Abstract In female mammals, Xist, the master regulator of X-chromosome inactivation (XCI), is expressed monoallelically. This pattern is established during early embryonic development, when the active Xist allele is chosen at random in each cell. How this choice is made remains incompletely understood. Combining knock-down and overexpression strategies in differentiating mouse embryonic stem cells, which recapitulate the onset of random XCI, we identify a role for the repressive chromatin mark H3K9me3 in the XCI initiation. We show that H3K9me3 accumulates at the promoter-proximal region of the silent Xist allele in female cells as monoallelic expression is established. Unexpectedly, this accumulation requires prior transcription of Xist itself, likely during the initial phase of upregulation, when Xist is frequently transcribed in male cells and from both X chromosomes in females. A repressive function of Xist-dependent H3K9me3 accumulation is supported by our finding that premature, transient Xist overexpression primes an allele for future silencing and skews the choice of the inactive X. Xist-dependent H3K9me3 recruitment does not require its antisense transcript Tsix, which can nonetheless enhance subsequent maintenance of the mark. In addition, the X-linked Xist activator RNF12 counteracts H3K9me3 formation, independently of its known target REX1. Our results thus point to facultative heterochromatin formation as a key contributor to choice at the onset of XCI, where activating and repressing mechanisms are intertwined to establish monoallelic Xist expression.

The capacity of cells to retain a memory of previous signals enables acquisition of unique fates and adaptation to their environment. The underlying gene expression memory can arise from mutual repression of two genes, forming a toggle switch. Mutual repression can occur at antisense loci, where convergent genes repress each other in cis. The conditions for generating expression memory via antisense transcription remain poorly understood. To address this question, we combine mathematical modeling, genomics and a synthetic biology approach. Simulations demonstrate stable memory emergence when both genes in an antisense pair transcribe via the convergent promoter and induce a stable repressive chromatin state. Genome-wide analysis of nascent transcription supports antisense-mediated promoter repression, since promoter-overlapping antisense gene pairs exhibit mutually exclusive expression. Through constructing a synthetic antisense locus in mESCs, we demonstrate that antisense transcription can induce stable repression, a key prerequisite for memory. Repression stability increases during mESC differentiation, highlighting cell type-specific epigenetic memory. Our work establishes a quantitative framework which predicts that antisense-mediated cis-memory can arise within physiologically relevant conditions, and shows that a biological phenomenon with kinetics in the range of weeks can emerge from the interplay of multiple faster molecular processes. This framework, combined with our experimental findings, demonstrates how antisense transcription can encode stable gene expression states. Our discovery that stem cells adjust their memory capacity during differentiation may clarify mechanisms underlying stemness maintenance.

Many mammals can temporally uncouple conception from parturition by pacing down their development around the blastocyst stage. In mice, this dormant state is achieved by decreasing the activity of the growth-regulating mTOR signaling pathway. It is unknown whether this ability is conserved in mammals in general and in humans in particular. Here, we show that decreasing the activity of the mTOR signaling pathway induces human pluripotent stem cells (hPSCs) and blastoids to enter a dormant state with limited proliferation, developmental progression, and capacity to attach to endometrial cells. These in vitro assays show that, similar to other species, the ability to enter dormancy is active in human cells around the blastocyst stage and is reversible at both functional and molecular levels. The pacing of human blastocyst development has potential implications for reproductive therapies.

X-chromosome inactivation (XCI) balances gene expression between the sexes in female mammals. Shortly after fertilization, upregulation of Xist RNA from one X chromosome initiates XCI, leading to chromosome-wide gene silencing. XCI is maintained in all cell types, except the germ line and the pluripotent state where XCI is reversed. The mechanisms triggering Xist upregulation have remained elusive. Here we identify GATA transcription factors as potent activators of Xist. Through a pooled CRISPR activation screen in murine embryonic stem cells, we demonstrate that GATA1, as well as other GATA transcription factors can drive ectopic Xist expression. Moreover, we describe GATA-responsive regulatory elements in the Xist locus bound by different GATA factors. Finally, we show that GATA factors are essential for XCI induction in mouse preimplantation embryos. Deletion of GATA1/4/6 or GATA-responsive Xist enhancers in mouse zygotes effectively prevents Xist upregulation. We propose that the activity or complete absence of various GATA family members controls initial Xist upregulation, XCI maintenance in extra-embryonic lineages and XCI reversal in the epiblast.

