Mechanisms of Choice in X-Chromosome Inactivation

Early in development, placental and marsupial mammals harbouring at least two X chromosomes per nuclei are faced with a choice that affects the rest of their lives: which of those X chromosomes to transcriptionally inactivate. This choice underlies phenotypical diversity in the composition of tissues and organs and in their response to environment, and can determine whether an individual will be healthy or affected by an X-linked disease. Here, we review our current understanding of the process of choice during X-chromosome inactivation and its implications, focusing on the strategies evolved by different mammalian lineages and on the known and unknown molecular mechanisms and players involved. We also call for a revised manner in which to think about choice during random X-inactivation.

The lncRNAs at X Chromosome Inactivation Center: Not Just a Matter of Sex Dosage Compensation

Non-coding RNAs (ncRNAs) constitute the majority of the transcriptome, as the result of pervasive transcription of the mammalian genome. Different RNA species, such as lncRNAs, miRNAs, circRNA, mRNAs, engage in regulatory networks based on their reciprocal interactions, often in a competitive manner, in a way denominated “competing endogenous RNA (ceRNA) networks” (“ceRNET”): miRNAs and other ncRNAs modulate each other, since miRNAs can regulate the expression of lncRNAs, which in turn regulate miRNAs, titrating their availability and thus competing with the binding to other RNA targets. The unbalancing of any network component can derail the entire regulatory circuit acting as a driving force for human diseases, thus assigning “new” functions to “old” molecules. This is the case of XIST, the lncRNA characterized in the early 1990s and well known as the essential molecule for X chromosome inactivation in mammalian females, thus preventing an imbalance of X-linked gene expression between females and males. Currently, literature concerning XIST biology is becoming dominated by miRNA associations and they are also gaining prominence for other lncRNAs produced by the X-inactivation center. This review discusses the available literature to explore possible novel functions related to ceRNA activity of lncRNAs produced by the X-inactivation center, beyond their role in dosage compensation, with prospective implications for emerging gender-biased functions and pathological mechanisms.

Single-cell landscape of nuclear configuration and gene expression during stem cell differentiation and X inactivation

Background Mammalian development is associated with extensive changes in gene expression, chromatin accessibility, and nuclear structure. Here, we follow such changes associated with mouse embryonic stem cell differentiation and X inactivation by integrating, for the first time, allele-specific data from these three modalities obtained by high-throughput single-cell RNA-seq, ATAC-seq, and Hi-C. Results Allele-specific contact decay profiles obtained by single-cell Hi-C clearly show that the inactive X chromosome has a unique profile in differentiated cells that have undergone X inactivation. Loss of this inactive X-specific structure at mitosis is followed by its reappearance during the cell cycle, suggesting a “bookmark” mechanism. Differentiation of embryonic stem cells to follow the onset of X inactivation is associated with changes in contact decay profiles that occur in parallel on both the X chromosomes and autosomes. Single-cell RNA-seq and ATAC-seq show evidence of a delay in female versus male cells, due to the presence of two active X chromosomes at early stages of differentiation. The onset of the inactive X-specific structure in single cells occurs later than gene silencing, consistent with the idea that chromatin compaction is a late event of X inactivation. Single-cell Hi-C highlights evidence of discrete changes in nuclear structure characterized by the acquisition of very long-range contacts throughout the nucleus. Novel computational approaches allow for the effective alignment of single-cell gene expression, chromatin accessibility, and 3D chromosome structure. Conclusions Based on trajectory analyses, three distinct nuclear structure states are detected reflecting discrete and profound simultaneous changes not only to the structure of the X chromosomes, but also to that of autosomes during differentiation. Our study reveals that long-range structural changes to chromosomes appear as discrete events, unlike progressive changes in gene expression and chromatin accessibility.

Intelligence Quotient Variability in Klinefelter Syndrome Is Associated With GTPBP6 Expression Under Regulation of X-Chromosome Inactivation Pattern

Klinefelter syndrome (KS) displays a broad dysmorphological, endocrinological, and neuropsychological clinical spectrum. We hypothesized that the neurocognitive dysfunction present in KS relies on an imbalance in X-chromosome gene expression. Thus, the X-chromosome inactivation (XCI) pattern and neurocognitive X-linked gene expression were tested and correlated with intelligence quotient (IQ) scores. We evaluated 11 KS patients by (a) IQ assessment, (b) analyzing the XCI patterns using both HUMARA and ZDHHC15 gene assays, and (c) blood RT-qPCR to investigate seven X-linked genes related to neurocognitive development (GTPBP6, EIF2S3, ITM2A, HUWE1, KDM5C, GDI1, and VAMP7) and XIST in comparison with 14 (male and female) controls. Considering IQ 80 as the standard minimum reference, we verified that the variability in IQ scores in KS patients seemed to be associated with the XCI pattern. Seven individuals in the KS group presented a random X-inactivation (RXI) and lower average IQ than the four individuals who presented a skewed X-inactivation (SXI) pattern. The evaluation of gene expression showed higher GTPBP6 expression in KS patients with RXI than in controls (p = 0.0059). Interestingly, the expression of GTPBP6 in KS patients with SXI did not differ from that observed in controls. Therefore, our data suggest for the first time that GTPBP6 expression is negatively associated with full-scale IQ under the regulation of the type of XCI pattern. The SXI pattern may regulate GTPBP6 expression, thereby dampening the impairment in cognitive performance and playing a role in intelligence variability in individuals with KS, which warrants further mechanistic investigations.

A temporally controlled sequence of X-chromosome inactivation and reactivation defines female mouse in vitro germ cells with meiotic potential

The early mammalian germ cell lineage is characterized by extensive epigenetic reprogramming, which is required for the maturation into functional eggs and sperm. In particular, the epigenome needs to be reset before parental marks can be established and then transmitted to the next generation. In the female germ line, reactivation of the inactive X chromosome is one of the most prominent epigenetic reprogramming events, and despite its scale involving an entire chromosome affecting hundreds of genes, very little is known about its kinetics and biological function. Here we investigate X-chromosome inactivation and reactivation dynamics by employing a tailor-made in vitro system to visualize the X-status during differentiation of primordial germ cell-like cells (PGCLCs) from female mouse embryonic stem cells (ESCs). We find that the degree of X-inactivation in PGCLCs is moderate when compared to somatic cells and characterized by a large number of genes escaping full inactivation. Nevertheless, PGCLCs that fail to undergo X-inactivation show an abnormal gene expression signature and deficiencies in meiotic entry. Subsequent to X-inactivation we observe gradual step-wise X-reactivation, which is mostly completed by the end of meiotic prophase I. Cells deviating from these progressive kinetics and undergoing X-reactivation too rapidly fail to enter a meiotic trajectory. Our data reveals that a fine-tuned X-inactivation and -reactivation cycle is a critical feature of female germ cell developmental competence towards meiosis and oogenesis