Elucidating the interplay between nuclear compartments and transcriptional dynamics during differentiation

Project Information

DynaDiff

Grant agreement ID: 101040548

DOI

10.3030/101040548

EC signature date 16 September 2022

Start date 1 October 2022

End date 30 September 2027

Funded under

• European Research Council (ERC)

Total cost

€ 1 498 372,00

EU contribution

€ 1 498 372,00

Coordinated by

HELMHOLTZ ZENTRUM MUENCHEN DEUTSCHES FORSCHUNGSZENTRUM FUER GESUNDHEIT UND UMWELT GMBH
Germany

Periodic Reporting for period 1 - DynaDiff (Elucidating the interplay between nuclear compartments and transcriptional dynamics during differentiation)

Reporting period: 2022-10-01 to 2025-03-31

Summary of the context and overall objectives of the project

Cellular differentiation, the specialization of cells into distinct cell types, is a fundamental biological process governed by intricate regulatory networks. Despite significant advancements in the field, our understanding of the precise mechanisms underlying these networks remains incomplete. Key challenges in unraveling these mechanisms are the difficulty in measuring transcriptional dynamics with sufficient temporal resolution, and understanding transcriptional output in the within the context of the complex nuclear compartmentalization.

While we know that nuclear compartments, such as nucleoli, promyelocytic leukaemia nuclear body/bodies (PML-NBs), and nuclear speckles (NSs), play crucial roles in various cellular processes, including gene expression and chromatin organization, their specific contributions to cell fate decisions and the coordination of developmental transcriptional programs remain elusive. DynaDiff aims to address these knowledge gaps by developing innovative single-cell omics techniques and employing advanced microscopy methods to investigate the interplay between nuclear compartments, chromatin organization, and transcriptional dynamics during early cell development. By deciphering these mechanisms, we will gain valuable insights into the fundamental principles governing cellular differentiation, paving the way for future advancements in regenerative medicine.

DynaDiff will have a profound impact on the field of developmental biology and regenerative medicine, as it will provide insights into how cells make fate decisions, which could lead to novel strategies for reprogramming cells and regenerating tissues. Additionally, understanding the role of nuclear compartments in regulating gene expression could open new avenues for therapeutic interventions.

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

So far, my team and I have established the technical pipelines for three state-of-the-art technologies that are of outmost importance for the proceeding of DynaDiff: scVASA-seq, multiplexing immunofluorescence, and the measurement of higher order chromatin interactions.
Since DynaDiff was associated with relocation and establishing my group, one key achievement so far is the establishment of a state-of-the-art laboratory equipped to conduct cutting-edge single-cell
-omics and high-throughput imaging experiments. Further, the team has optimized protocols and reagents for multiplexed fluorescence imaging and successfully implemented single-cell total transcriptomics and metabolic labeling techniques that are key for sequencing of true nascent RNA in single cells. In addition, we formed a robust collaboration network with leading institutions to facilitate knowledge exchange and joint research endeavors.
A major technical highlight during the first reporting period was the development of a versatile single-cell -omics platform capable of performing various assays, including scRNA-seq, scDNA-seq, ATAC-seq, as well as single cell cut-and-run. This will enable us to comprehensively analyze cellular heterogeneity and gene expression patterns, leading to insights into fundamental biological processes, disease mechanisms, and potential therapeutic strategies. By integrating high-throughput imaging with single-cell omics, the DynaDiff is set to dissect complex biological systems and uncover key regulatory pathways.
We are currently expanding our research towards exploring cellular heterogeneity in human differentiation, using single-cell data from various models like embryoid bodies and potentially gastruloids and organoids. We are also developing novel single-cell analysis methods, including temporal resolved -omics, to map gene expression dynamics.

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)

My team and I have made significant progress in establishing and advancing VASA-seq to study gene expression at the single-cell level. This was a major step forward and now allows us to measure newly synthesized RNA molecules with unprecedented precision, including non-polyadenylated transcripts. This breakthrough opens new possibilities for understanding how genes are activated and regulated over time, advancing our knowledge of cellular processes and disease mechanisms.