Dynamic reversal of random X-Chromosome inactivation during iPSC reprogramming.

Induction and reversal of chromatin silencing is critical for successful development, tissue homeostasis, and the derivation of induced pluripotent stem cells (iPSCs). X-Chromosome inactivation (XCI) and reactivation (XCR) in female cells represent chromosome-wide transitions between active and inactive chromatin states. Although XCI has long been studied, providing important insights into gene regulation, the dynamics and mechanisms underlying the reversal of stable chromatin silencing of X-linked genes are much less understood. Here, we use allele-specific transcriptomics to study XCR during mouse iPSC reprogramming in order to elucidate the timing and mechanisms of chromosome-wide reversal of gene silencing. We show that XCR is hierarchical, with subsets of genes reactivating early, late, and very late during reprogramming. Early genes are activated before the onset of late pluripotency genes activation. Early genes are located genomically closer to genes that escape XCI, unlike genes reactivating late. Early genes also show increased pluripotency transcription factor (TF) binding. We also reveal that histone deacetylases (HDACs) restrict XCR in reprogramming intermediates and that the severe hypoacetylation state of the inactive X Chromosome (Xi) persists until late reprogramming stages. Altogether, these results reveal the timing of transcriptional activation of monoallelically repressed genes during iPSC reprogramming, and suggest that allelic activation involves the combined action of chromatin topology, pluripotency TFs, and chromatin regulators. These findings are important for our understanding of gene silencing, maintenance of cell identity, reprogramming, and disease.

Tox4 modulates cell fate reprogramming.

Reprogramming to induced pluripotency induces the switch of somatic cell identity to induced pluripotent stem cells (iPSCs). However, the mediators and mechanisms of reprogramming remain largely unclear. To elucidate the mediators and mechanisms of reprogramming, we used a siRNA-mediated knockdown approach for selected candidate genes during the conversion of somatic cells into iPSCs. We identified Tox4 as a novel factor that modulates cell fate through an assay that determined the efficiency of iPSC reprogramming. We found that Tox4 is needed early in reprogramming to efficiently generate early reprogramming intermediates, irrespective of the reprogramming conditions used. Tox4 enables proper exogenous reprogramming factor expression, and the closing and opening of putative somatic and pluripotency enhancers early during reprogramming, respectively. We show that the TOX4 protein assembles into a high molecular form. Moreover, Tox4 is also required for the efficient conversion of fibroblasts towards the neuronal fate, suggesting a broader role of Tox4 in modulating cell fate. Our study reveals Tox4 as a novel transcriptional modulator of cell fate that mediates reprogramming from the somatic state to the pluripotent and neuronal fate.This article has an associated First Person interview with the first author of the paper.

Modeling human extraembryonic mesoderm cells using naive pluripotent stem cells.

A hallmark of primate postimplantation embryogenesis is the specification of extraembryonic mesoderm (EXM) before gastrulation, in contrast to rodents where this tissue is formed only after gastrulation. Here, we discover that naive human pluripotent stem cells (hPSCs) are competent to differentiate into EXM cells (EXMCs). EXMCs are specified by inhibition of Nodal signaling and GSK3B, are maintained by mTOR and BMP4 signaling activity, and their transcriptome and epigenome closely resemble that of human and monkey embryo EXM. EXMCs are mesenchymal, can arise from an epiblast intermediate, and are capable of self-renewal. Thus, EXMCs arising via primate-specific specification between implantation and gastrulation can be modeled in vitro. We also find that most of the rare off-target cells within human blastoids formed by triple inhibition (Kagawa et al., 2021) correspond to EXMCs. Our study impacts our ability to model and study the molecular mechanisms of early human embryogenesis and related defects.

Integrated multi-omics reveal polycomb repressive complex 2 restricts human trophoblast induction.

