Chromatin 3D architecture in Archaea

Project Information

3DchromArchaea

Grant agreement ID: 101076671

DOI

10.3030/101076671

EC signature date 2 February 2023

Start date 1 April 2023

End date 31 March 2028

Funded under

• European Research Council (ERC)

Total cost

€ 1 494 500,00

EU contribution

€ 1 494 500,00

Coordinated by

EUROPEAN MOLECULAR BIOLOGY LABORATORY
Germany

Periodic Reporting for period 1 - 3DchromArchaea (Chromatin 3D architecture in Archaea)

Reporting period: 2023-04-01 to 2025-09-30

Summary of the context and overall objectives of the project

This project brings together the field of chromatin evolution and state-of-the-art structural biology to advance our understanding of a fundamental question: origin of chromatin structural and regulatory complexity.
Eukaryotes and most groups of archaea organize their genomes in the form of histone-based chromatin. Conservation of histones across the tree of life goes beyond protein sequence and histone fold. Tertiary arrangement of histones and DNA geometry in archaeal nucleosomes resemble those in eukaryotes; however, archaea can form special hypernucleosomes and “slinky”-like arrangements. Similarly to eukaryotes, some archaea have multiple histone variants and extended histone tails, although it is unclear whether their structural and regulatory roles are conserved. Eukaryotes inherited histone-based chromatin from archaea, however, the origins of eukaryotic chromatin complexity are enigmatic.
Therefore, this proposal addresses the 3D organization of chromatin in archaea to advance the understanding of chromatin evolution. We will test the following hypotheses: archaeal chromatin along with hypernucleosomes contains multiple open structures to maintain DNA accessibility and allow polymerase passage; histone variant exchange and histone tails in archaea play an important role in chromatin compaction similarly to eukaryotes. To test our hypotheses, we will synergistically apply state-of-the-art cryo- electron microscopy (cryo-EM) in situ and in vitro to selected archaeal systems. In situ cryo-EM will provide structural information about chromatin in native context, while cryo-EM of in vitro reconstituted chromatin will provide high-resolution structural information. Structural analysis complemented with biochemical, biophysical characterization and nucleosome positioning data will provide insights into 3D chromatin architecture in archaea in the context of eukaryotic chromatin evolution.

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

Over the two years of ongoing ERC funding, we have made substantial progress in investigating the 3D architecture of archaeal chromatin. We have successfully laid the groundwork for all our project aims and advanced significantly in multiple directions.
Work performed:
For aims focused on studying chromatin organization in situ:
• Successfully established in-house cultures of several model archaeal species.
• Developed and optimized cryo-grid preparation, FIB-milling and cryo-ET workflows for selected samples, and acquired tomographic datasets.
• Formed key collaborations with leading experts in relevant fields.
• Obtained initial insights into chromatin organization in model archaeal species.
For aims focused on studying chromatin organization in vitro:
• Cloned, expressed, and purified numerous histone proteins from a variety of archaeal species, including both Euryarchaea and Asgard archaea. Prepared multiple DNA templates.
• Characterized their DNA-binding properties and reconstituted diverse histone–DNA complexes.
• Solved multiple novel structures using cryo-EM and single-particle analysis, and initiated their functional characterization.
• Through structural insights, targeted mutagenesis, and a combination of biochemical and biophysical assays, we have uncovered new details about archaeal chromatin organization in vitro.
Main Achievements
Over the course of the project, we have made substantial progress toward understanding chromatin organization in Archaea, both in situ and in vitro. Key achievements include:
• In-depth structural studies of chromatin from diverse archaeal groups, including Euryarchaeaota and beyond.
• Establishment of robust experimental pipelines, combining cryo-EM, cryo-ET, and advanced sample preparation methods (including FIB-milling) tailored for archaeal systems.
• Discovery of multiple novel chromatin structures, including those from Asgard archaea, expanding the known diversity and evolutionary landscape of archaeal chromatin beyond previously studied lineages.
• Reconstitution and structural resolution of diverse histone–DNA complexes, using histones from both Euryarchaea and Asgard archaea, revealing new modes of DNA packaging.
• Functional characterization through mutagenesis and biochemical assays, offering new insights into archaeal chromatin function.
• Strategic collaborations with field leaders, reinforcing interdisciplinary approaches and enhancing the impact of our findings.

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)

Our research has already yielded results that significantly extend the current boundaries of knowledge in the field of archaeal chromatin biology. One of the most impactful outcomes to date is our work on Asgard archaeal chromatin, which is now publicly available as a preprint (https://www.biorxiv.org/content/10.1101/2025.05.24.653377v1). This study presents the first-ever structural insights into chromatin architecture within Asgard archaea—a major and previously unexplored branch of the archaeal domain. Until our work, structural knowledge of archaeal chromatin was confined exclusively to Euryarchaeota, and even within that group, only two structural studies had been published.
By leveraging cryo-electron microscopy and related structural techniques, we were able to identify and characterize multiple distinct chromatin structures based on Asgard histones. Remarkably, some of these structures show conservation with those found in other archaeal groups, suggesting shared evolutionary mechanisms. Others, however, appear to be completely novel, hinting at lineage-specific adaptations in chromatin organization. This diversity challenges existing paradigms and provides a much richer framework for understanding the evolution of chromatin across the tree of life.
In parallel, our investigations into chromatin organization in other archaeal lineages are progressing well. We are applying both in situ (cryo-electron tomography) and in vitro (biochemical and structural) approaches to obtain a comprehensive view of chromatin architecture in various archaeal species. Early findings from these ongoing studies are promising and suggest that the diversity of chromatin structures across Archaea is even greater than previously anticipated.
Once these additional results are ready for publication, they are expected to contribute significantly to a holistic and integrative understanding of archaeal chromatin. These insights will not only illuminate fundamental aspects of archaeal biology but may also have broader implications for the study of chromatin evolution, the origin of eukaryotic chromatin, and the design of synthetic nucleoprotein systems.
Taken together, the work funded by this ERC grant is already pushing the boundaries of what is known in the field and is poised to have a lasting impact on how we understand genome organization in the archaeal domain and beyond.