The Molecular and Nuclear Dynamics of X-Chromosome Inactivation

Abstract

In female eutherian mammals, dosage compensation of X-linked gene expression is achieved during development through transcriptional silencing of one of the two X chromosomes. Following X chromosome inactivation (XCI), the inactive X chromosome remains faithfully silenced throughout somatic cell divisions. XCI is dependent on Xist, a long noncoding RNA that coats and silences the X chromosome from which it is transcribed. Xist coating triggers a cascade of chromosome-wide changes occurring at the levels of transcription, chromatin composition, chromosome structure, and spatial organization within the nucleus. XCI has emerged as a paradigm for the study of such crucial nuclear processes and the dissection of their functional interplay. In the past decade, the advent of tools to characterize and perturb these processes have provided an unprecedented understanding into their roles during XCI. The mechanisms orchestrating the initiation of XCI as well as its maintenance are thus being unraveled, although many questions still remain. Here, we introduce key aspects of the XCI process and review the recent discoveries about its molecular basis.

Figure 1.

Dynamics of X chromosome inactivation (XCI) during early mouse development. (A) Following fertilization and zygotic genome activation, both X chromosomes are expressed. (B) At the four-cell stage, imprinted XCI initiates, and (C) by the early blastocyst stage, all cells have inactivated the paternal X (Xp). The Xp remains inactivated in extraembryonic cells (trophectoderm and hypoblast). (D) On the other hand, the Xp becomes reactivated in cells that will later give rise to the epiblast (embryo proper). (E) In these cells, a second wave of XCI (random) initiates at the late blastocyst stage. Hence, after implantation and clonal propagation of XCI status, half of the cells of the embryo proper will have inactivated the Xp, and the other half the maternal X (Xm). (F) Random XCI hence results in cellular mosaicism with respect to X-linked allelic expression, which can, for example, translate into mosaic hair phenotypes.

Figure 2.

The Xist lncRNA coats the X chromosome and triggers gene silencing in cis. (A) The position of the X inactivation center (Xic, highlighted in red), is shown relative to the mouse X chromosome. (B) The Xist transcription unit (located within the Xic) is shown with exons corresponding to the predominant transcript isoform represented by blue rectangles. Xist RNA is capped, spliced, and polyadenylated to yield a 15-kb-long noncoding RNA. Xist is composed of several repeat elements (labeled A–F), some of which are important for Xist localization (blue) and Xist-mediated gene silencing (black). (C) (Left panel) Before X chromosome inactivation (XCI), both X chromosomes are expressed and, consequently, the X-linked gene Huwe1 (green rectangle) is biallelically expressed (two green foci of nascent transcription are detected by RNA FISH in murine embryonic stem cells (mESCs). XCI initiates with the up-regulation of Xist on one of the two X chromosomes. (Middle panel) Xist RNA coats the chromosome from which it is expressed, forming a detectable Xist cloud (red domain detected by RNA FISH). (Right panel) Xist coating triggers cis-transcriptional silencing of virtually all genes across the X chromosome, with Huwe1 transcription being restricted to the active (i.e., non-Xist-coated) X chromosome. (Image courtesy of the authors. N.B. some images were modified for illustration purposes.) (D) Xist does not spread uniformly through time across the X chromosome. Instead, Xist first spreads into regions termed Xist entry sites (shown in blue), which are spatially proximal to the site of Xist transcription.

Figure 3.

cis- and trans-regulation of Xist expression before and after exit from pluripotency. (A) At the pluripotent state, Xist expression is repressed by the combination of cis- and trans-regulators. (A1) Tsix transcription, which is promoted by its Xite and DXPas34 enhancer elements, represses Xist expression in cis. Tsix expression is driven by several key pluripotency factors. Conversely, those same pluripotency factors also repress Xist in trans. (A2) The Xic is spatially partitioned into the Xist and Tsix-TADs (topologically associating domains), which contain their respective cis-activators. Linx represses Xist expression, in cis, across the TADs boundary. Rnf12 is also repressed in trans by several pluripotency factors. (B) Upon pluripotency exit, pluripotency factors are depleted, and their trans-repressive activity on Xist and Rnf12 is relieved. (B1) Furthermore, RNF12 ubiquitinates REX1, triggering its degradation. Xist expression is further activated in trans by YY1, and (B2) in cis by Jpx, Ftx, Xert, and XistAR.

Figure 4.

Positioning of the inactive X chromosome at the nuclear periphery and/or proximal to the nucleolus. Through its RS motif, LBR (the lamin B receptor) directly binds Xist and recruits the X chromosome to the nuclear lamina. Targeting the Xi to the perinucleolar space requires Xist and Firre, although the mechanisms involved are unknown.

Figure 5.

Spatial and structural organization of the inactive X chromosome. (A) Following coating by Xist, the X chromosome forms a compartment within which RNA polymerase II (RNAPII) concentration is significantly lower than in the nucleoplasm. As X chromosome inactivation (XCI) proceeds, silenced genes become localized inside this compartment, while escapees remain outside or at the periphery. (B) The inactive X chromosome adopts a peculiar structure, characterized by two megadomains separated by the Dxz4 boundary element, harboring strong CTCF binding. While the megadomains show poor local structure, some level of organization is retained at clusters of escaping genes, which form topologically associating domain (TAD)-like structures.

Figure 6.

Xist interactors implicated in gene silencing during X chromosome inactivation (XCI). (A) Several factors implicated in Xist-mediated gene silencing were identified when characterizing the Xist protein interactome. Of note, RBM15 and SPEN both bind the A-repeat of Xist while hnRNPK interacts with a sequence region comprising the B- and part of the C-repeat, termed PID (Polycomb interaction domain). (B) Upon binding Xist A-repeat, RBM15 recruits the m⁶A RNA methylation machinery to Xist. m⁶A sites on Xist then recruit the YTHDC1 reader protein, which has been proposed to play a role in gene silencing during XCI. (C) Upon binding Xist, hnRNPK recruits noncanonical PRC1 by interacting directly with PCGF3/5. RING1B-mediated ubiquitination of H2AK119 stimulates PRC2 recruitment, through JARID2 binding to H2AK119ub. EZH2 then catalyzes H3K27 trimethylation, which enables the recruitment of canonical PRC1. (D) Immediately upon Xist up-regulation, SPEN's RNA recognition motifs (RRMs) bind the A-repeat of Xist RNA. Xist then targets SPEN to the promoters and enhancers of active X-linked genes, where SPOC engages with NCoR/HDAC3, NuRD, and RNAPII to promote transcriptional repression. Following gene silencing, SPEN disengages from silenced chromatin.