Céline Morey

Molecular Coupling of Xist Regulation and Pluripotency

During mouse embryogenesis, reversion of imprinted X chromosome inactivation in the pluripotent inner cell mass of the female blastocyst is initiated by the repression of Xist from the paternal X chromosome. Here we report that key factors supporting pluripotency—Nanog, Oct3/4, and Sox2—bind within Xist intron 1 in undifferentiated embryonic stem (ES) cells. Whereas Nanog null ES cells display a reversible and moderate up-regulation of Xist in the absence of any apparent modification of Oct3/4 and Sox2 binding, the drastic release of all three factors from Xist intron 1 triggers rapid ectopic accumulation of Xist RNA. We conclude that the three main genetic factors underlying pluripotency cooperate to repress Xist and thus couple X inactivation reprogramming to the control of pluripotency during embryogenesis.

Employment opportunities for non-coding RNAs

Analysis of the genomes of several higher eukaryotic organisms, including mouse and human, has reached the striking conclusion that the mammalian transcriptome is constituted in large part of non‐protein‐coding transcripts. Conversely, the number of protein‐coding genes was initially at least overestimated. A growing number of studies report the involvement of non‐coding transcripts in a large variety of regulatory processes. This review examines the different types of non‐coding RNAs (ncRNAs) and discusses their putative mode of action with particular reference to large ncRNAs and their role in epigenetic regulation.

The region 3′ to Xist mediates X chromosome counting and H3 Lys‐4 dimethylation within the Xist gene

A counting process senses the X chromosome/autosome ratio and ensures that X chromosome inactivation (XCI) initiates in the female (XX) but not in the male (XY) mouse embryo. Counting is regulated by the X‐inactivation centre, which contains the Xist gene. Deleting 65 kb 3′ to Xist in XO embryonic stem (ES) cells affects counting and results in inappropriate XCI upon differentiation. We show here that normal counting can be rescued in these deleted ES cells using cre/loxP re‐insertion, and refine the location of elements controlling counting within a 20 kb bipartite domain. Furthermore, we show that the 65 kb deletion also leads to inappropriate XCI in XY differentiated ES cells, which excludes the involvement of sex‐specific mechanisms in the initiation of XCI. At the chromatin level, we have found that the Xist gene corresponds to a peak of H3 Lys‐4 dimethylation, which is dramatically and specifically affected by the deletion 3′ to Xist. Our results raise the possibility that H3 Lys‐4 dimethylation within Xist may be functionally implicated in the counting process.

The Demoiselle of X-Inactivation: 50 Years Old and As Trendy and Mesmerising As Ever

In humans, sexual dimorphism is associated with the presence of two X chromosomes in the female, whereas males possess only one X and a small and largely degenerate Y chromosome. How do men cope with having only a single X chromosome given that virtually all other chromosomal monosomies are lethal? Ironically, or even typically many might say, women and more generally female mammals contribute most to the job by shutting down one of their two X chromosomes at random. This phenomenon, called X-inactivation, was originally described some 50 years ago by Mary Lyon and has captivated an increasing number of scientists ever since. The fascination arose in part from the realisation that the inactive X corresponded to a dense heterochromatin mass called the “Barr body” whose number varied with the number of Xs within the nucleus and from the many intellectual questions that this raised: How does the cell count the X chromosomes in the nucleus and inactivate all Xs except one? What kind of molecular mechanisms are able to trigger such a profound, chromosome-wide metamorphosis? When is X-inactivation initiated? How is it transmitted to daughter cells and how is it reset during gametogenesis? This review retraces some of the crucial findings, which have led to our current understanding of a biological process that was initially considered as an exception completely distinct from conventional regulatory systems but is now viewed as a paradigm “par excellence” for epigenetic regulation.

Genetics and epigenetics of the X chromosome

A consequence of Mendelian inheritance of X‐linked traits is that women are more than equal to men in the face of X‐linked diseases, protected as they are by the presence of two X chromosomes in their genome. This potentially beneficial inequality is diminished by the molecular mechanism known as X‐chromosome inactivation (XCI), which triggers the transcriptional silencing of one of the X chromosomes in each female cell. The determination of which X to inactivate, a process that occurs during early embryogenesis, is random and clonally inherited. As a result, females are mosaic for the expression of X‐linked genes. XCI is a highly regulated process involving large noncoding RNAs, chromatin remodeling, and nuclear reorganization of the X chromosome. It is a paradigm for epigenetic regulation and is frequently used as a biomarker for monitoring long‐range gene reprogramming during cell differentiation and dedifferentiation. Our review analyses how XCI affects the expression of X‐linked mutations, describes some of the most recent discoveries on the molecular mechanisms triggering XCI, and explores the therapeutic potentialities of the XCI process per se.

