In somatic cells of female placental mammals, one of the two X chromosomes is transcriptionally silenced to accomplish an equal dose of X-encoded gene products in males and females. Initiation of random X chromosome inactivation (XCI) is thought to be regulated by X-encoded activators and autosomally encoded suppressors controlling Xist. Spreading of Xist RNA leads to silencing of the X chromosome in cis. Here, we demonstrate that the dose dependent X-encoded XCI activator RNF12/RLIM acts in trans and activates Xist. We did not find evidence for RNF12-mediated regulation of XCI through Tsix or the Xist intron 1 region, which are both known to be involved in inhibition of Xist. In addition, we found that Xist intron 1, which contains a pluripotency factor binding site, is not required for suppression of Xist in undifferentiated ES cells. Analysis of female Rnf12⁻/⁻ knockout ES cells showed that RNF12 is essential for initiation of XCI and is mainly involved in the regulation of Xist. We conclude that RNF12 is an indispensable factor in up-regulation of Xist transcription, thereby leading to initiation of random XCI.

DNA replication occurs in a defined temporal order known as the replication-timing (RT) program. RT is regulated during development in discrete chromosomal units, coordinated with transcriptional activity and 3D genome organization. Here, we derived distinct cell types from F1 hybrid musculus × castaneus mouse crosses and exploited the high single-nucleotide polymorphism (SNP) density to characterize allelic differences in RT (Repli-seq), genome organization (Hi-C and promoter-capture Hi-C), gene expression (total nuclear RNA-seq), and chromatin accessibility (ATAC-seq). We also present HARP, a new computational tool for sorting SNPs in phased genomes to efficiently measure allele-specific genome-wide data. Analysis of six different hybrid mESC clones with different genomes (C57BL/6, 129/sv, and CAST/Ei), parental configurations, and gender revealed significant RT asynchrony between alleles across ∼12% of the autosomal genome linked to subspecies genomes but not to parental origin, growth conditions, or gender. RT asynchrony in mESCs strongly correlated with changes in Hi-C compartments between alleles but not as strongly with SNP density, gene expression, imprinting, or chromatin accessibility. We then tracked mESC RT asynchronous regions during development by analyzing differentiated cell types, including extraembryonic endoderm stem (XEN) cells, four male and female primary mouse embryonic fibroblasts (MEFs), and neural precursor cells (NPCs) differentiated in vitro from mESCs with opposite parental configurations. We found that RT asynchrony and allelic discordance in Hi-C compartments seen in mESCs were largely lost in all differentiated cell types, accompanied by novel sites of allelic asynchrony at a considerably smaller proportion of the genome, suggesting that genome organization of homologs converges to similar folding patterns during cell fate commitment.

The directed differentiation of patient-derived induced pluripotent stem cells into cell-type specific neurons has inspired the development of therapeutic discovery for neurodegenerative diseases. Many forms of ataxia result from degeneration of cerebellar Purkinje cells, but thus far it has not been possible to efficiently generate Purkinje neuron (PN) progenitors from human or mouse pluripotent stem cells, let alone to develop a methodology for in vivo transplantation in the adult cerebellum. Here, we present a protocol to obtain an expandable population of cerebellar neuron progenitors from mouse embryonic stem cells. Our protocol is characterized by applying factors that promote proliferation of cerebellar progenitors. Cerebellar progenitors isolated in culture from cell aggregates contained a stable subpopulation of PN progenitors that could be expanded for up to 6 passages. When transplanted into the adult cerebellum of either wild-type mice or a strain lacking Purkinje cells (L7cre-ERCC1 knockout), GFP-labeled progenitors differentiated in vivo to establish a population of calbindin-positive cells in the molecular layer with dendritic trees typical of mature PNs. We conclude that this protocol may be useful for the generation and maturation of PNs, highlighting the potential for development of a regenerative medicine approach to the treatment of cerebellar neurodegenerative diseases.

X chromosome inactivation (XCI) is the mammalian mechanism that compensates for the difference in gene dosage between XX females and XY males. Genetic and epigenetic regulatory mechanisms induce transcriptional silencing of one X chromosome in female cells. In mouse embryos, XCI is initiated at the preimplantation stage following early whole-genome activation. It is widely thought that human embryos do not employ XCI prior to implantation. Here, we show that female preimplantation embryos have a progressive accumulation of XIST RNA on one of the two X chromosomes, starting around the 8-cell stage. XIST RNA accumulates at the morula and blastocyst stages and is associated with transcriptional silencing of the XIST-coated chromosomal region. These findings indicate that XCI is initiated in female human preimplantation-stage embryos and suggest that preimplantation dosage compensation is evolutionarily conserved in placental mammals.

