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X-chromosome inactivation (XCI) is the epigenetic mechanism that ensures X-linked dosage compensation between cells of females (XX karyotype) and males (XY). XCI is essential for female embryos to survive through development and requires the accurate spatiotemporal regulation of many different factors to achieve remarkable chromosome-wide gene silencing. As a result of XCI, the active and inactive X chromosomes are functionally and structurally different, with the inactive X chromosome undergoing a major conformational reorganization within the nucleus. In this Review, we discuss the multiple layers of genetic and epigenetic regulation that underlie initiation of XCI during development and then maintain it throughout life, in light of the most recent findings in this rapidly advancing field. We discuss exciting new insights into the regulation of X inactive-specific transcript (XIST), the trigger and master regulator of XCI, and into the mechanisms and dynamics that underlie the silencing of nearly all X-linked genes. Finally, given the increasing interest in understanding the impact of chromosome organization on gene regulation, we provide an overview of the factors that are thought to reshape the 3D structure of the inactive X chromosome and of the relevance of such structural changes for XCI establishment and maintenance.X chromosome inactivation in mammals involves chromosome-wide gene silencing at one X chromosome in cells of females, a process that requires complex spatiotemporal regulation. Recent findings provide new insights into the mechanisms and dynamics of X chromosome inactivation and the accompanying 3D reshaping of the chromosome.

In mammals, dosage compensation of sex chromosomal genes between females (XX) and males (XY) is achieved through X-chromosome inactivation (XCI). The X-linked X-inactive-specific transcript (Xist) long noncoding RNA is indispensable for XCI and initiates the process early during development by spreading in cis across the X chromosome from which it is transcribed. During XCI, Xist RNA triggers gene silencing, recruits a plethora of chromatin modifying factors, and drives a major structural reorganization of the X chromosome. Here, we review our knowledge of the multitude of epigenetic events orchestrated by Xist RNA to allow female mammals to survive through embryonic development by establishing and maintaining proper dosage compensation. In particular, we focus on recent studies characterizing the interaction partners of Xist RNA, and we discuss how they have affected the field by addressing long-standing controversies or by giving rise to new research perspectives that are currently being explored. This review is dedicated to the memory of Denise Barlow, pioneer of genomic imprinting and functional long noncoding RNAs (lncRNAs), whose work has revolutionized the epigenetics field and continues to inspire generations of scientists.

Understanding the factors involved in chromatin dynamics is crucial for the study of biochemical processes in which distant genomic regions need to come in close proximity. Previous single locus tracking studies suggest that chromatin dynamics are linked to active transcription, but studies which compare the chromatin dynamics between different locations within a defined chromatin domain are still very limited. Here we used the ANCHOR3 DNA labeling system to track multiple cis -regulatory elements and non-regulatory control regions at different positions in the mouse Klf4 locus. We observe homogeneous chromatin motion of cis -regulatory elements and non-regulatory control regions in Klf4 transcribing mESCs and their non-transcribing EpiLCs daughter cells. These observations challenge the notion that active transcription has a major effect on the locus dynamics of mammalian genes.

Xist is indispensable for X chromosome inactivation. However, how Xist RNA directs chromosome-wide silencing and why some regions are more efficiently silenced than others remains unknown. Here, we explore the function of Xist by inducing ectopic Xist expression from multiple different X-linked and autosomal loci in mouse aneuploid and female diploid embryonic stem cells in which Xist-mediated silencing does not lead to lethal functional monosomy. We show that ectopic Xist expression faithfully recapitulates endogenous X chromosome inactivation from any location on the X chromosome, whereas long-range silencing of autosomal genes is less efficient. Long interspersed elements facilitate inactivation of genes located far away from the Xist transcription locus, and genes escaping X chromosome inactivation show enrichment of CTCF on X chromosomal but not autosomal loci. Our findings highlight important genomic and epigenetic features acquired during sex chromosome evolution to facilitate an efficient X chromosome inactivation process. Xist RNA is required for X chromosome inactivation but it is not well understood how Xist silences some regions more efficiently than others. Here, the authors induce ectopic Xist expression from multiple different X-linked and autosomal loci in cells to explore Xist function.

X chromosome inactivation (XCI) is a mammalian specific, developmentally regulated process relying on several mechanisms including antisense transcription, non-coding RNA-mediated silencing, and recruitment of chromatin remodeling complexes. In vitro modeling of XCI, through differentiation of embryonic stem cells (ESCs), provides a powerful tool to study the dynamics of XCI, overcoming the need for embryos, and facilitating genetic modification of key regulatory players. However, to date, robust initiation of XCI in vitro has been mostly limited to mouse pluripotent stem cells. Here, we adapted existing protocols to establish a novel monolayer differentiation protocol for rat ESCs to study XCI. We show that differentiating rat ESCs properly downregulate pluripotency factor genes, and present female specific Xist RNA accumulation and silencing of X-linked genes. We also demonstrate that RNF12 seems to be an important player in regulation of initiation of XCI in rat, acting as an Xist activator. Our work provides the basis to investigate the mechanisms directing the XCI process in a model organism different from the mouse.

