A long noncoding RNA influences the choice of the X chromosome to be inactivated

X chromosome inactivation (XCI) is the process of silencing one of the X chromosomes in cells of the female mammal which ensures dosage compensation between the sexes. Although theoretically random in somatic tissues, the choice of which X chromosome is chosen to be inactivated can be biased in mice by genetic element(s) associated with the so-called X-controlling element (Xce). Although the Xce was first described and genetically localized nearly 40 y ago, its mode of action remains elusive. In the approach presented here, we identify a single long noncoding RNA (lncRNA) within the Xce locus, Lppnx, which may be the driving factor in the choice of which X chromosome will be inactivated in the developing female mouse embryo. Comparing weak and strong Xce alleles we show that Lppnx modulates the expression of Xist lncRNA, one of the key factors in XCI, by controlling the occupancy of pluripotency factors at Intron1 of Xist. This effect is counteracted by enhanced binding of Rex1 in DxPas34, another key element in XCI regulating the activity of Tsix lncRNA, the main antagonist of Xist, in the strong but not in the weak Xce allele. These results suggest that the different susceptibility for XCI observed in weak and strong Xce alleles results from differential transcription factor binding of Xist Intron 1 and DxPas34, and that Lppnx represents a decisive factor in explaining the action of the Xce

Variability of Sequence Surrounding the Xist Gene in Rodents Suggests Taxon-Specific Regulation of X Chromosome Inactivation

One of the two X chromosomes in female mammalian cells is subject to inactivation (XCI) initiated by the Xist gene. In this study, we examined in rodents (voles and rat) the conservation of the microsatellite region DXPas34, the Tsix gene (antisense counterpart of Xist), and enhancer Xite that have been shown to flank Xist and regulate XCI in mouse. We have found that mouse regions of the Tsix gene major promoter and minisatellite repeat DXPas34 are conserved among rodents. We have also shown that in voles and rat the region homologous to the mouse Tsix major promoter, initiates antisense to Xist transcription and terminates around the Xist gene start site as is observed with mouse Tsix. A conservation of Tsix expression pattern in voles, rat and mice suggests a crucial role of the antisense transcription in regulation of Xist and XIC in rodents. Most surprisingly, we have found that voles lack the regions homologous to the regulatory element Xite, which is instead replaced with the Slc7a3 gene that is unassociated with the X-inactivation centre in any other eutherians studied. Furthermore, we have not identified any transcription that could have the same functions as murine Xite in voles. Overall, our data show that not all the functional elements surrounding Xist in mice are well conserved even within rodents, thereby suggesting that the regulation of XCI may be at least partially taxon-specific

A Novel RNA Transcript with Antiapoptotic Function Is Silenced in Fragile X Syndrome

Several genome-wide transcriptomics efforts have shown that a large percentage of the mammalian genome is transcribed into RNAs, however, only a small percentage (1–2%) of these RNAs is translated into proteins. Currently there is an intense interest in characterizing the function of the different classes of noncoding RNAs and their relevance to human disease. Using genomic approaches we discovered FMR4, a primate-specific noncoding RNA transcript (2.4 kb) that resides upstream and likely shares a bidirectional promoter with FMR1. FMR4 is a product of RNA polymerase II and has a similar half-life to FMR1. The CGG expansion in the 5′ UTR of FMR1 appears to affect transcription in both directions as we found FMR4, similar to FMR1, to be silenced in fragile X patients and up-regulated in premutation carriers. Knockdown of FMR4 by several siRNAs did not affect FMR1 expression, nor vice versa, suggesting that FMR4 is not a direct regulatory transcript for FMR1. However, FMR4 markedly affected human cell proliferation in vitro; siRNAs knockdown of FMR4 resulted in alterations in the cell cycle and increased apoptosis, while the overexpression of FMR4 caused an increase in cell proliferation. Collectively, our results demonstrate an antiapoptotic function of FMR4 and provide evidence that a well-studied genomic locus can show unexpected functional complexity. It cannot be excluded that altered FMR4 expression might contribute to aspects of the clinical presentation of fragile X syndrome and/or related disorders

Tsx Produces a Long Noncoding RNA and Has General Functions in the Germline, Stem Cells, and Brain

The Tsx gene resides at the X-inactivation center and is thought to encode a protein expressed in testis, but its function has remained mysterious. Given its proximity to noncoding genes that regulate X-inactivation, here we characterize Tsx and determine its function in mice. We find that Tsx is actually noncoding and the long transcript is expressed robustly in meiotic germ cells, embryonic stem cells, and brain. Targeted deletion of Tsx generates viable offspring and X-inactivation is only mildly affected in embryonic stem cells. However, mutant embryonic stem cells are severely growth-retarded, differentiate poorly, and show elevated cell death. Furthermore, male mice have smaller testes resulting from pachytene-specific apoptosis and a maternal-specific effect results in slightly smaller litters. Intriguingly, male mice lacking Tsx are less fearful and have measurably enhanced hippocampal short-term memory. Combined, our study indicates that Tsx performs general functions in multiple cell types and links the noncoding locus to stem and germ cell development, learning, and behavior in mammals