Human pluripotent stem cells (hPSCs) are of fundamental relevance in regenerative medicine. Naïve hPSCs hold promise to overcome some of the limitations of conventional (primed) hPSCs, including recurrent epigenetic anomalies. Naïve-to-primed transition (capacitation) follows transcriptional dynamics of human embryonic epiblast and is necessary for somatic differentiation from naïve hPSCs. We found that capacitated hPSCs are transcriptionally closer to postimplantation epiblast than conventional hPSCs. This prompted us to comprehensively study epigenetic and related transcriptional changes during capacitation. Our results show that CpG islands, gene regulatory elements, and retrotransposons are hotspots of epigenetic dynamics during capacitation and indicate possible distinct roles of specific epigenetic modifications in gene expression control between naïve and primed hPSCs. Unexpectedly, PRC2 activity appeared to be dispensable for the capacitation. We find that capacitated hPSCs acquire an epigenetic state similar to conventional hPSCs. Significantly, however, the X chromosome erosion frequently observed in conventional female hPSCs is reversed by resetting and subsequent capacitation.

For a short period during early development of mammalian embryos, both X chromosomes in females are active, before dosage compensation is ensured through X‐chromosome inactivation. In female mouse embryonic stem cells (mESCs), which carry two active X chromosomes, increased X‐dosage affects cell signaling and impairs differentiation. The underlying mechanisms, however, remain poorly understood. To dissect X‐dosage effects on the signaling network in mESCs, we combine systematic perturbation experiments with mathematical modeling. We quantify the response to a variety of inhibitors and growth factors for cells with one (XO) or two X chromosomes (XX). We then build models of the signaling networks in XX and XO cells through a semi‐quantitative modeling approach based on modular response analysis. We identify a novel negative feedback in the PI3K/AKT pathway through GSK3. Moreover, the presence of a single active X makes mESCs more sensitive to the differentiation‐promoting Activin A signal and leads to a stronger RAF1‐mediated negative feedback in the FGF‐triggered MAPK pathway. The differential response to these differentiation‐promoting pathways can explain the impaired differentiation propensity of female mESCs.

SUMMARY Many mammals can control the timing of gestation and birth by pausing embryonic development at the blastocyst stage. It is unknown whether the capacity to pause development is conserved, in general across mammals, and more specifically in humans. Activity of the growth regulating mTOR pathway governs developmental pausing in the mouse ( 1 ). Here we show a stage-specific capacity to delay the progression of human development via mTOR inhibition. In this context, human blastoids and pluripotent stem cells in naïve and naïve-like, but not primed, states can be induced to enter a dormant state, which is reversible at the functional and molecular level. Comparative analysis of mouse and human naïve cells’ longitudinal response to mTORi revealed distinct temporal dynamics and metabolic requirements of dormancy in each species. Mouse and human blastocysts show similar tissue-specific patterns of mTOR pathway activity, suggesting that the mTOR pathway may be a conserved regulator of blastocyst development and timing in both species. Our results raise the possibility that the developmental timing of the human embryo may be controllable, with implications for reproductive therapies.

Developmental genes such as Xist, which initiates X chromosome inactivation, are controlled by complex cis-regulatory landscapes, which decode multiple signals to establish specific spatiotemporal expression patterns. Xist integrates information on X chromosome dosage and developmental stage to trigger X inactivation in the epiblast specifically in female embryos. Through a pooled CRISPR screen in differentiating mouse embryonic stem cells, we identify functional enhancer elements of Xist at the onset of random X inactivation. Chromatin profiling reveals that X-dosage controls the promoter-proximal region, while differentiation cues activate several distal enhancers. The strongest distal element lies in an enhancer cluster associated with a previously unannotated Xist-enhancing regulatory transcript, which we named Xert. Developmental cues and X-dosage are thus decoded by distinct regulatory regions, which cooperate to ensure female-specific Xist upregulation at the correct developmental time. With this study, we start to disentangle how multiple, functionally distinct regulatory elements interact to generate complex expression patterns in mammals.

