartment into the B compartment. The newly formed Xi predominantly adopts a repressed, B-like structure. In the early stages of X chromosome inactivation (XCI), Xist RNA selectively coats active regions, recruiting them into a B1-like subcompartment via Polycomb-associated chromatin modifications. Subsequently, SMCHD1 is recruited to the Xi, facilitating the partial merging of B subcompartments. This allows Xist RNA to further spread into the B4-like subcompartment, resulting in nearly complete silencing of the X chromosome. Escape regions, however, remain in the A-like compartment, preserving euchromatin and forming large chromatin loops

Fig. 3

Xist-mediated phase separation and protein recruitment on the inactive X chromosome (Xi). A Mechanism of phase separation driven by weak, multivalent interactions between nucleic acids and proteins. These interactions facilitate the formation of membrane-less condensates through phase separation. Condensate-promoting features are illustrated. Scaffold RNAs, such as Xist, initiate condensate assembly by recruiting proteins containing intrinsically disordered regions (IDRs), resulting in dynamic, reversible molecular environments. B Illustration of Xist RNA localization to the Xi in a population of cells, emphasizing its role in molecular crowding and the formation of Xist condensates. C Detailed view of Xist RNA with its characteristic repeats (A–F) interacting with various proteins known to harbor iIDRs. Key protein interactors include SPEN, RBM15, HNRNPK, TDP-43, CELF1, and MATR3. Dashed lines represent specific RNA–protein interactions mediated by the Xist transcript

Fig. 4

Therapeutic strategies for X-linked disorders. These methodologies include both gene function restoration and targeted editing, as well as reactivation of the inactive X chromosome. A Functional restoration via AAV (adeno‑associated viral)‑mediated gene therapy, mRNA‑based treatments, and protein replacement. B DNA and RNA editing approaches, including CRISPR‑Cas9 genome editing, base editing, and RNA editing. C Nonsense read‑through therapies to bypass premature stop codons. D Xi reactivation strategies employing antisense oligonucleotides (ASOs), small‑molecule modulators, short hairpin RNAs (shRNAs), and disruption of liquid–liquid phase separation (LLPS)

Fig. 5

Modulation of Xist condensate for therapeutic intervention. Schematic representation of possible strategies to target the Xist condensate formation to reactivate gene expression from the inactive X chromosome (Xi). Top, Targeting the scaffold (Xist): Modifying the structure or interaction valency of the Xist RNA scaffold using small molecules, aptamers, or peptides. These changes can alter Xist structural properties or weaken specific molecular interactions (e.g., electrostatic interactions), leading to reduced condensate stability. Middle, Targeting a condensate client: Disrupting the interactions between the client molecule and the scaffold (e.g., via small molecules, aptamers, or PROTACs) can result in the exclusion or mispartitioning of specific condensate components, thereby weakening the condensate. Bottom, Targeting condensate activity: Modulating protein–protein interactions or enzymatic activity within the condensate can interfere with its regulatory mechanisms. This approach alters condensate function and may promote reactivation of X-linked gene expression. All strategies aim to destabilize the Xist condensate, enabling the reactivation of previously silenced X-linked genes

Fig. 6

Strategies for the development of therapeutics targeting Xist condensates. Potential therapeutic approaches for targeting Xist condensates to achieve gene reactivation on the Xi. This figure represents just some of the possibilities for leveraging condensate biology as a therapeutic strategy. At the center, the general concept is depicted: targeting the Xist condensate to promote Xi reactivation. A Top square: Xist scaffold modulation through structural modification. Small molecules or RNA aptamers can directly bind the Xist RNA scaffold, altering its structure and multivalency. These modifications can disrupt RNA–protein interactions, destabilize the condensate, and promote gene reactivation. Bottom square: Xist scaffold modulation through m⁶A-dependent regulation. Inhibitors of the m⁶A writer METTL3 can reduce Xist methylation, impairing its ability to recruit m⁶A readers such as YTHDC1. Alternatively, tethering YTHDC1 directly to Xist can bypass the need for m⁶A, restoring condensate function and enabling precise control of gene silencing. B Top square: Client exclusion. Targeting client proteins recruited by Xist, such as SPEN (harboring an intrinsically disordered region, IDR), can disrupt essential protein interactions within the condensate, destabilizing it and enabling gene reactivation. Bottom square: De novo partitioning. Activators of gene expression can be selectively recruited to the condensate, promoting transcription and gene reactivation