DNA methylation is a well-known epigenetic modification that plays a crucial role in gene regulation, but genome-wide analysis of DNA methylation remains technically challenging and costly. DNA methylation-dependent restriction enzymes can be used to restrict CpG methylation analysis to methylated regions of the genome only, which significantly reduces the required sequencing depth and simplifies subsequent bioinformatics analysis. Unfortunately, this approach has been hampered by complete digestion of DNA in CpG methylation-dense regions, resulting in fragments that are too small for accurate mapping. Here, we show that the activity of DNA methylation-dependent enzyme, LpnPI, is blocked by a fragment size smaller than 32 bp. This unique property prevents complete digestion of methylation-dense DNA and allows accurate genome-wide analysis of CpG methylation at single-nucleotide resolution. Methylated DNA sequencing (MeD-seq) of LpnPI digested fragments revealed highly reproducible genome-wide CpG methylation profiles for >50% of all potentially methylated CpGs, at a sequencing depth less than one-tenth required for whole-genome bisulfite sequencing (WGBS). MeD-seq identified a high number of patient and tissue-specific differential methylated regions (DMRs) and revealed that patient-specific DMRs observed in both blood and buccal samples predict DNA methylation in other tissues and organs. We also observed highly variable DNA methylation at gene promoters on the inactive X Chromosome, indicating tissue-specific and interpatient-specific escape of X Chromosome inactivation. These findings highlight the potential of MeD-seq for high-throughput epigenetic profiling.

Approximately one-third of the patients with well-differentiated liposarcoma (WDLPS) will develop a local recurrence. Not much is known about the molecular relationship between the primary tumor and the recurrent tumor, which is important to reveal potential drivers of recurrence. Here we investigated the biology of recurrent WDLPS by comparing paired primary and recurrent WDLPS using microRNA profiling and genome-wide DNA methylation analyses. In total, 27 paired primary and recurrent WDLPS formalin-fixed and paraffin-embedded tumor samples were collected. MicroRNA expression profiles were determined using TaqMan® Low Density Array (TLDA) cards. Genome-wide DNA methylation and differentially methylated regions (DMRs) were assessed by methylated DNA sequencing (MeD-seq). A supervised cluster analysis based on differentially expressed microRNAs between paired primary and recurrent WDLPS did not reveal a clear cluster pattern separating the primary from the recurrent tumors. The clustering was also not based on tumor localization, time to recurrence, age or status of the resection margins. Changes in DNA methylation between primary and recurrent tumors were extremely variable, and no consistent DNA methylation changes were found. As a result, a supervised clustering analysis based on DMRs between primary and recurrent tumors did not show a distinct cluster pattern based on any of the features. Subgroup analysis for tumors localized in the extremity or the retroperitoneum also did not yield a clear distinction between primary and recurrent WDLPS samples. In conclusion, microRNA expression profiles and DNA methylation profiles do not distinguish between primary and recurrent WDLPS and no putative common drivers could be identified.

Uveal melanoma (UM) is an aggressive intra-ocular cancer with a strong tendency to metastasize. Metastatic UM is associated with mutations in BAP1 and SF3B1, however only little is known about the epigenetic modifications that arise in metastatic UM. In this study we aim to unravel epigenetic changes contributing to UM metastasis using a new genome-wide methylation analysis technique that covers over 50% of all CpG's. We identified aberrant methylation contributing to BAP1 and SF3B1-mediated UM metastasis. The methylation data was integrated with expression data and surveyed in matched UM metastases from the liver, skin and bone. UM metastases showed no commonly shared novel epigenetic modifications, implying that epigenetic changes contributing to metastatic spreading and colonization in distant tissues occur early in the development of UM and epigenetic changes that occur after metastasis are mainly patient-specific. Our findings reveal a plethora of epigenetic modifications in metastatic UM and its metastases, which could subsequently result in aberrant repression or activation of many tumor-related genes. This observation points towards additional layers of complexity at the level of gene expression regulation, which may explain the low mutational burden of UM.

In mammals, dosage compensation of X-linked gene expression between males and females is achieved by inactivation of a single X chromosome in females, while upregulation of the single active X in males and females leads to X:autosome dosage balance. Studies in human embryos revealed that random X chromosome inactivation starts at the preimplantation stage and is not complete by day 12 of development. Alternatively, others proposed that dosage compensation in human preimplantation embryos is achieved by dampening expression from the two X chromosomes in females. Here, we characterize X-linked dosage compensation in another primate, the marmoset (Callithrix jacchus). Analyzing scRNA-seq data from preimplantation embryos, we detected upregulation of XIST at the morula stage, where female embryos presented a significantly higher expression of XIST than males. Moreover, we show an increase of X-linked monoallelically expressed genes in female embryos between the morula and late blastocyst stages, indicative of XCI. Nevertheless, dosage compensation was not achieved by the late blastocyst stage. Finally, we show that X:autosome dosage compensation is achieved at the 8-cell stage, and demonstrate that X chromosome dampening in females does not take place in the marmoset. Our work contributes to the elucidation of primate X-linked dosage compensation.