The coronavirus disease (COVID-19) caused by SARS-CoV-2 is creating tremendous human suffering. To date, no effective drug is available to directly treat the disease. In a search for a drug against COVID-19, we have performed a high-throughput x-ray crystallographic screen of two repurposing drug libraries against the SARS-CoV-2 main protease (M pro ), which is essential for viral replication. In contrast to commonly applied x-ray fragment screening experiments with molecules of low complexity, our screen tested already-approved drugs and drugs in clinical trials. From the three-dimensional protein structures, we identified 37 compounds that bind to M pro . In subsequent cell-based viral reduction assays, one peptidomimetic and six nonpeptidic compounds showed antiviral activity at nontoxic concentrations. We identified two allosteric binding sites representing attractive targets for drug development against SARS-CoV-2.

To ensure dosage compensation between the sexes, one randomly chosen X chromosome is silenced in each female cell in the process of X-chromosome inactivation (XCI). XCI is initiated during early development through upregulation of the long non-coding RNA Xist, which mediates chromosome-wide gene silencing. Cell differentiation, Xist upregulation and gene silencing are thought to be coupled at multiple levels to ensure inactivation of exactly one out of two X chromosomes. Here we perform an integrated analysis of all three processes through allele-specific single-cell RNA-sequencing. Specifically, we assess the onset of random XCI in differentiating mouse embryonic stem cells, and develop dedicated analysis approaches. By exploiting the inter-cellular heterogeneity of XCI onset, we identify putative Xist regulators. Moreover, we show that transient Xist upregulation from both X chromosomes results in biallelic gene silencing right before transitioning to the monoallelic state, confirming a prediction of the stochastic model of XCI. Finally, we show that genetic variation modulates the XCI process at multiple levels, providing a potential explanation for the long-known X-controlling element (Xce) effect, which leads to preferential inactivation of a specific X chromosome in inter-strain crosses. We thus draw a detailed picture of the different levels of regulation that govern the initiation of XCI. The experimental and computational strategies we have developed here will allow us to profile random XCI in more physiological contexts, including primary human cells in vivo.

X-chromosomal genes contribute to sex differences, in particular during early development, when both X chromosomes are active in females. Here, double X-dosage shifts female pluripotent cells towards the naive stem cell state by increasing pluripotency factor expression, inhibiting the differentiation-promoting MAP kinase (MAPK) signalling pathway and delaying differentiation. To identify the genetic basis of these sex differences, we have performed a series of CRISPR knockout screens in murine embryonic stem cells to comprehensively identify X-linked genes that cause the female pluripotency phenotype. We found multiple genes that act in concert, among which Klhl13 plays a central role. We show that this E3 ubiquitin ligase substrate adaptor protein promotes pluripotency factor expression, delays differentiation and represses MAPK target genes, and we identify putative substrates. We thus elucidate the mechanisms that drive sex-induced differences in pluripotent cells with implications for gender medicine in the context of induced pluripotent stem cell based therapies. ### Competing Interest Statement The authors have declared no competing interest.

Background X-chromosomal genes contribute to sex differences, in particular during early development, when both X chromosomes are active in females. Double X-dosage shifts female pluripotent cells towards the naive stem cell state by increasing pluripotency factor expression, inhibiting the differentiation-promoting MAP kinase (MAPK) signaling pathway, and delaying differentiation. Results To identify the genetic basis of these sex differences, we use a two-step CRISPR screening approach to comprehensively identify X-linked genes that cause the female pluripotency phenotype in murine embryonic stem cells. A primary chromosome-wide CRISPR knockout screen and three secondary screens assaying for different aspects of the female pluripotency phenotype allow us to uncover multiple genes that act in concert and to disentangle their relative roles. Among them, we identify Dusp9 and Klhl13 as two central players. While Dusp9 mainly affects MAPK pathway intermediates, Klhl13 promotes pluripotency factor expression and delays differentiation, with both factors jointly repressing MAPK target gene expression. Conclusions Here, we elucidate the mechanisms that drive sex-induced differences in pluripotent cells and our approach serves as a blueprint to discover the genetic basis of the phenotypic consequences of other chromosomal effects.

