Periodic Reporting for period 1 - EpiDevoTimeMachine (Connecting the dots going backwards: An epigenetic memory recorder to trace ancestry during mouse gastrulation.)
Reporting period: 2023-09-01 to 2025-08-31
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.
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.
Objective 1: Simultaneous quantification of transcription and PcG proteins in single-cells
during gastruloid differentiation.
The initial phase of the project focused on establishing robust protocols for gastruloid formation using mouse embryonic stem cells (ESCs). Standard protocols, when applied to female ESCs, did not yield any elongated structures, highlighting a significant challenge in recapitulating efficient gastruloid development in this context. Through systematic optimization efforts, including adjustments to culture conditions and timing of CHIR pulse, the elongation efficiency for female cells was increased to approximately 30%. Despite this improvement, the efficiency remained markedly below the field standard of 80–90%, prompting a strategic decision to shift the focus to male ESCs, which are more commonly utilized in gastruloid studies and have a proven track record of high efficiency.
Upon transitioning to male ESCs, the optimized protocol consistently achieved elongation efficiencies of 80–90%, thus meeting the established benchmarks and providing a reliable platform for downstream analyses. In these cells, doxycycline-inducible expression constructs encoding Dam fusions to Ezh2 (a core component of PRC2), Ring1b (a key member of PRC1), and Laminb1 were successfully integrated. The correct genomic integration and inducible expression of these constructs were thoroughly verified, ensuring their suitability for high-resolution single-cell analyses of protein-DNA interactions and transcriptional states during gastruloid differentiation.
To interrogate the functional role of PRC2 during gastrulation, two complementary approaches were employed. First, the auxin-inducible degron system was used to target Suz12, a core PRC2 subunit, enabling rapid and specific depletion of PRC2 activity. Second, pharmacological inhibition of Ezh2, the catalytic subunit of PRC2, was implemented. Both strategies resulted in a pronounced reduction in the elongation capacity of gastruloids and induced a bias toward ectodermal fate, thereby demonstrating the essential role of PRC2 in orchestrating proper lineage specification and morphogenesis during early development.
Collectively, these activities led to several key achievements. The project established and validated a robust protocol for high-efficiency gastruloid formation using male ESCs, overcoming the limitations encountered with female lines. The generation and verification of dox-inducible Dam fusion constructs for critical chromatin regulators and nuclear envelope proteins provided the necessary tools for advanced single-cell epigenomic and transcriptomic investigations. Furthermore, functional perturbation experiments unequivocally demonstrated that PRC2 activity is indispensable for normal gastruloid elongation and balanced germ layer specification, as its loss results in impaired morphogenesis and a shift toward ectodermal differentiation. The established cell lines and optimized protocols now serve as a solid foundation for the high-resolution mapping of PcG protein dynamics and transcriptional changes during early developmental processes, positioning the project for impactful scientific contributions in the field of developmental epigenetics
Objective 2: Development of an epigenetic memory recorder system to record past epigenetic states.
The second objective of the project centered on the development of a molecular memory system to record past epigenetic states at single-cell resolution, with the aim of directly linking the binding history of key chromatin regulators to current transcriptional identity. The initial phase involved the successful cloning and integration of constructs encoding the bacterial DNA cytosine methyltransferase (Dcm) fused to Ezh2 (a core PRC2 component), Ring1b (a key PRC1 member), and Laminb1 into the target cell lines. These fusion proteins were designed to deposit Dcm methylation marks at specific genomic loci, thereby providing a record of chromatin regulator occupancy over time.
Following integration, the specificity of the Dcm fusion proteins was rigorously validated through bulk sequencing approaches, which confirmed their correct localization and the targeted deposition of methylation marks. This validation established the feasibility of the Dcm-based memory system in principle. However, subsequent functional testing, complemented by unpublished data from collaborators, revealed a critical limitation: the dynamic range of Dcm methylation was insufficient to capture the quantitative differences inherent to many epigenetic marks. This constraint rendered the system unsuitable for recording nuanced, quantitative variations in chromatin regulator binding, necessitating a strategic shift in approach.
In response to these findings, the project pivoted towards establishing a memory system based on DamID technology. This entailed the design and optimization of doxycycline-inducible DamID constructs, with particular emphasis on minimizing background methylation and enabling precise temporal control using the auxin-inducible degron system. Multiple Dam methyltransferase variants were engineered and systematically tested for their ability to maintain N6-methyladenine (m6A) marks through successive cell divisions, a prerequisite for stable epigenetic memory recording.