During early embryonic development, one of the two X chromosomes in mammalian female cells is inactivated to compensate for a potential imbalance in transcript levels with male cells, which contain a single X chromosome. Here, we use mouse female embryonic stem cells (ESCs) with non-random X chromosome inactivation (XCI) and polymorphic X chromosomes to study the dynamics of gene silencing over the inactive X chromosome by high-resolution allele-specific RNA-seq.

In placental mammals, balanced expression of X-linked genes is accomplished by X chromosome inactivation (XCI) in female cells. In humans, random XCI is initiated early during embryonic development. To investigate whether reprogramming of female human fibroblasts into induced pluripotent stem cells (iPSCs) leads to reactivation of the inactive X chromosome (Xi), we have generated iPSC lines from fibroblasts heterozygous for large X-chromosomal deletions. These fibroblasts show completely skewed XCI of the mutated X chromosome, enabling monitoring of X chromosome reactivation (XCR) and XCI using allele-specific single-cell expression analysis. This approach revealed that XCR is robust under standard culture conditions, but does not prevent reinitiation of XCI, resulting in a mixed population of cells with either two active X chromosomes (Xas) or one Xa and one Xi. This mixed population of XaXa and XaXi cells is stabilized in naive human stem cell medium, allowing expansion of clones with two Xas.

In the nuclei of most mammalian cells, pericentric heterochromatin is characterized by DNA methylation, histone modifications such as H3K9me3 and H4K20me3, and specific binding proteins like heterochromatin-binding protein 1 isoforms (HP1 isoforms). Maintenance of this specialized chromatin structure is of great importance for genome integrity and for the controlled repression of the repetitive elements within the pericentric DNA sequence. Here we have studied histone modifications at pericentric heterochromatin during primordial germ cell (PGC) development using different fixation conditions and fluorescent immunohistochemical and immunocytochemical protocols.

The long noncoding RNA Xist is critical for initiation and establishment of X-chromosome inactivation during embryogenesis in mammals, but it is unclear whether its continued expression is required for maintaining X-inactivation in vivo.

Chimpanzees and humans are genetically very similar, with the striking exception of their Y chromosomes, which have diverged tremendously. The male-specific region (MSY), representing the greater part of the Y chromosome, is inherited from father to son in a clonal fashion, with natural selection acting on the MSY as a unit. Positive selection might involve the performance of the MSY in spermatogenesis. Chimpanzees have a highly polygamous mating behavior, so that sperm competition is thought to provide a strong selective force acting on the Y chromosome in the chimpanzee lineage. In consequence of evolution of the heterologous sex chromosomes in mammals, meiotic sex chromosome inactivation (MSCI) results in a transcriptionally silenced XY body in male meiotic prophase, and subsequently also in postmeiotic repression of the sex chromosomes in haploid spermatids. This has evolved to a situation where MSCI has become a prerequisite for spermatogenesis. Here, by analysis of microarray testicular expression data representing a small number of male chimpanzees and men, we obtained information indicating that meiotic and postmeiotic X chromosome silencing might be more effective in chimpanzee than in human spermatogenesis. From this, we suggest that the remarkable reorganization of the chimpanzee Y chromosome, compared to the human Y chromosome, might have an impact on its meiotic interactions with the X chromosome and thereby on X chromosome silencing in spermatogenesis. Further studies will be required to address comparative functional aspects of MSCI in chimpanzee, human, and other placental mammals.

X chromosome inactivation (XCI) is a dynamically regulated developmental process with inactivation and reactivation accompanying the loss and gain of pluripotency, respectively. A functional relationship between pluripotency and lack of XCI has been suggested, whereby pluripotency transcription factors repress the master regulator of XCI, the noncoding transcript Xist, by binding to its first intron (intron 1). To test this model, we have generated intron 1 mutant embryonic stem cells (ESCs) and two independent mouse models. We found that Xist's repression in ESCs, its transcriptional upregulation upon differentiation, and its silencing upon reprogramming to pluripotency are not dependent on intron 1. Although we observed subtle effects of intron 1 deletion on the randomness of XCI and in the absence of the antisense transcript Tsix in differentiating ESCs, these have little relevance in vivo because mutant mice do not deviate from Mendelian ratios of allele transmission. Altogether, our findings demonstrate that intron 1 is dispensable for the developmental dynamics of Xist expression.

