To achieve a balanced gene expression dosage between males (XY) and females (XX), mammals have evolved a compensatory mechanism to randomly inactivate one of the female X chromosomes. Despite this chromosome-wide silencing, a number of genes escape X inactivation: in women about 15% of X-linked genes are bi-allelically expressed and in mice, about 3%. Expression from the inactive X allele varies from a few percent of that from the active allele to near equal expression. While most genes have a stable inactivation pattern, a subset of genes exhibit tissue-specific differences in escape from X inactivation. Escape genes appear to be protected from the repressive chromatin modifications associated with X inactivation. Differences in the identity and distribution of escape genes between species and tissues suggest a role for these genes in the evolution of sex differences in specific phenotypes. The higher expression of escape genes in females than in males implies that they may have female-specific roles and may be responsible for some of the phenotypes observed in X aneuploidy.

X chromosome inactivation takes place in the early development of female mammals and depends on the Xist gene expression. The mechanisms of Xist expression regulation have not been well understood so far. In this work, we compared Xist promoter region of vole Microtus rossiaemeridionalis and other mammalian species. We observed three conserved regions which were characterized by computational analysis, DNaseI in vitro footprinting, and reporter construct assay. Regulatory factors potentially involved in Xist activation and repression in voles were determined. The role of CpG methylation in vole Xist expression regulation was established. A CTCF binding site was found in the 5′ flanking region of the Xist promoter on the active X chromosome in both males and females. We suggest that CTCF acts as an insulator which defines an inactive Xist domain on the active X chromosome in voles.

Rett syndrome (RTT) is a neurodevelopmental disorder that affects girls due primarily to heterozygous mutations in the X-linked gene encoding methyl-CpG binding protein 2 (MECP2). Random X-chromosome inactivation (XCI) results in cellular mosaicism in which some cells express wild-type (WT) MECP2 while other cells express mutant MECP2. The generation of patient-specific human induced pluripotent stem cells (hiPSCs) facilitates the production of RTT-hiPSC-derived neurons in vitro to investigate disease mechanisms and identify novel drug treatments. The generation of RTT-hiPSCs has been reported by many laboratories, however, the XCI status of RTT-hiPSCs has been inconsistent. Some report RTT-hiPSCs retain the inactive X-chromosome (post-XCI) of the founder somatic cell allowing isogenic RTT-hiPSCs that express only the WT or mutant MECP2 allele to be isolated from the same patient. Post-XCI RTT-hiPSCs-derived neurons retain this allele-specific expression pattern of WT or mutant MECP2. Conversely, others report RTT-hiPSCs in which the inactive X-chromosome of the founder somatic cell reactivates (pre-XCI) upon reprogramming into RTT-hiPSCs. Pre-XCI RTT-hiPSC-derived neurons exhibit random XCI resulting in cellular mosaicism with respect to WT and mutant MECP2 expression. Here we review and attempt to interpret the inconsistencies in XCI status of RTT-hiPSCs generated to date by comparison to other pluripotent systems in vitro and in vivo and the methods used to analyze XCI. Finally, we discuss the relative strengths and weaknesses of post- and pre-XCI hiPSCs in the context of RTT, and other X-linked and autosomal disorders for translational medicine.

To achieve dosage balance of X-linked genes between mammalian males and females, one female X chromosome becomes inactivated. However, approximately 15% of genes on this inactivated chromosome escape X chromosome inactivation (XCI). Here, using a chromosome-wide analysis of primate X-linked orthologs, we test a hypothesis that such genes evolve under a unique selective pressure. We find that escape genes are subject to stronger purifying selection than inactivated genes and that positive selection does not significantly affect the evolution of these genes. The strength of selection does not differ between escape genes with similar versus different expression levels in males versus females. Intriguingly, escape genes possessing Y homologs evolve under the strongest purifying selection. We also found evidence of stronger conservation in gene expression levels in escape than inactivated genes. We hypothesize that divergence in function and expression between X and Y gametologs is driving such strong purifying selection for escape genes.