Spontaneous Reactivation of Clusters of X‐Linked Genes Is Associated with the Plasticity of X‐Inactivation in Mouse Trophoblast Stem Cells

Random epigenetic silencing of the X‐chromosome in somatic tissues of female mammals equalizes the dosage of X‐linked genes between the sexes. Unlike this form of X‐inactivation that is essentially irreversible, the imprinted inactivation of the paternal X, which characterizes mouse extra‐embryonic tissues, appears highly unstable in the trophoblast giant cells of the placenta. Here, we wished to determine whether such instability is already present in placental progenitor cells prior to differentiation toward lineage‐specific cell types. To this end, we analyzed the behavior of a GFP transgene on the paternal X both in vivo and in trophoblast stem (TS) cells derived from the trophectoderm of XXGFP blastocysts. Using single‐cell studies, we show that not only the GFP transgene but also a large number of endogenous genes on the paternal X are subject to orchestrated cycles of reactivation/de novo inactivation in placental progenitor cells. This reversal of silencing is associated with local losses of histone H3 lysine 27 trimethylation extending over several adjacent genes and with the topological relocation of the hypomethylated loci outside of the nuclear compartment of the inactive X. The “reactivated” state is maintained through several cell divisions. Our study suggests that this type of “metastable epigenetic” states may underlie the plasticity of TS cells and predispose specific genes to relaxed regulation in specific subtypes of placental cells. Stem Cells 2014;32:377–390

Lineage-specific regulation of imprinted X inactivation in extraembryonic endoderm stem cells

BackgroundSilencing of the paternal X chromosome (Xp), a phenomenon known as imprinted X-chromosome inactivation (I-XCI), characterises, amongst mouse extraembryonic lineages, the primitive endoderm and the extraembryonic endoderm (XEN) stem cells derived from it.ResultsUsing a combination of chromatin immunoprecipitation characterisation of histone modifications and single-cell expression studies, we show that whilst the Xp in XEN cells, like the inactive X chromosome in other cell types, globally accumulates the repressive histone mark H3K27me3, a large number of Xp genes locally lack H3K27me3 and escape from I-XCI. In most cases this escape is specific to the XEN cell lineage. Importantly, the degree of escape and the genes concerned remain unchanged upon XEN conversion into visceral endoderm, suggesting stringent control of I-XCI in XEN derivatives. Surprisingly, chemical inhibition of EZH2, a member of the Polycomb repressive complex 2 (PRC2), and subsequent loss of H3K27me3 on the Xp, do not drastically perturb the pattern of silencing of Xp genes in XEN cells.ConclusionsThe observations that we report here suggest that the maintenance of gene expression profiles of the inactive Xp in XEN cells involves a tissue-specific mechanism that acts partly independently of PRC2 catalytic activity.

A rapid passage through a two-active-X-chromosome state accompanies the switch of imprinted X-inactivation patterns in mouse trophoblast stem cells

BackgroundIn female mice, while the presence of two-active X-chromosomes characterises pluripotency, it is not tolerated in most other cellular contexts. In particular, in the trophoblastic lineage, impairment of paternal X (XP) inactivation results in placental defects.ResultsHere, we show that Trophoblast Stem (TS) cells can undergo a complete reversal of imprinted X-inactivation without detectable change in cell-type identity. This reversal occurs through a reactivation of the XP leading to TS clones showing two active Xs. Intriguingly, within such clones, all the cells rapidly and homogeneously either re-inactivate the XP or inactivate, de novo, the XM.ConclusionThis secondary non-random inactivation suggests that the two-active-X states in TS and in pluripotent contexts are epigenetically distinct. These observations also reveal a pronounced plasticity of the TS epigenome allowing TS cells to dramatically and accurately reprogram gene expression profiles. This plasticity may serve as a back-up system when X-linked mono-allelic gene expression is perturbed.

Antagonist Xist and Tsix co-transcription during mouse oogenesis and maternal Xist expression during pre-implantation development calls into question the nature of the maternal imprint on the X chromosome

During the first divisions of the female mouse embryo, the paternal X-chromosome is coated by Xist non-coding RNA and gradually silenced. This imprinted X-inactivation principally results from the apposition, during oocyte growth, of an imprint on the X-inactivation master control region: the X-inactivation center (Xic). This maternal imprint of yet unknown nature is thought to prevent Xist upregulation from the maternal X (XM) during early female development. In order to provide further insight into the XM imprinting mechanism, we applied single-cell approaches to oocytes and pre-implantation embryos at different stages of development to analyze the expression of candidate genes within the Xic. We show that, unlike the situation pertaining in most other cellular contexts, in early-growing oocytes, Xist and Tsix sense and antisense transcription occur simultaneously from the same chromosome. Additionally, during early development, Xist appears to be transiently transcribed from the XM in some blastomeres of late 2-cell embryos concomitant with the general activation of the genome indicating that XM imprinting does not completely suppress maternal Xist transcription during embryo cleavage stages. These unexpected transcriptional regulations of the Xist locus call for a re-evaluation of the early functioning of the maternal imprint on the X-chromosome and suggest that Xist/Tsix antagonist transcriptional activities may participate in imprinting the maternal locus as described at other loci subject to parental imprinting.