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Random X-chromosome inactivation (XCI) is a hallmark of female mammalian somatic cells. This epigenetic mechanism, mediated by the long non-coding RNAXist, occurs in the epiblast and is stably maintained to ensure proper dosage compensation of X-linked genes during life. However, this silencing is lost during primordial germ cell (PGC) development. Using a combination of single-cell allele-specific RNA sequencing and low-input chromatin profiling in developingin vivoPGC, we provide unprecedented detailed maps of gene reactivation. We demonstrated that PGC still carry a fully silent X chromosome on embryonic day (E) 9.5, despite the loss ofXistexpression. X-linked genes are then gradually reactivated outside theXistfirst-bound regions. At E12.5, a significant part of the inactive X chromosome (Xi) still resists reactivation, carrying an epigenetic memory of its silencing. Late-reactivated genes are enriched in repressive chromatin marks, including DNA methylation and H3K27me3 marks. Our results define the timing of reactivation of the silent X chromosome a key event in female PGC reprogramming with direct implications for reproduction.

Somatic DNA methylation is established early during mammalian development, as embryonic cells transition from naive to primed pluripotency. This precedes the emergence of the three somatic germ layers, but also the segregation of the germline that undergoes genome-wide DNA demethylation after specification. While DNA methylation is essential for embryogenesis, the point at which it becomes critical during differentiation and whether all lineages equally depend on it is unclear. Using culture modeling of cellular transitions, we found that DNA methylation-free embryonic stem cells (ESCs) with a triple DNA methyltransferase knockout (TKO) normally progressed through the continuum of pluripotency states, but demonstrated skewed differentiation abilities towards neural versus other somatic lineages. More saliently, TKO ESCs were fully competent for establishing primordial germ cell-like cells (PGCLCs), even showing temporally extended and self-sustained capacity for the germline fate. By mapping chromatin states, we found that the neural and germline lineages are linked by a similar enhancer dynamics during priming, defined by common sets of methyl-sensitive transcription factors that fail to be decommissioned in absence of DNA methylation. We propose that DNA methylation controls the temporality of a coordinated neural-germline axis of preferred differentiation route during early development.

Quiescence is a hallmark of CD4+ T cells latently infected with human immunodeficiency virus 1 (HIV-1). While reversing this quiescence is an effective approach to reactivate latent HIV from T cells in culture, it can cause deleterious cytokine dysregulation in patients. As a key regulator of T-cell quiescence, FOXO1 promotes latency and suppresses productive HIV infection. We report that, in resting T cells, FOXO1 inhibition impaired autophagy and induced endoplasmic reticulum (ER) stress, thereby activating two associated transcription factors: activating transcription factor 4 (ATF4) and nuclear factor of activated T cells (NFAT). Both factors associate with HIV chromatin and are necessary for HIV reactivation. Indeed, inhibition of protein kinase R-like ER kinase, an ER stress sensor that can mediate the induction of ATF4, and calcineurin, a calcium-dependent regulator of NFAT, synergistically suppressed HIV reactivation induced by FOXO1 inhibition. Thus, our studies uncover a link of FOXO1, ER stress and HIV infection that could be therapeutically exploited to selectively reverse T-cell quiescence and reduce the size of the latent viral reservoir.

SUMMARYDuring early mammalian development, the genome undergoes profound transitions in chromatin states, topological organization and recruitment ofcisregulatory factors involved in transcriptional control. How these three layers of gene regulation interact is the matter of intense research. TheZdbf2gene—which is involved in growth control—provides a valuable model to study this question: upon exit from naïve pluripotency and prior to tissue differentiation, it undergoes a switch in usage from a distal to a proximal promoter, along with a switch in chromatin states, from polycomb to DNA methylation occupancy. Using an embryonic stem cell (ESC) culture system to mimic this period, we show here that four enhancers contribute to theZdbf2promoter switch, concomitantly with dynamic changes in chromosome architecture. Indeed, CTCF plays a key role in partitioning the locus in ESCs, to facilitate enhancer contact with the distalZdbf2promoter only. Partition relieving enhances proximalZdbf2promoter activity, as observed during differentiation or with mutants that lack local CTCF-based partition. Importantly, we show that CTCF-based regulation occurs independently of the polycomb and DNA methylation pathways. Our study reveals the importance of multi-layered regulatory frameworks to ensure proper spatio-temporal activation of developmentally important genes.

SARS-CoV-2 is the causative agent behind the COVID-19 pandemic, responsible for over 170 million infections, and over 3.7 million deaths worldwide. Efforts to test, treat and vaccinate against this pathogen all benefit from an improved understanding of the basic biology of SARS-CoV-2. Both viral and cellular proteases play a crucial role in SARS-CoV-2 replication. Here, we study proteolytic cleavage of viral and cellular proteins in two cell line models of SARS-CoV-2 replication using mass spectrometry to identify protein neo-N-termini generated through protease activity. We identify previously unknown cleavage sites in multiple viral proteins, including major antigens S and N: the main targets for vaccine and antibody testing efforts. We discover significant increases in cellular cleavage events consistent with cleavage by SARS-CoV-2 main protease, and identify 14 potential high-confidence substrates of the main and papain-like proteases. We show that siRNA depletion of these cellular proteins inhibits SARS-CoV-2 replication, and that drugs targeting two of these proteins: the tyrosine kinase SRC and Ser/Thr kinase MYLK, show a dose-dependent reduction in SARS-CoV-2 titres. Overall, our study provides a powerful resource to understand proteolysis in the context of viral infection, and to inform the development of targeted strategies to inhibit SARS-CoV-2 and treat COVID-19., During SARS-CoV-2 replication, viral and cellular proteases play crucial roles and have been shown to be promising anti-viral targets. Here, Meyer et al. apply mass spectrometry to characterize the proteolytic cleavage profile of viral and cellular proteins in vitro.