Mutations in the F-box only protein 7 gene (FBXO7) cause PARK15, an autosomal recessive neurodegenerative disease presenting with severe levodopa-responsive parkinsonism and pyramidal disturbances. Understanding the PARK15 pathogenesis might thus provide clues on the mechanisms of maintenance of brain dopaminergic neurons, the same which are lost in Parkinson's disease. The protein(s) encoded by FBXO7 remain very poorly characterized. Here, we show that two protein isoforms are expressed from the FBXO7 gene in normal human cells. The isoform 1 is more abundant, particularly in primary skin fibroblasts. Both isoforms are undetectable in cell lines from the PARK15 patient of an Italian family; the isoform 1 is undetectable and the isoform 2 is severely decreased in the patients from a Dutch PARK15 family. In human cell lines and mouse primary neurons, the endogenous or over-expressed, wild type FBXO7 isoform 1 displays mostly a diffuse nuclear localization. An intact N-terminus is needed for the nuclear FBXO7 localization, as N-terminal modification by PARK15-linked missense mutation, or N-terminus tag leads to cytoplasmic mislocalization. Furthermore, the N-terminus of wild type FBXO7 (but not of mutant FBXO7) is able to confer nuclear localization to profilin (a cytoplasmic protein). Our data also suggest that overexpressed mutant FBXO7 proteins (T22M, R378G and R498X) have decreased stability compared to their wild type counterpart. In human brain, FBXO7 immunoreactivity was highest in the nuclei of neurons throughout the cerebral cortex, intermediate in the globus pallidum and the substantia nigra, and lowest in the hippocampus and cerebellum. In conclusion, the common cellular abnormality found in the PARK15 patients from the Dutch and Italian families is the depletion of the FBXO7 isoform 1, which normally localizes in the cell nucleus. The activity of FBXO7 in the nucleus appears therefore crucial for the maintenance of brain neurons and the pathogenesis of PARK15.

In mammals, gene dosage of X-chromosomal genes is equalized between the sexes by random inactivation of either one of the two X chromosomes in female cells. X chromosome inactivation (XCI) is initiated early during female embryonic development and can be recapitulated upon differentiation of female embryonic stem cells (ES). In this chapter, we provide a comprehensive review of XCI regulation in mammals, but also briefly highlight different dosage compensation strategies that evolved in other organisms. Important cis and trans acting regulators of XCI are presented, as well as factors involved in the maintenance of XCI. We also discuss different models that have been postulated to explain initiation of XCI in female cells. Finally, we describe X chromosome reactivation (XCR) in mouse development and the potential of induced pluripotent stem cells (iPSCs) as a model system to study the XCI process in mouse and human.

In placental females, one copy of the two X chromosomes is largely silenced during a narrow developmental time window, in a process mediated by the non-coding RNA Xist . Here, we demonstrate that Xist can initiate X-chromosome inactivation (XCI) well beyond early embryogenesis. By modifying its endogenous level, we show that Xist has the capacity to actively silence genes that escape XCI both in neuronal progenitor cells (NPCs) and , in mouse embryos. We also show that Xist plays a direct role in eliminating TAD-like structures associated with clusters of escapee genes on the inactive X chromosome, and that this is dependent on Xist's XCI initiation partner, SPEN . We further demonstrate that Xist's function in suppressing gene expression of escapees and topological domain formation is reversible for up to seven days post-induction, but that sustained Xist up-regulation leads to progressively irreversible silencing and CpG island DNA methylation of facultative escapees. Thus, the distinctive transcriptional and regulatory topologies of the silenced X chromosome is actively, directly - and reversibly - controlled by Xist RNA throughout life.

Unravelling how gene regulatory networks are remodelled during evolution is crucial to understand how species adapt to environmental changes. We addressed this question for X-chromosome inactivation, a process essential to female development that is governed, in eutherians, by the XIST lncRNA and its cis-regulators. To reach high resolution, we studied closely related primate species, spanning 55 million years of evolution. We show that the XIST regulatory circuitry has diversified extensively over such an evolutionary timeframe. The insertion of a HERVK transposon has reshuffled XIST 3D interaction network in macaque embryonic stem cells (ESC) and XIST expression is maintained by the additive effects of the JPX lncRNA gene and a macaque specific enhancer. In contrast, JPX is the main contributor to XIST expression in human ESCs but is not significantly involved in XIST regulation in marmoset ESCs. None of these entities are however under purifying selection, which suggests that neutrally evolving non-coding elements harbour high adaptive potentials.Competing Interest StatementThe authors have declared no competing interest.