A Dual Origin of the Xist Gene from a Protein-Coding Gene and a Set of Transposable Elements

X-chromosome inactivation, which occurs in female eutherian mammals is controlled by a complex X-linked locus termed the X-inactivation center (XIC). Previously it was proposed that genes of the XIC evolved, at least in part, as a result of pseudogenization of protein-coding genes. In this study we show that the key XIC gene Xist, which displays fragmentary homology to a protein-coding gene Lnx3, emerged de novo in early eutherians by integration of mobile elements which gave rise to simple tandem repeats. The Xist gene promoter region and four out of ten exons found in eutherians retain homology to exons of the Lnx3 gene. The remaining six Xist exons including those with simple tandem repeats detectable in their structure have similarity to different transposable elements. Integration of mobile elements into Xist accompanies the overall evolution of the gene and presumably continues in contemporary eutherian species. Additionally we showed that the combination of remnants of protein-coding sequences and mobile elements is not unique to the Xist gene and is found in other XIC genes producing non-coding nuclear RNA

The X-inactivation trans-activator Rnf12 is negatively regulated by pluripotency factors in embryonic stem cells

X-inactivation, the molecular mechanism enabling dosage compensation in mammals, is tightly controlled during mouse early embryogenesis. In the morula, X-inactivation is imprinted with exclusive silencing of the paternally inherited X-chromosome. In contrast, in the post-implantation epiblast, X-inactivation affects randomly either the paternal or the maternal X-chromosome. The transition from imprinted to random X-inactivation takes place in the inner cell mass (ICM) of the blastocyst from which embryonic stem (ES) cells are derived. The trigger of X-inactivation, Xist, is specifically downregulated in the pluripotent cells of the ICM, thereby ensuring the reactivation of the inactive paternal X-chromosome and the transient presence of two active X-chromosomes. Moreover, Tsix, a critical cis-repressor of Xist, is upregulated in the ICM and in ES cells where it imposes a particular chromatin state at the Xist promoter that ensures the establishment of random X-inactivation upon differentiation. Recently, we have shown that key transcription factors supporting pluripotency directly repress Xist and activate Tsix and thus couple Xist/Tsix control to pluripotency. In this manuscript, we report that Rnf12, a third X-linked gene critical for the regulation of X-inactivation, is under the control of Nanog, Oct4 and Sox2, the three factors lying at the heart of the pluripotency network. We conclude that in mouse ES cells the pluripotency-associated machinery exerts an exhaustive control of X-inactivation by taking over the regulation of all three major regulators of X-inactivation: Xist, Tsix, and Rnf12

Conformation Regulation of the X Chromosome Inactivation Center: A Model

X-Chromosome Inactivation (XCI) is the process whereby one, randomly chosen X becomes transcriptionally silenced in female cells. XCI is governed by the Xic, a locus on the X encompassing an array of genes which interact with each other and with key molecular factors. The mechanism, though, establishing the fate of the X's, and the corresponding alternative modifications of the Xic architecture, is still mysterious. In this study, by use of computer simulations, we explore the scenario where chromatin conformations emerge from its interaction with diffusing molecular factors. Our aim is to understand the physical mechanisms whereby stable, non-random conformations are established on the Xic's, how complex architectural changes are reliably regulated, and how they lead to opposite structures on the two alleles. In particular, comparison against current experimental data indicates that a few key cis-regulatory regions orchestrate the organization of the Xic, and that two major molecular regulators are involved

Sexually dimorphic gene expression emerges with embryonic genome activation and is dynamic throughout development

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public DomainVKR is supported by grants from the Biotechnology and Biological Sciences Research Council, UK (BB/M012494/1), VKR and CG by (BB/G00711/X/1). MLH is supported by a Research Council UK Academic Fellowship. RL is supported by EU-FP7 BLUEPRINT

Genes flanking Xist in mouse and human are separated on the X chromosome in American marsupials

X inactivation, the transcriptional silencing of one of the two X chromosomes in female mammals, achieves dosage compensation of X-linked genes relative to XY males. In eutherian mammals X inactivation is regulated by the X-inactive specific transcript (Xist), a cis-acting non-coding RNA that triggers silencing of the chromosome from which it is transcribed. Marsupial mammals also undergo X inactivation but the mechanism is relatively poorly understood. We set out to analyse the X chromosome in Monodelphis domestica and Didelphis virginiana, focusing on characterizing the interval defined by the Chic1 and Slc16a2 genes that in eutherians flank the Xist locus. The synteny of this region is retained on chicken chromosome 4 where other loci belonging to the evolutionarily ancient stratum of the human X chromosome, the so-called X conserved region (XCR), are also located. We show that in both M. domestica and D. virginiana an evolutionary breakpoint has separated the Chic1 and Slc16a2 loci. Detailed analysis of opossum genomic sequences revealed linkage of Chic1 with the Lnx3 gene, recently proposed to be the evolutionary precursor of Xist, and Fip1, the evolutionary precursor of Tsx, a gene located immediately downstream of Xist in eutherians. We discuss these findings in relation to the evolution of Xist and X inactivation in mammals