The coronavirus disease (COVID-19) caused by SARS-CoV-2 is creating tremendous health problems and economical challenges for mankind. To date, no effective drug is available to directly treat the disease and prevent virus spreading. In a search for a drug against COVID-19, we have performed a massive X-ray crystallographic screen of repurposing drug libraries containing 5953 individual compounds against the SARS-CoV-2 main protease (Mpro), which is a potent drug target as it is essential for the virus replication. In contrast to commonly applied X-ray fragment screening experiments with molecules of low complexity, our screen tested already approved drugs and drugs in clinical trials. From the three-dimensional protein structures, we identified 37 compounds binding to Mpro. In subsequent cell-based viral reduction assays, one peptidomimetic and five non-peptidic compounds showed antiviral activity at non-toxic concentrations. Interestingly, two compounds bind outside the active site to the native dimer interface in close proximity to the S1 binding pocket. Another compound binds in a cleft between the catalytic and dimerization domain of Mpro. Neither binding site is related to the enzymatic active site and both represent attractive targets for drug development against SARS-CoV-2. This X-ray screening approach thus has the potential to help deliver an approved drug on an accelerated time-scale for this and future pandemics.

Here we present the crystal structure of SARS-CoV-2 main protease (M pro ) covalently bound to 2-methyl-1-tetralone. This complex was obtained by co-crystallization of M pro with HEAT (2-(((4-hydroxyphenethyl)amino)methyl)-3,4-dihydronaphthalen-1(2H)-one) in the framework of a large X-ray crystallographic screening project of M pro against a drug repurposing library, consisting of 5632 approved drugs or compounds in clinical phase trials. Further investigations showed that HEAT is cleaved by M pro in an E1cB-like reaction mechanism into 2-methylene-1-tetralone and tyramine. The catalytic Cys145 subsequently binds covalently in a Michael addition to the methylene carbon atom of 2-methylene-1-tetralone. According to this postulated model HEAT is acting in a pro-drug-like fashion. It is metabolized by M pro , followed by covalent binding of one metabolite to the active site. The structure of the covalent adduct elucidated in this study opens up a new path for developing non-peptidic inhibitors.

The coronavirus disease (COVID-19) caused by SARS-CoV-2 is creating tremendous health problems and economical challenges for mankind. To date, no effective drug is available to directly treat the disease and prevent virus spreading. In a search for a drug against COVID-19, we have performed a massive X-ray crystallographic screen of two repurposing drug libraries against the SARS-CoV-2 main protease (M pro ), which is essential for the virus replication and, thus, a potent drug target. In contrast to commonly applied X-ray fragment screening experiments with molecules of low complexity, our screen tested already approved drugs and drugs in clinical trials. From the three-dimensional protein structures, we identified 37 compounds binding to M pro . In subsequent cell-based viral reduction assays, one peptidomimetic and five non-peptidic compounds showed antiviral activity at non-toxic concentrations. We identified two allosteric binding sites representing attractive targets for drug development against SARS-CoV-2.

To ensure dosage compensation between the sexes, one randomly chosen X chromosome is silenced in each female cell in the process of X-chromosome inactivation (XCI). XCI is initiated during early development through upregulation of the long non-coding RNA Xist, which mediates chromosome-wide gene silencing. Cell differentiation, Xist upregulation and silencing are thought to be coupled at multiple levels to ensure inactivation of exactly one out of two X chromosomes. Here we perform an integrated analysis of all three processes through allele-specific single-cell RNA-sequencing. Specifically, we assess the onset of random XCI with high temporal resolution in differentiating mouse embryonic stem cells, and develop dedicated analysis approaches. By exploiting the inter-cellular heterogeneity of XCI onset, we identify Nanog downregulation as its main trigger and discover additional putative Xist regulators. Moreover, we confirm several predictions of the stochastic model of XCI where monoallelic silencing is thought to be ensured through negative feedback regulation. Finally, we show that genetic variation modulates the XCI process at multiple levels, providing a potential explanation for the long-known Xce effect, which leads to preferential inactivation of a specific X chromosome in inter-strain crosses. We thus draw a detailed picture of the different levels of regulation that govern the initiation of XCI. The experimental and computational strategies we have developed here will allow us to profile random XCI in more physiological contexts, including primary human cells in vivo.