Rett syndrome (RTT) is a neurodevelopmental autism spectrum disorder that affects girls due primarily to mutations in the gene encoding methyl-CpG binding protein 2 (MECP2). The majority of RTT patients carry missense and nonsense mutations leading to a hypomorphic MECP2, while null mutations leading to the complete absence of a functional protein are rare. MECP2 is an X-linked gene subject to random X-chromosome inactivation resulting in mosaic expression of mutant MECP2. The lack of human brain tissue motivates the need for alternative human cellular models to study RTT. Here we report the characterization of a MECP2 mutation in a classic female RTT patient involving rearrangements that remove exons 3 and 4 creating a functionally null mutation. To generate human neuron models of RTT, we isolated human induced pluripotent stem (hiPS) cells from RTT patient fibroblasts. RTT-hiPS cells retained the MECP2 mutation, are pluripotent and fully reprogrammed, and retained an inactive X-chromosome in a nonrandom pattern. Taking advantage of the latter characteristic, we obtained a pair of isogenic wild-type and mutant MECP2 expressing RTT-hiPS cell lines that retained this MECP2 expression pattern upon differentiation into neurons. Phenotypic analysis of mutant RTT-hiPS cell-derived neurons demonstrated a reduction in soma size compared with the isogenic control RTT-hiPS cell-derived neurons from the same RTT patient. Analysis of isogenic control and mutant hiPS cell-derived neurons represents a promising source for understanding the pathogenesis of RTT and the role of MECP2 in human neurons.

What genomic landmarks render most genes silent while leaving others expressed on the inactive X chromosome in mammalian females? To date, signals determining expression status of genes on the inactive X remain enigmatic despite the availability of complete genomic sequences. Long interspersed repeats (L1s), particularly abundant on the X, are hypothesized to spread the inactivation signal and are enriched in the vicinity of inactive genes. However, both L1s and inactive genes are also more prevalent in ancient evolutionary strata. Did L1s accumulate there because of their role in inactivation or simply because they spent more time on the rarely recombining X? Here we utilize an experimentally derived inactivation profile of the entire human X chromosome to uncover sequences important for its inactivation, and to predict expression status of individual genes. Focusing on Xp22, where both inactive and active genes reside within evolutionarily young strata, we compare neighborhoods of genes with different inactivation states to identify enriched oligomers. Occurrences of such oligomers are then used as features to train a linear discriminant analysis classifier. Remarkably, expression status is correctly predicted for 84% and 91% of active and inactive genes, respectively, on the entire X, suggesting that oligomers enriched in Xp22 capture most of the genomic signal determining inactivation. To our surprise, the majority of oligomers associated with inactivated genes fall within L1 elements, even though L1 frequency in Xp22 is low. Moreover, these oligomers are enriched in parts of L1 sequences that are usually underrepresented in the genome. Thus, our results strongly support the role of L1s in X inactivation, yet indicate that a chromatin microenvironment composed of multiple genomic sequence elements determines expression status of X chromosome genes.

Synopsis

To match the amount of gene product produced in males (XY), most genes in mammalian females (XX) are active on one X chromosome and inactivated on the other. However, some genes “escape” inactivation and are expressed from both X chromosomes. This study investigates sequences that may control whether a gene undergoes or escapes X chromosome inactivation, including DNA sequences previously thought of as non-functional or “junk.” Earlier work suggested that one such sequence, L1 interspersed repeats, may be associated with inactivation, but the extent of such association, and whether it represented a consequence of the evolutionary history of X, remained unclear. This study utilized recently generated chromosome-wide data on sequence and gene expression for human X, with a particular focus on the Xp22 region, which is evolutionarily young and has had no time to accumulate many L1 elements. A rigorous statistical analysis identified with high accuracy a set of short sequences that discriminate between genes undergoing and those escaping X chromosome inactivation. Interestingly, the majority of such sequences enriched in the vicinity of inactivated genes were found within L1s. These results strengthen the case for an involvement of L1s in X chromosome inactivation and suggest other DNA elements that might also play a role.