EpiDevoTimeMachine · Connecting the dots going backwards: An epigenetic memory recorder to trace ancestry during mouse gastrulation.

Connecting the dots going backwards: An epigenetic memory recorder to trace ancestry during mouse gastrulation.

Scientific Background During early mammalian embryonic development, pluripotent epiblast cells possess the remarkable capacity to generate all three definitive germ layers: ectoderm, endoderm, and mesoderm. This process, termed gastrulation, is characterized by a progressive restriction of developmental potential, as cells acquire specialized fates and establish the blueprint of the adult organism. The orchestration of these fate decisions is governed by a complex interplay between transcription factors and epigenetic modifiers, notably the polycomb-group (PcG) proteins. While single-cell RNA sequencing has provided critical insights into the transcriptional landscape of gastrulation, comparatively little is known about the quantitative dynamics and functional roles of epigenetic regulators, particularly PRC1 and PRC2, during this pivotal developmental window. Recent studies have underscored the essential role of PcG proteins in lineage commitment, with perturbations in these complexes resulting in profound developmental phenotypes. However, the field currently lacks a comprehensive understanding of the temporal dynamics of PRC1 and PRC2 during gastrulation, and, crucially, no approach exists to directly record the ancestral epigenetic state of a cell. This gap in knowledge limits our ability to decipher the causal relationships between epigenetic regulation and cell fate specification. Rationale and Innovation Building on recent advances in single-cell genomics, the proposed project seeks to address these challenges by developing and applying a molecular memory system capable of recording the past epigenetic state of single cells, while simultaneously capturing their current transcriptional identity. By leveraging the unique properties of Dcm methylation and the TAPS sequencing methodology, this system could enable the stable transmission and detection of epigenetic marks over cell generations. Gastruloids—three-dimensional aggregates of embryonic stem cells that recapitulate key features of gastrulation—will serve as a robust in vitro model, allowing for high-resolution temporal sampling and manipulation. Objectives The project comprises two main objectives: Objective 1: Simultaneous quantification of transcription and PcG proteins in single cells during gastruloid differentiation. This will be achieved by employing scDam&T using established Dam-Rnf2 and Dam-Ezh2 fusion cell lines, enabling the construction of a detailed roadmap of PRC1 and PRC2 dynamics in the context of transcriptional changes. Objective 2: Development of an epigenetic memory recorder system to record past epigenetic states. The system will utilize a bacterial Dcm methylase fused to PcG proteins, with Dcm methylation maintained by DNMT1, and read out via TAPS and CEL-seq2. This approach will allow for the direct linking of past epigenetic states to current lineage identity at single-cell resolution. Methodological Approach Integration of Technologies: The methodology integrates state-of-the-art single-cell approaches, including scDam&T and TAPS, with advanced cell culture models and inducible degron systems to achieve precise temporal control over protein activity. Data Analysis: The project will leverage expertise in bioinformatics to analyze multi-modal single-cell data, enabling the reconstruction of epigenetic trajectories and identification of molecular signatures associated with lineage commitment. Open Science Practices: All sequencing data, code, and cell lines generated will be made publicly available through repositories such as GEO, GitHub, and Addgene, ensuring transparency and fostering collaboration within the scientific community. Expected Scientific Impact Fundamental Insights: The project will provide the first comprehensive, quantitative maps of PcG protein dynamics during gastruloid differentiation at single-cell resolution, elucidating the interplay between epigenetic regulation and cell fate decisions. Technological Advancement: The development of an epigenetic memory recorder system represents a significant methodological innovation, enabling retrospective analysis of epigenetic states and offering broad applicability to diverse proteins and developmental contexts. Catalyzing Future Research: By openly sharing tools and resources, the project will facilitate the adoption of these methodologies by the wider research community, accelerating discoveries in developmental biology, epigenetics, and related fields. Scale and Significance Global Relevance: The approaches and findings will be of broad interest to researchers worldwide, transcending disciplinary boundaries and providing a unique toolbox for the study of cellular decision-making. Resource Sharing: The deposition of cell lines, plasmids, and sequencing data in public repositories will maximize the utility and impact of the project outputs. Capacity Building: Training in advanced single-cell and epigenomic techniques will contribute to the development of expertise and scientific excellence within the field.

Data: CORDIS, © European Union