Human naive pluripotent stem cells have unrestricted lineage potential. Underpinning this property, naive cells are thought to lack chromatin-based lineage barriers. However, this assumption has not been tested. Here we define the chromatin-associated proteome, histone post-translational modifications and transcriptome of human naive and primed pluripotent stem cells. Our integrated analysis reveals differences in the relative abundance and activities of distinct chromatin modules. We identify a strong enrichment of polycomb repressive complex 2 (PRC2)-associated H3K27me3 in the chromatin of naive pluripotent stem cells and H3K27me3 enrichment at promoters of lineage-determining genes, including trophoblast regulators. PRC2 activity acts as a chromatin barrier restricting the differentiation of naive cells towards the trophoblast lineage, whereas inhibition of PRC2 promotes trophoblast-fate induction and cavity formation in human blastoids. Together, our results establish that human naive pluripotent stem cells are not epigenetically unrestricted, but instead possess chromatin mechanisms that oppose the induction of alternative cell fates.

Enhanced chromatin accessibility contributes to X chromosome dosage compensation in mammals.

BACKGROUND: Precise gene dosage of the X chromosomes is critical for normal development and cellular function. In mice, XX female somatic cells show transcriptional X chromosome upregulation of their single active X chromosome, while the other X chromosome is inactive. Moreover, the inactive X chromosome is reactivated during development in the inner cell mass and in germ cells through X chromosome reactivation, which can be studied in vitro by reprogramming of somatic cells to pluripotency. How chromatin processes and gene regulatory networks evolved to regulate X chromosome dosage in the somatic state and during X chromosome reactivation remains unclear. RESULTS: Using genome-wide approaches, allele-specific ATAC-seq and single-cell RNA-seq, in female embryonic fibroblasts and during reprogramming to pluripotency, we show that chromatin accessibility on the upregulated mammalian active X chromosome is increased compared to autosomes. We further show that increased accessibility on the active X chromosome is erased by reprogramming, accompanied by erasure of transcriptional X chromosome upregulation and the loss of increased transcriptional burst frequency. In addition, we characterize gene regulatory networks during reprogramming and X chromosome reactivation, revealing changes in regulatory states. Our data show that ZFP42/REX1, a pluripotency-associated gene that evolved specifically in placental mammals, targets multiple X-linked genes, suggesting an evolutionary link between ZFP42/REX1, X chromosome reactivation, and pluripotency. CONCLUSIONS: Our data reveal the existence of intrinsic compensatory mechanisms that involve modulation of chromatin accessibility to counteract X-to-Autosome gene dosage imbalances caused by evolutionary or in vitro X chromosome loss and X chromosome inactivation in mammalian cells.

PU.1 drives specification of pluripotent stem cell-derived endothelial cells to LSEC-like cells.

To date, there is no representative in vitro model for liver sinusoidal endothelial cells (LSECs), as primary LSECs dedifferentiate very fast in culture and no combination of cytokines or growth factors can induce an LSEC fate in (pluripotent stem cell (PSC)-derived) endothelial cells (ECs). Furthermore, the transcriptional programmes driving an LSEC fate have not yet been described. Here, we first present a computational workflow (CenTFinder) that can identify transcription factors (TFs) that are crucial for modulating pathways involved in cell lineage specification. Using CenTFinder, we identified several novel LSEC-specific protein markers, such as FCN2 and FCN3, which were validated by analysis of previously published single-cell RNAseq data. We also identified PU.1 (encoded by the SPI1 gene) as a major regulator of LSEC-specific immune functions. We show that SPI1 overexpression (combined with the general EC TF ETV2) in human PSCs induces ECs with an LSEC-like phenotype. The ETV2-SPI1-ECs display increased expression of LSEC markers, such as CD32B and MRC1, as well as several of the proposed novel markers. More importantly, ETV2-SPI1-ECs acquire LSEC functions, including uptake of FSA-FITC, as well as labelled IgG. In conclusion, we present the CenTFinder computational tool to identify key regulatory TFs within specific pathways, in this work pathways of lineage specification, and we demonstrate its use by the identification and validation of PU.1 as a master regulator for LSEC fating.

