The project comprised three work packages, all completed, along with a major additional line of investigation.
In WP1, a comprehensive inventory of histone variants and post-translational modifications (PTMs) was established for three red algae. Histones were analyzed by mass spectrometry. The results confirmed that extremophilic red algae carry a radically simplified set of chromatin marks. The absence of the transcription elongation mark H3K36me3 in Cyanidioschyzon was confirmed, while Galdieria surprisingly retained this modification despite losing its canonical writer enzyme. Bisulfite sequencing confirmed negligible DNA methylation across four extremophilic species. A comparative genomic survey across approximately 50 plant and algal species reconstructed how gene losses, duplications, and transfers shaped chromatin during adaptation to extreme environments.
In WP2, genome-wide chromatin maps were generated using ChIP-Seq. More than 15 histone modifications and variants were identified in all three species. These datasets revealed distinct chromatin states associated with specific genomic elements. Comparison between extremophilic and mesophilic species identified chromatin patterns unique to extreme-environment adaptation, including distinct enrichment of repressive marks H3K27me3 and H3K9me3 at transposable elements. These states were compared with published maps of land plants and bryophytes, placing red algal chromatin in a broader evolutionary context.
In WP3, genetic engineering and experimental evolution tested the functional impact of chromatin on adaptation. Chromatin regulators were introduced or removed in extremophilic species, and engineered strains were subjected to growth assays under multiple conditions. Barcoded lineage-tracking systems monitored fitness changes over hundreds of generations, providing direct measurements of how chromatin composition affects adaptability.
Beyond the original scope, findings in red algae motivated investigation of the deep origins of chromatin regulation. Chromatin proteins were traced across 107 taxa spanning the tree of life, with a focus on Asgard archaea. A landmark result was the discovery that SIR2 deacetylase proteins from Lokiarchaeota and Hodarchaeota can functionally replace yeast SIR2 in chromatin silencing, demonstrated using a yeast reporter assay. Structural analysis confirmed highly conserved catalytic domains with near-identical three-dimensional structures (approximately 1 angstrom deviation). These results demonstrate that histone-based genome regulation predates eukaryotic origins, establishing a proto-regulatory chromatin layer later elaborated into modern epigenetic systems.
Results were disseminated through international conferences, preprints on bioRxiv, open-access publications, and public engagement events. All sequencing data were deposited in public repositories following FAIR principles.
