"Be sustainable": EOSC-Life recommendations for implementation of FAIR principles in life science data handling.

The main goals and challenges for the life science communities in the Open Science framework are to increase reuse and sustainability of data resources, software tools, and workflows, especially in large-scale data-driven research and computational analyses. Here, we present key findings, procedures, effective measures and recommendations for generating and establishing sustainable life science resources based on the collaborative, cross-disciplinary work done within the EOSC-Life (European Open Science Cloud for Life Sciences) consortium. Bringing together 13 European life science research infrastructures, it has laid the foundation for an open, digital space to support biological and medical research. Using lessons learned from 27 selected projects, we describe the organisational, technical, financial and legal/ethical challenges that represent the main barriers to sustainability in the life sciences. We show how EOSC-Life provides a model for sustainable data management according to FAIR (findability, accessibility, interoperability, and reusability) principles, including solutions for sensitive- and industry-related resources, by means of cross-disciplinary training and best practices sharing. Finally, we illustrate how data harmonisation and collaborative work facilitate interoperability of tools, data, solutions and lead to a better understanding of concepts, semantics and functionalities in the life sciences.

Cohesin controls X chromosome structure remodeling and X-reactivation during mouse iPSC-reprogramming.

Reactivation of the inactive X chromosome is a hallmark epigenetic event during reprogramming of mouse female somatic cells to induced pluripotent stem cells (iPSCs). This involves global structural remodeling from a condensed, heterochromatic into an open, euchromatic state, thereby changing a transcriptionally inactive into an active chromosome. Despite recent advances, very little is currently known about the molecular players mediating this process and how this relates to iPSC-reprogramming in general. To gain more insight, here we perform a RNAi-based knockdown screen during iPSC-reprogramming of mouse fibroblasts. We discover factors important for X chromosome reactivation (XCR) and iPSC-reprogramming. Among those, we identify the cohesin complex member SMC1a as a key molecule with a specific function in XCR, as its knockdown greatly affects XCR without interfering with iPSC-reprogramming. Using super-resolution microscopy, we find SMC1a to be preferentially enriched on the active compared with the inactive X chromosome and that SMC1a is critical for the decompacted state of the active X. Specifically, depletion of SMC1a leads to contraction of the active X both in differentiated and in pluripotent cells, where it normally is in its most open state. In summary, we reveal cohesin as a key factor for remodeling of the X chromosome from an inactive to an active structure and that this is a critical step for XCR during iPSC-reprogramming.

Controlled X-chromosome dynamics defines meiotic potential of female mouse in vitro germ cells.

The mammalian germline is characterized by extensive epigenetic reprogramming during its development into functional eggs and sperm. Specifically, the epigenome requires resetting before parental marks can be established and transmitted to the next generation. In the female germline, X-chromosome inactivation and reactivation are among the most prominent epigenetic reprogramming events, yet very little is known about their kinetics and biological function. Here, we investigate X-inactivation and reactivation dynamics using a tailor-made in vitro system of primordial germ cell-like cell (PGCLC) differentiation from mouse embryonic stem cells. We find that X-inactivation in PGCLCs in vitro and in germ cell-competent epiblast cells in vivo is moderate compared to somatic cells, and frequently characterized by escaping genes. X-inactivation is followed by step-wise X-reactivation, which is mostly completed during meiotic prophase I. Furthermore, we find that PGCLCs which fail to undergo X-inactivation or reactivate too rapidly display impaired meiotic potential. Thus, our data reveal fine-tuned X-chromosome remodelling as a critical feature of female germ cell development towards meiosis and oogenesis.

Epigenetic gene silencing by heterochromatin primes fungal resistance.

Heterochromatin that depends on histone H3 lysine 9 methylation (H3K9me) renders embedded genes transcriptionally silent1-3. In the fission yeast Schizosaccharomyces pombe, H3K9me heterochromatin can be transmitted through cell division provided the counteracting demethylase Epe1 is absent4,5. Heterochromatin heritability might allow wild-type cells under certain conditions to acquire epimutations, which could influence phenotype through unstable gene silencing rather than DNA change6,7. Here we show that heterochromatin-dependent epimutants resistant to caffeine arise in fission yeast grown with threshold levels of caffeine. Isolates with unstable resistance have distinct heterochromatin islands with reduced expression of embedded genes, including some whose mutation confers caffeine resistance. Forced heterochromatin formation at implicated loci confirms that resistance results from heterochromatin-mediated silencing. Our analyses reveal that epigenetic processes promote phenotypic plasticity, letting wild-type cells adapt to unfavourable environments without genetic alteration. In some isolates, subsequent or coincident gene-amplification events augment resistance. Caffeine affects two anti-silencing factors: Epe1 is downregulated, reducing its chromatin association, and a shortened isoform of Mst2 histone acetyltransferase is expressed. Thus, heterochromatin-dependent epimutation provides a bet-hedging strategy allowing cells to adapt transiently to insults while remaining genetically wild type. Isolates with unstable caffeine resistance show cross-resistance to antifungal agents, suggesting that related heterochromatin-dependent processes may contribute to resistance of plant and human fungal pathogens to such agents.