Evaluating totipotency using criteria of increasing stringency.

Totipotency is the ability of a single cell to give rise to all of the differentiated cell types that build the conceptus, yet how to capture this property in vitro remains incompletely understood. Defining totipotency relies on a variety of assays of variable stringency. Here, we describe criteria to define totipotency. We explain how distinct criteria of increasing stringency can be used to judge totipotency by evaluating candidate totipotent cell types in mice, including early blastomeres and expanded or extended pluripotent stem cells. Our data challenge the notion that expanded or extended pluripotent states harbour increased totipotent potential relative to conventional embryonic stem cells under in vitro and in vivo conditions.

Unraveling Assemblage, Functions and Stability of the Gut Microbiota of Blattella germanica by Antibiotic Treatment.

Symbiosis between prokaryotes and eukaryotes is a widespread phenomenon that has contributed to the evolution of eukaryotes. In cockroaches, two types of symbionts coexist: an endosymbiont in the fat body (Blattabacterium), and a rich gut microbiota. The transmission mode of Blattabacterium is vertical, while the gut microbiota of a new generation is mainly formed by bacterial species present in feces. We have carried out a metagenomic analysis of Blattella germanica populations, treated and non-treated with two antibiotics (vancomycin and ampicillin) over two generations to (1) determine the core of bacterial communities and potential functions of the gut microbiota and (2) to gain insights into the mechanisms of resistance and resilience of the gut microbiota. Our results indicate that the composition and functions of the bacteria were affected by treatment, more severely in the case of vancomycin. Further results demonstrated that in an untreated second-generation population that comes from antibiotic-treated first-generation, the microbiota is not yet stabilized at nymphal stages but can fully recover in adults when feces of a control population were added to the diet. This signifies the existence of a stable core in either composition and functions in lab-reared populations. The high microbiota diversity as well as the observed functional redundancy point toward the microbiota of cockroach hindguts as a robust ecosystem that can recover from perturbations, with recovery being faster when feces are added to the diet.

Recent Advances in Understanding the Reversal of Gene Silencing During X Chromosome Reactivation.

Dosage compensation between XX female and XY male cells is achieved by a process known as X chromosome inactivation (XCI) in mammals. XCI is initiated early during development in female cells and is subsequently stably maintained in most somatic cells. Despite its stability, the robust transcriptional silencing of XCI is reversible, in the embryo and also in a number of reprogramming settings. Although XCI has been intensively studied, the dynamics, factors, and mechanisms of X chromosome reactivation (XCR) remain largely unknown. In this review, we discuss how new sequencing technologies and reprogramming approaches have enabled recent advances that revealed the timing of transcriptional activation during XCR. We also discuss the factors and chromatin features that might be important to understand the dynamics and mechanisms of the erasure of transcriptional gene silencing on the inactive X chromosome (Xi).

X-Chromosome Dosage Modulates Multiple Molecular and Cellular Properties of Mouse Pluripotent Stem Cells Independently of Global DNA Methylation Levels.

Reprogramming female mouse somatic cells into induced pluripotent stem cells (iPSCs) leads to X-chromosome reactivation. The extent to which increased X-chromosome dosage (X-dosage) in female iPSCs compared with male iPSCs leads to differences in the properties of iPSCs is still unclear. We show that chromatin accessibility in mouse iPSCs is modulated by X-dosage. Specific sets of transcriptional regulator motifs are enriched in chromatin with increased accessibility in XX or XY iPSCs. The transcriptome, growth and pluripotency exit are also modulated by X-dosage in iPSCs. To understand how increased X-dosage modulates the properties of mouse pluripotent stem cells, we used heterozygous deletions of the X-linked gene Dusp9. We show that X-dosage regulates the transcriptome, open chromatin landscape, growth, and pluripotency exit largely independently of global DNA methylation. Our results provide insights into how gene dosage modulates the epigenetic and genetic mechanisms that regulate cell identity.