DNA methylation detection in liquid biopsies provides a highly promising and much needed means for real-time monitoring of disease load in advanced cancer patient care. Compared to the often-used somatic mutations, tissue- and cancer-type specific epigenetic marks affect a larger part of the cancer genome and generally have a high penetrance throughout the tumour. Here, we describe the successful application of the recently described MeD-seq assay for genome-wide DNA methylation profiling on cell-free DNA (cfDNA). The compatibility of the MeD-seq assay with different types of blood collection tubes, cfDNA input amounts, cfDNA isolation methods, and vacuum concentration of samples was evaluated using plasma from both metastatic cancer patients and healthy blood donors (HBDs). To investigate the potential value of cfDNA methylation profiling for tumour load monitoring, we profiled paired samples from 8 patients with resectable colorectal liver metastases (CRLM) before and after surgery.

The X and Y sex chromosomes of placental mammals show hallmarks of a tumultuous evolutionary past. The X Chromosome has a rich and conserved gene content, while the Y Chromosome has lost most of its genes. In the Transcaucasian mole vole Ellobius lutescens, the Y Chromosome including Sry has been lost, and both females and males have a 17,X diploid karyotype. Similarly, the closely related Ellobius talpinus, has a 54,XX karyotype in both females and males. Here, we report the sequencing and assembly of the E. lutescens and E. talpinus genomes. The results indicate that the loss of the Y Chromosome in E. lutescens and E. talpinus occurred in two independent events. Four functional homologs of mouse Y-Chromosomal genes were detected in both female and male E. lutescens, of which three were also detected in the E. talpinus genome. One of these is Eif2s3y, known as the only Y-derived gene that is crucial for successful male meiosis. Female and male E. lutescens can carry one and the same X Chromosome with a largely conserved gene content, including all genes known to function in X Chromosome inactivation. The availability of the genomes of these mole vole species provides unique models to study the dynamics of sex chromosome evolution.

In female mammals, X chromosome inactivation (XCI) is a key process in the control of gene dosage compensation between X-linked genes and autosomes. Xist and Tsix, two overlapping antisense-transcribed noncoding genes, are central elements of the X inactivation center (Xic) regulating XCI. Xist upregulation results in the coating of the entire X chromosome by Xist RNA in cis, whereas Tsix transcription acts as a negative regulator of Xist Here, we generated Xist and Tsix reporter mouse embryonic stem (ES) cell lines to study the genetic and dynamic regulation of these genes upon differentiation. Our results revealed mutually antagonistic roles for Tsix on Xist and vice versa and indicate the presence of semistable transcriptional states of the Xic locus predicting the outcome of XCI. These transcriptional states are instructed by the X-to-autosome ratio, directed by regulators of XCI, and can be modulated by tissue culture conditions.

The majority of desmoid-type fibromatosis (DTF) tumors harbor a β-catenin mutation, affecting specific codons in CTNNB1 exon 3. S45F tumors are reported to have a higher chance of recurrence after surgery and more resistance to systemic treatments compared to wild-type (WT) and T41A tumors. The aim of this pilot study was to examine the genome-wide DNA methylation profiles of S45F and T41A mutated DTF, to explain the observed differences in clinical behavior between these DTF subtypes.

In mice, imprinted X chromosome inactivation (iXCI) of the paternal X in the pre-implantation embryo and extraembryonic tissues is followed by X reactivation in the inner cell mass (ICM) of the blastocyst to facilitate initiation of random XCI (rXCI) in all embryonic tissues. RNF12 is an E3 ubiquitin ligase that plays a key role in XCI. RNF12 targets pluripotency protein REX1 for degradation to initiate rXCI in embryonic stem cells (ESCs) and loss of the maternal copy of Rnf12 leads to embryonic lethality due to iXCI failure. Here, we show that loss of Rex1 rescues the rXCI phenotype observed in Rnf12-/- ESCs, and that REX1 is the prime target of RNF12 in ESCs. Genetic ablation of Rex1 in Rnf12-/- mice rescues the Rnf12-/- iXCI phenotype, and results in viable and fertile Rnf12-/-:Rex1-/- female mice displaying normal iXCI and rXCI. Our results show that REX1 is the critical target of RNF12 in XCI.

In spite of the emergence of genome editing tools, ES cell mediated transgenesis remains the most controllable way of creating genetically modified animals. Although tetraploid (4N) complementation of 4N host embryos and ES cells, is the only method guaranteeing that offspring are entirely ES cell derived, this technique is challenging, not always successful and difficult to implement in some laboratory settings. The current study shows that pretreatment of host blastocysts with FGF4 prior to ES cell injection can provide an alternative method for the generation of animals displaying high rates of chimaerism. Chimaerism assessment in E11 fetuses and born pups shows that a large percentage of resulting conceptuses show a high ES cell contribution from implantation onwards and that developing pups do not necessitate c-section for delivery.