Epigenetics. Restricted epigenetic inheritance of H3K9 methylation.

Posttranslational histone modifications are believed to allow the epigenetic transmission of distinct chromatin states, independently of associated DNA sequences. Histone H3 lysine 9 (H3K9) methylation is essential for heterochromatin formation; however, a demonstration of its epigenetic heritability is lacking. Fission yeast has a single H3K9 methyltransferase, Clr4, that directs all H3K9 methylation and heterochromatin. Using releasable tethered Clr4 reveals that an active process rapidly erases H3K9 methylation from tethering sites in wild-type cells. However, inactivation of the putative histone demethylase Epe1 allows H3K9 methylation and silent chromatin maintenance at the tethering site through many mitotic divisions, and transgenerationally through meiosis, after release of tethered Clr4. Thus, H3K9 methylation is a heritable epigenetic mark whose transmission is usually countered by its active removal, which prevents the unauthorized inheritance of heterochromatin.

A nucleosome turnover map reveals that the stability of histone H4 Lys20 methylation depends on histone recycling in transcribed chromatin.

Nucleosome composition actively contributes to chromatin structure and accessibility. Cells have developed mechanisms to remove or recycle histones, generating a landscape of differentially aged nucleosomes. This study aimed to create a high-resolution, genome-wide map of nucleosome turnover in Schizosaccharomyces pombe. The recombination-induced tag exchange (RITE) method was used to study replication-independent nucleosome turnover through the appearance of new histone H3 and the disappearance or preservation of old histone H3. The genome-wide location of histones was determined by chromatin immunoprecipitation-exonuclease methodology (ChIP-exo). The findings were compared with diverse chromatin marks, including histone variant H2A.Z, post-translational histone modifications, and Pol II binding. Finally, genome-wide mapping of the methylation states of H4K20 was performed to determine the relationship between methylation (mono, di, and tri) of this residue and nucleosome turnover. Our analysis showed that histone recycling resulted in low nucleosome turnover in the coding regions of active genes, stably expressed at intermediate levels. High levels of transcription resulted in the incorporation of new histones primarily at the end of transcribed units. H4K20 was methylated in low-turnover nucleosomes in euchromatic regions, notably in the coding regions of long genes that were expressed at low levels. This transcription-dependent accumulation of histone methylation was dependent on the histone chaperone complex FACT. Our data showed that nucleosome turnover is highly dynamic in the genome and that several mechanisms are at play to either maintain or suppress stability. In particular, we found that FACT-associated transcription conserves histones by recycling them and is required for progressive H4K20 methylation.

Distinct roles for Sir2 and RNAi in centromeric heterochromatin nucleation, spreading and maintenance.

Epigenetically regulated heterochromatin domains govern essential cellular activities. A key feature of heterochromatin domains is the presence of hypoacetylated nucleosomes, which are methylated on lysine 9 of histone H3 (H3K9me). Here, we investigate the requirements for establishment, spreading and maintenance of heterochromatin using fission yeast centromeres as a paradigm. We show that establishment of heterochromatin on centromeric repeats is initiated at modular 'nucleation sites' by RNA interference (RNAi), ensuring the mitotic stability of centromere-bearing minichromosomes. We demonstrate that the histone deacetylases Sir2 and Clr3 and the chromodomain protein Swi6(HP1) are required for H3K9me spreading from nucleation sites, thus allowing formation of extended heterochromatin domains. We discovered that RNAi and Sir2 along with Swi6(HP1) operate in two independent pathways to maintain heterochromatin. Finally, we demonstrate that tethering of Sir2 is pivotal to the maintenance of heterochromatin at an ectopic locus in the absence of RNAi. These analyses reveal that Sir2, together with RNAi, are sufficient to ensure heterochromatin integrity and provide evidence for sequential establishment, spreading and maintenance steps in the assembly of centromeric heterochromatin.