SUMMARY Differentiating pluripotent epiblast cells in eutherians undergo random X-inactivation, which equalizes X-linked gene expression between the sexes by silencing one of the two X-chromosomes in females. Tsix RNA is believed to orchestrate the initiation of X-inactivation, influencing the choice of which X remains active by preventing expression of the antisense Xist RNA, which is required to silence the inactive-X. Here we profile X-chromosome activity in Tsix-mutant (XΔTsix) mouse embryonic epiblasts, epiblast stem cells, and embryonic stem cells. Unexpectedly, we find that Xist is stably repressed on the XΔTsix in both sexes in undifferentiated epiblast cells in vivo and in vitro, resulting in stochastic X-inactivation in females despite Tsix-heterozygosity. Tsix is instead required to silence Xist on the active-X as epiblast cells differentiate in both males and females. Thus, Tsix is not required at the onset of random X-inactivation; instead, it protects the active-X from ectopic silencing once X-inactivation has commenced.

Birds and other vertebrates display stunning variation in pigmentation patterning, yet the genes controlling this diversity remain largely unknown. Rock pigeons (Columba livia) are fundamentally one of four color pattern phenotypes, in decreasing order of melanism: T-check, checker, bar (ancestral), or barless. Using whole-genome scans, we identified NDP as a candidate gene for this variation. Allele-specific expression differences in NDP indicate cis-regulatory divergence between ancestral and melanistic alleles. Sequence comparisons suggest that derived alleles originated in the speckled pigeon (Columba guinea), providing a striking example of introgression. In contrast, barless rock pigeons have an increased incidence of vision defects and, like human families with hereditary blindness, carry start-codon mutations in NDP. In summary, we find that both coding and regulatory variation in the same gene drives wing pattern diversity, and post-domestication introgression supplied potentially advantageous melanistic alleles to feral populations of this ubiquitous urban bird.

Imprinted X-inactivation is a paradigm of mammalian transgenerational epigenetic regulation resulting in silencing of genes on the paternally-inherited X-chromosome. The pre-programmed fate of the X-chromosomes is thought to be controlled in cis by the parent-of-origin-specific expression of two long non-coding RNAs, Tsix and Xist, in mice. Exclusive expression of Tsix from the maternal–X has implicated it as the instrument through which the maternal germline prevents inactivation of the maternal–X in the offspring. Here, we show that Tsix is dispensable for inhibiting Xist and X-inactivation in the early embryo and in cultured stem cells of extra-embryonic lineages. Tsix is instead required to prevent Xist expression as trophectodermal progenitor cells differentiate. Despite induction of wild-type Xist RNA and accumulation of histone H3-K27me3, many Tsix-mutant X-chromosomes fail to undergo ectopic X-inactivation. We propose a novel model of lncRNA function in imprinted X-inactivation that may also apply to other genomically imprinted loci.

The domestic rock pigeon (Columba livia) is among the most widely distributed and phenotypically diverse avian species. C. livia is broadly studied in ecology, genetics, physiology, behavior, and evolutionary biology, and has recently emerged as a model for understanding the molecular basis of anatomical diversity, the magnetic sense, and other key aspects of avian biology. Here we report an update to the C. livia genome reference assembly and gene annotation dataset. Greatly increased scaffold lengths in the updated reference assembly, along with an updated annotation set, provide improved tools for evolutionary and functional genetic studies of the pigeon, and for comparative avian genomics in general.

X-inactive specific transcript (Xist) long noncoding RNA (lncRNA) is thought to catalyze silencing of X-linked genes in cis during X-chromosome inactivation, which equalizes X-linked gene dosage between male and female mammals. To test the impact of Xist RNA on X-linked gene silencing, we ectopically induced endogenous Xist by ablating the antisense repressor Tsix in mice. We find that ectopic Xist RNA induction and subsequent X-linked gene silencing is sex specific in embryos and in differentiating embryonic stem cells (ESCs) and epiblast stem cells (EpiSCs). A higher frequency of X ΔTsix Y male cells displayed ectopic Xist RNA coating compared with X ΔTsix X female cells. This increase reflected the inability of X ΔTsix Y cells to efficiently silence X-linked genes compared with X ΔTsix X cells, despite equivalent Xist RNA induction and coating. Silencing of genes on both Xs resulted in significantly reduced proliferation and increased cell death in X ΔTsix X female cells relative to X ΔTsix Y male cells. Thus, whereas Xist RNA can inactivate the X chromosome in females it may not do so in males. We further found comparable silencing in differentiating X ΔTsix Y and 39,X ΔTsix (X ΔTsix O) ESCs, excluding the Y chromosome and instead implicating the X-chromosome dose as the source of the sexspecific differences. Because X ΔTsix X female embryonic epiblast cells and EpiSCs harbor an inactivated X chromosome prior to ectopic inactivation of the active X ΔTsix X chromosome, we propose that the increased expression of one or more X-inactivation escapees activates Xist and, separately, helps trigger X-linked gene silencing.inactivation represents a paradigm of epigenetic regulation and long noncoding RNA (lncRNA) function. In XX female cells, one of the two X chromosomes undergoes transcriptional silencing (1). Moreover, replicated copies of the active and inactive X chromosomes faithfully maintain their respective transcriptional states through many cell division cycles (2-5).X inactivation requires the X-inactive specific transcript (Xist) (6-8), a lncRNA that is selectively expressed from and physically coats the future inactive X chromosome (9-12). Xist RNA enables X-linked gene silencing by recruiting protein complexes to the inactive X (13-15). Female mouse embryos that inherit a paternal Xist mutation die due to defects in imprinted X inactivation of the paternal X chromosome in extraembryonic tissues (8,16,17). Xist is also required in the epiblast-derived embryonic cells, which undergo random X inactivation of either the maternal or the paternal X chromosome. Xist heterozygote fetal cells exhibit inactivation of only the X chromosome with an intact Xist locus, suggesting that Xist is necessary to choose the X chromosome to be inactivated (7,18,19). That the Xist-mutant X chromosome is not selected for inactivation, however, precludes assigning to Xist RNA a gene silencing role in the epiblast lineage.Ectopic expression studies have, however, demonstrated that Xist RNA can silence genes, albeit in a context-...