Background Understanding the transcriptome is critical for explaining the functional as well as regulatory roles of genomic regions. Current methods for the identification of transcription units (TUs) use RNA-seq that, however, require large quantities of mRNA rendering the identification of inherently unstable TUs, e.g. miRNA precursors, difficult. This problem can be alleviated by chromatin-based approaches due to a correlation between histone modifications and transcription. Results Here, we introduce EPIGENE, a novel chromatin segmentation method for the identification of active TUs using transcription-associated histone modifications. Unlike the existing chromatin segmentation approaches, EPIGENE uses a constrained, semi-supervised multivariate hidden Markov model (HMM) that models the observed combination of histone modifications using a product of independent Bernoulli random variables, to identify active TUs. Our results show that EPIGENE can identify genome-wide TUs in an unbiased manner. EPIGENE-predicted TUs show an enrichment of RNA Polymerase II at the transcription start site and in gene body indicating that they are indeed transcribed. Comprehensive validation using existing annotations revealed that 93% of EPIGENE TUs can be explained by existing gene annotations and 5% of EPIGENE TUs in HepG2 can be explained by microRNA annotations. EPIGENE outperformed the existing RNA-seq-based approaches in TU prediction precision across human cell lines. Finally, we identified 232 novel TUs in K562 and 43 novel cell-specific TUs all of which were supported by RNA Polymerase II ChIP-seq and Nascent RNA-seq data. Conclusion We demonstrate the applicability of EPIGENE to identify genome-wide active TUs and to provide valuable information about unannotated TUs. EPIGENE is an open-source method and is freely available at: https://githu b.com/imbbLab/EPIGENE..

Gene-regulatory networks control the establishment and maintenance of alternative gene-expression states during development. A particular challenge is the acquisition of opposing states by two copies of the same gene, as in the case of the long non-coding RNA Xist in mammals at the onset of random X-chromosome inactivation (XCI). The regulatory principles that lead to stable mono-allelic expression of Xist remain unknown. Here, we uncover the minimal regulatory network that can ensure female-specific and mono-alleleic upregulation of Xist, by combining mathematical modeling and experimental validation of central model predictions. We identify a symmetric toggle switch as the basis for random mono-allelic upregulation of Xist, which reproduces data from several mutant, aneuploid and polyploid mouse cell lines with various Xist expression patterns. Moreover, this toggle switch explains the diversity of strategies employed by different species at the onset of XCI. In addition to providing a unifying conceptual framework with which to explore XCI across mammals, our study sets the stage for identifying the molecular mechanisms needed to initiate random XCI.

MicroRNAs (miRNAs) play an important role in guiding development and maintaining function of the human heart. Dysregulation of miRNAs has been linked to various congenital heart diseases including Tetralogy of Fallot (TOF), which represents the most common cyanotic heart malformation in humans. Several studies have identified dysregulated miRNAs in right ventricular (RV) tissues of TOF patients. In this study, we profiled genome-wide the whole transcriptome and analyzed the relationship of miRNAs and mRNAs of RV tissues of a homogeneous group of 22 non-syndromic TOF patients. Observed profiles were compared to profiles obtained from right and left ventricular tissue of normal hearts. To reduce the commonly observed large list of predicted target genes of dysregulated miRNAs, we applied a stringent target prediction pipeline integrating probabilities for miRNA-mRNA interaction. The final list of disease-related miRNA-mRNA pairs comprises novel as well as known miRNAs including miR-1 and miR-133, which are essential to cardiac development and function by regulating KCNJ2, FBN2, SLC38A3 and TNNI1. Overall, our study provides additional insights into post-transcriptional gene regulation of malformed hearts of TOF patients.

To initiate X-Chromosome inactivation (XCI), the long noncoding RNA Xist mediates chromosome-wide gene silencing of one X Chromosome in female mammals to equalize gene dosage between the sexes. The efficiency of gene silencing is highly variable across genes, with some genes even escaping XCI in somatic cells. A gene's susceptibility to Xist-mediated silencing appears to be determined by a complex interplay of epigenetic and genomic features; however, the underlying rules remain poorly understood. We have quantified chromosome-wide gene silencing kinetics at the level of the nascent transcriptome using allele-specific Precision nuclear Run-On sequencing (PRO-seq). We have developed a Random Forest machine-learning model that can predict the measured silencing dynamics based on a large set of epigenetic and genomic features and tested its predictive power experimentally. The genomic distance to the Xist locus, followed by gene density and distance to LINE elements, are the prime determinants of the speed of gene silencing. Moreover, we find two distinct gene classes associated with different silencing pathways: a class that requires Xist-repeat A for silencing, which is known to activate the SPEN pathway, and a second class in which genes are premarked by Polycomb complexes and tend to rely on the B repeat in Xist for silencing, known to recruit Polycomb complexes during XCI. Moreover, a series of features associated with active transcriptional elongation and chromatin 3D structure are enriched at rapidly silenced genes. Our machine-learning approach can thus uncover the complex combinatorial rules underlying gene silencing during X inactivation.