While the technical milestones of generating and validating inducible DamID constructs with improved controllability were achieved, ongoing experiments have yet to conclusively demonstrate the reliable maintenance of m6A marks across cell generations. The results to date remain inconclusive, indicating that further optimization and testing are required to fully realize the potential of this approach.
In summary, the project has made significant progress towards the development of an epigenetic memory recorder system. The initial Dcm-based strategy provided valuable insights, ultimately guiding the transition to a DamID-based approach that offers enhanced temporal control and reduced background. Although the establishment of a robust, heritable epigenetic memory system remains a work in progress, the advances in construct design and validation achieved thus far lay a strong foundation for future efforts in single-cell epigenetic lineage tracing.
during gastruloid differentiation.
The initial phase of the project focused on establishing robust protocols for gastruloid formation using mouse embryonic stem cells (ESCs). Standard protocols, when applied to female ESCs, did not yield any elongated structures, highlighting a significant challenge in recapitulating efficient gastruloid development in this context. Through systematic optimization efforts, including adjustments to culture conditions and timing of CHIR pulse, the elongation efficiency for female cells was increased to approximately 30%. Despite this improvement, the efficiency remained markedly below the field standard of 80–90%, prompting a strategic decision to shift the focus to male ESCs, which are more commonly utilized in gastruloid studies and have a proven track record of high efficiency.
Upon transitioning to male ESCs, the optimized protocol consistently achieved elongation efficiencies of 80–90%, thus meeting the established benchmarks and providing a reliable platform for downstream analyses. In these cells, doxycycline-inducible expression constructs encoding Dam fusions to Ezh2 (a core component of PRC2), Ring1b (a key member of PRC1), and Laminb1 were successfully integrated. The correct genomic integration and inducible expression of these constructs were thoroughly verified, ensuring their suitability for high-resolution single-cell analyses of protein-DNA interactions and transcriptional states during gastruloid differentiation.
To interrogate the functional role of PRC2 during gastrulation, two complementary approaches were employed. First, the auxin-inducible degron system was used to target Suz12, a core PRC2 subunit, enabling rapid and specific depletion of PRC2 activity. Second, pharmacological inhibition of Ezh2, the catalytic subunit of PRC2, was implemented. Both strategies resulted in a pronounced reduction in the elongation capacity of gastruloids and induced a bias toward ectodermal fate, thereby demonstrating the essential role of PRC2 in orchestrating proper lineage specification and morphogenesis during early development.
Collectively, these activities led to several key achievements. The project established and validated a robust protocol for high-efficiency gastruloid formation using male ESCs, overcoming the limitations encountered with female lines. The generation and verification of dox-inducible Dam fusion constructs for critical chromatin regulators and nuclear envelope proteins provided the necessary tools for advanced single-cell epigenomic and transcriptomic investigations. Furthermore, functional perturbation experiments unequivocally demonstrated that PRC2 activity is indispensable for normal gastruloid elongation and balanced germ layer specification, as its loss results in impaired morphogenesis and a shift toward ectodermal differentiation. The established cell lines and optimized protocols now serve as a solid foundation for the high-resolution mapping of PcG protein dynamics and transcriptional changes during early developmental processes, positioning the project for impactful scientific contributions in the field of developmental epigenetics
Objective 2: Development of an epigenetic memory recorder system to record past epigenetic states.
The second objective of the project centered on the development of a molecular memory system to record past epigenetic states at single-cell resolution, with the aim of directly linking the binding history of key chromatin regulators to current transcriptional identity. The initial phase involved the successful cloning and integration of constructs encoding the bacterial DNA cytosine methyltransferase (Dcm) fused to Ezh2 (a core PRC2 component), Ring1b (a key PRC1 member), and Laminb1 into the target cell lines. These fusion proteins were designed to deposit Dcm methylation marks at specific genomic loci, thereby providing a record of chromatin regulator occupancy over time.
Following integration, the specificity of the Dcm fusion proteins was rigorously validated through bulk sequencing approaches, which confirmed their correct localization and the targeted deposition of methylation marks. This validation established the feasibility of the Dcm-based memory system in principle. However, subsequent functional testing, complemented by unpublished data from collaborators, revealed a critical limitation: the dynamic range of Dcm methylation was insufficient to capture the quantitative differences inherent to many epigenetic marks. This constraint rendered the system unsuitable for recording nuanced, quantitative variations in chromatin regulator binding, necessitating a strategic shift in approach.