Gene drives are genetic systems designed to efficiently spread a modification through a population. They have been designed almost exclusively in eukaryotic species, especially in insects. We recently developed a CRISPR-based gene drive system in herpesviruses that relies on similar mechanisms and could efficiently spread into a population of wild-type viruses. A common consequence of gene drives in insects is the appearance and selection of drive-resistant sequences that are no longer recognized by CRISPR-Cas9. In this study, we analyzed in cell culture experiments the evolution of resistance in a viral gene drive against human cytomegalovirus. We report that after an initial invasion of the wild-type population, a drive-resistant population is positively selected over time and outcompetes gene drive viruses. However, we show that targeting evolutionarily conserved sequences ensures that drive-resistant viruses acquire long-lasting mutations and are durably attenuated. As a consequence, and even though engineered viruses do not stably persist in the viral population, remaining viruses have a replication defect, leading to a long-term reduction of viral levels. This marks an important step toward developing effective gene drives in herpesviruses, especially for therapeutic applications. IMPORTANCE The use of defective viruses that interfere with the replication of their infectious parent after coinfecting the same cells—a therapeutic strategy known as viral interference—has recently generated a lot of interest. The CRISPR-based system that we recently reported for herpesviruses represents a novel interfering strategy that causes the conversion of wild-type viruses into new recombinant viruses and drives the native viral population to extinction. In this study, we analyzed how targeted viruses evolved resistance against the technology. Through numerical simulations and cell culture experiments with human cytomegalovirus, we showed that after the initial propagation, a resistant viral population is positively selected and outcompetes engineered viruses over time. We show, however, that targeting evolutionarily conserved sequences ensures that resistant viruses are mutated and attenuated, which leads to a long-term reduction of viral levels. This marks an important step toward the development of novel therapeutic strategies against herpesviruses.

Databases are one of the great success stories in IT. However, they have been continuously increasing in complexity, hampering operation, maintenance, and upgrades. To face this complexity, sophisticated methods for schema summarization, data cleaning, information integration, and many more have been devised that usually rely on data profiles, such as data statistics, signatures, and integrity constraints. Such data profiles are often extracted by automatic algorithms, which entails various problems: The profiles can be unfiltered and huge in volume; different profile types require different complex data structures; and the various profile types are not integrated with each other. We introduce Metacrate, a system to store, organize, and analyze data profiles of relational databases, thereby following the proven design of databases. In particular, we (i) propose a logical and a physical data model to store all kinds of data profiles in a scalable fashion; (ii) describe an analytics layer to query, integrate, and analyze the profiles efficiently; and (iii) implement on top a library of established algorithms to serve use cases, such as schema discovery, database refactoring, and data cleaning.

The potential for early embryonic events to program epigenetic states that influence adult physiology remains an important question in health and development. Using the imprinted Zdbf2 locus as a paradigm for the early programming of phenotypes, we demonstrate here that chromatin changes that occur in the pluripotent embryo can be dispensable for embryogenesis but instead signal essential regulatory information in the adult. The Liz (long isoform of Zdbf2) transcript is transiently expressed in early embryos and embryonic stem cells (ESCs). This transcription locally promotes de novo DNA methylation upstream of the Zdbf2 promoter, which antagonizes Polycomb-mediated repression of Zdbf2. Strikingly, mouse embryos deficient for Liz develop normally but fail to activate Zdbf2 in the postnatal brain and show indelible growth reduction, implying a crucial role for a Liz-dependent epigenetic switch. This work provides evidence that transcription during an early embryonic timeframe can program a stable epigenetic state with later physiological consequences.

DNA methylation is essential for protecting the mammalian germline against transposons. When DNA methylation-based transposon control is defective, meiotic chromosome pairing is consistently impaired during spermatogenesis: How and why meiosis is vulnerable to transposon activity is unknown. Using two DNA methylation-deficient backgrounds, the Dnmt3L and Miwi2 mutant mice, we reveal that DNA methylation is largely dispensable for silencing transposons before meiosis onset. After this, it becomes crucial to back up to a developmentally programmed H3K9me2 loss. Massive retrotransposition does not occur following transposon derepression, but the meiotic chromatin landscape is profoundly affected. Indeed, H3K4me3 marks gained over transcriptionally active transposons correlate with formation of SPO11-dependent double-strand breaks and recruitment of the DMC1 repair enzyme in Dnmt3L−/− meiotic cells, whereas these features are normally exclusive to meiotic recombination hot spots. Here, we demonstrate that DNA methylation restrains transposons from adopting chromatin characteristics amenable to meiotic recombination, which we propose prevents the occurrence of erratic chromosomal events.