Epigenetics and System Biology of Stem cells and Reprogramming

Project Information

EpiSyStem

Grant agreement ID: 765966

DOI

10.3030/765966

Project closed

EC signature date 27 February 2018

Start date 1 March 2018

End date 31 August 2022

Funded under

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Total cost

€ 3 598 191,72

EU contribution

€ 3 598 191,72

Coordinated by

THE HEBREW UNIVERSITY OF JERUSALEM
Israel

Periodic Reporting for period 2 - EpiSyStem (Epigenetics and System Biology of Stem cells and Reprogramming)

Reporting period: 2020-03-01 to 2022-08-31

Summary of the context and overall objectives of the project

In eukaryotic cells, DNA is tightly packaged as chromatin in a form of nucleosomes, comprised of 147 bp of DNA wrapped around two copies each of the core histones H2A, H2B, H3 and H4. Nucleosomes are separated by linker DNA, bound by a linker histone, H1. Chromatin structure is controlled by protein complexes generally called chromatin modifiers, which affect DNA accessibility, DNA-protein interactions and chromatin organisation. Chromatin structure, regulation and plasticity dictate many of the nuclear processes, e.g. replication, transcription, cell cycle, protein dynamics and more - all of which ultimately facilitate or repress changes in transcription, either locally or globally. Chromatin is the basic regulatory unit of life and as such, it controls the developmental and functional states of all cells, including pluripotency. The aim of EpiSyStem was to concentrate on technological advances and state-of-the-art methods which enable a fresh and unbiased look at some of the long-standing questions in epigenetics. We combined chromatin biology in stem cells, early embryos and reprogramming (including the modelling of human neurological, chromatin-related, diseases) with bioinformatics, epigenetics/epigenomics, and systems biology at the population and the single-cell/single embryo level. This multidisciplinary approach enabled profound training with a deep, systems-level understanding of chromatin biology and epigenetic regulation of stem cells and reprogramming:
1. Histone dynamics and development of novel single-cell technologies: histone turnover in cell populations and single cells, to understand the consequences of cell-to-cell variability in the epigenome on phenotypic heterogeneity.
2. Identify the components and mechanisms that control chromatin regulation in early development: chromatin plasticity and epigenetic decisions in early mouse embryos, in embryonic stem cells using SNAP-tag technology, in later developmental stages during neurogenesis and neuronal/glial fate decisions, and, using specific chromatin-related antibodies and reagents.
3. Comprehensive mapping of chromatin structure and function during differentiation: multi-disciplinary state-of-the-art approaches in stem cell differentiation, including mass-spectrometry, CRISPR genome editing and live-cell imaging of endogenously tagged genomic loci, and single-cell transcriptomics and epigenomic technologies.
4. Study and understand chromatin regulation in selected disease models of iPSCs: induced pluripotent stem cell (iPSC) models of chromatin-related diseases caused by symmetric dosage imbalances in epigenetic regulators.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

WP1: IC analysed the regulatory pathways that target histone variants and particular RNA with histone chaperones to control the assembly line and its connecting network. DGN optimised the iDEAL protocol for high-input sample preparation for the proteomic analysis. RU developed a PCR-based method to amplify, methylate, biotinylate and immobilize genomic mouse DNA sequences. HUJI demonstrated the hypothesis that clones are composed of cells more similar to each other than cells picked at random. KNAW developed T-ChIC (Transcriptome + Chromatin ImmunoCleavage), a method that allows for the simultaneous acquisition of full-length transcripts and histone mark positions of the same single cell.
WP2: HMGU performed joint profiling of H3K4me1 and H3K9me3 and demonstrated that cell types within the myeloid lineage have distinct active chromatin but share similar myeloid-specific heterochromatin-repressed states. UMIL provided a tunable histone variant expression along with ‘in-cell’ fluorescent labelling. KNAW investigated the role of Tip60/Ep400 in 2-cell-like state.
WP3: RU created a list of potential chromatin regulators of ESC differentiation. HUJI concentrated on the multi-faceted nuclear oncogene called SET and found that knockdown for P53 in SET-KO ESCs partially rescues lineage-marker misregulation during differentiation. UMIL aimed to uncover for the first time the molecular role of YY1 with the application of relevant human in vitro systems for disease modelling of a paradigmatic neurodevelopmental disorder, Gabriele-de Vries syndrome and performed the analysis of sequencing data to evidence elements that are specifically enriched with H3 variants.
WP4: UMIL provided the first empirical validation of the self-domestication hypothesis and positioned BAZ1B as a master regulator of the modern human face. HUJI, following the work of UMIL, performed ChIP-seq profiling on chromatin regulator SET in KH2 cell lines, as a first step in determining the role of SET in early development and as a necessary step before experiments on patient-specific iPSC line, which will be determined in the future.
EpiSyStem yield at the end of the project, are 14 publications; of them, 9 are research papers and 5 reviews. In 7 of the papers, our ESRs were named as first authors or equal-contributing first authors, and more papers are in press. All publications are Open Access. We provided the ESRs with 8 workshops and courses, and supplementary online training and meetings during Covid. EpiSyStem organised 2 international conferences with a total of 500 on-site attendees.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

EpiSyStem provided multidisciplinary opportunities together with career development sessions that promote vast interactions between the members, the ESRs and the community, and of industry-oriented training activities. The structured exchange-of-ideas within EpiSyStem allowed collaborations that we expect to last beyond the training period and be developed into career interactions. Such associations reflect the basic idea of EpiSyStem: to expose the ESRs to a broad range of disciplines, methods and perspectives, as well as innovation beyond fundamental science and beyond the state-of-the-art.
In parallel, EpiSyStem provided a unique ground for learning, centred on the ESRs' training. The workshops boosted the knowledge of the young researchers, giving them innovative tools and expertise on state-of-the-art methods to implement in their research and future careers. The network enhanced their understanding of related fields of study while giving them a broader perspective on science and exposing them to both industrial and academic environments. EpiSyStem equipped the ESRs with the required knowledge and tools, opening up a wide range of possibilities and directions for their future.
Numerous training sessions in methods of chromatin assays, cell cultures of human and nonhuman organisms, single-cell technologies, and bioinformatics-related techniques were given. The ESRs learned to analyse large-scale data providing a systems-level view of stem cell epigenetics. We had multiple sessions aiming to improve the fellows' interrelations and encourage their entrepreneurship skills. We trust EpiSyStem improved their scientific and interpersonal skills and will make them better scientists, aimed at advancing research skills to the broader community in the future.
We participated in several outreach activities such as “Adopt a School”, Native Scientists, Women in science and more. Our Twitter account has 469 followers and constantly growing and we have a web of interactions and synergies with other MSCA networks.
EpiSyStem ESRs were trained to communicate their scientific results professionally to various audiences and were instructed on how to refine a message and how to amplify its impact on the general public.

An example from the work of ESR Adrianos Skaros