In response to these findings, the project pivoted towards establishing a memory system based on DamID technology. This entailed the design and optimization of doxycycline-inducible DamID constructs, with particular emphasis on minimizing background methylation and enabling precise temporal control using the auxin-inducible degron system. Multiple Dam methyltransferase variants were engineered and systematically tested for their ability to maintain N6-methyladenine (m6A) marks through successive cell divisions, a prerequisite for stable epigenetic memory recording.
While the technical milestones of generating and validating inducible DamID constructs with improved controllability were achieved, ongoing experiments have yet to conclusively demonstrate the reliable maintenance of m6A marks across cell generations. The results to date remain inconclusive, indicating that further optimization and testing are required to fully realize the potential of this approach.
In summary, the project has made significant progress towards the development of an epigenetic memory recorder system. The initial Dcm-based strategy provided valuable insights, ultimately guiding the transition to a DamID-based approach that offers enhanced temporal control and reduced background. Although the establishment of a robust, heritable epigenetic memory system remains a work in progress, the advances in construct design and validation achieved thus far lay a strong foundation for future efforts in single-cell epigenetic lineage tracing.
The project achieved substantial advances in both the quantitative mapping of polycomb-group (PcG) proteins during gastruloid differentiation and the development of a molecular memory system for recording past epigenetic states.
For the first objective, robust protocols for gastruloid formation were established. After initial challenges with female embryonic stem cells, which showed low elongation efficiency, the focus shifted to male cells, where optimized protocols consistently yielded 80–90% elongation—matching field standards. In these lines, doxycycline-inducible Dam fusion constructs for Ezh2 (PRC2), Ring1b (PRC1), and Laminb1 were successfully integrated and validated, enabling precise, single-cell mapping of protein-DNA interactions and transcriptional states. Functional studies using auxin-inducible degron and pharmacological inhibition approaches demonstrated that PRC2 activity is crucial for proper gastruloid elongation and balanced germ layer specification, as its loss led to impaired morphogenesis and a bias toward ectodermal fate.
For the second objective, the project initially developed and validated Dcm methyltransferase fusions to key chromatin regulators. While these constructs displayed correct targeting and specificity, functional testing revealed that Dcm methylation lacked the dynamic range needed for quantitative epigenetic memory. This led to a strategic pivot toward a DamID-based system. Optimized, inducible DamID constructs were engineered to reduce background methylation and allow temporal control. Multiple Dam variants were tested for their ability to maintain m6A marks across cell divisions, a prerequisite for stable epigenetic memory, though further optimization is ongoing.
These efforts represent a significant step beyond the current state of the art. The project will enable to provide the first comprehensive, single-cell maps of PcG protein dynamics during lineage commitment in gastruloids, and lays the groundwork for retrospective epigenetic lineage tracing. The technical advances—particularly in inducible Dam fusion lines and memory system design—offer new tools for the scientific community, with broad potential for adoption and further development. Future progress will depend on continued optimization of the memory system, broader demonstration in diverse contexts, and strategic collaborations to maximize scientific impact and uptake.
For the first objective, robust protocols for gastruloid formation were established. After initial challenges with female embryonic stem cells, which showed low elongation efficiency, the focus shifted to male cells, where optimized protocols consistently yielded 80–90% elongation—matching field standards. In these lines, doxycycline-inducible Dam fusion constructs for Ezh2 (PRC2), Ring1b (PRC1), and Laminb1 were successfully integrated and validated, enabling precise, single-cell mapping of protein-DNA interactions and transcriptional states. Functional studies using auxin-inducible degron and pharmacological inhibition approaches demonstrated that PRC2 activity is crucial for proper gastruloid elongation and balanced germ layer specification, as its loss led to impaired morphogenesis and a bias toward ectodermal fate.
For the second objective, the project initially developed and validated Dcm methyltransferase fusions to key chromatin regulators. While these constructs displayed correct targeting and specificity, functional testing revealed that Dcm methylation lacked the dynamic range needed for quantitative epigenetic memory. This led to a strategic pivot toward a DamID-based system. Optimized, inducible DamID constructs were engineered to reduce background methylation and allow temporal control. Multiple Dam variants were tested for their ability to maintain m6A marks across cell divisions, a prerequisite for stable epigenetic memory, though further optimization is ongoing.
These efforts represent a significant step beyond the current state of the art. The project will enable to provide the first comprehensive, single-cell maps of PcG protein dynamics during lineage commitment in gastruloids, and lays the groundwork for retrospective epigenetic lineage tracing. The technical advances—particularly in inducible Dam fusion lines and memory system design—offer new tools for the scientific community, with broad potential for adoption and further development. Future progress will depend on continued optimization of the memory system, broader demonstration in diverse contexts, and strategic collaborations to maximize scientific impact and uptake.