Three-dimensional genome organisation reveals spatially insulated outlier chromosomes in Ostreococcus tauri

Abstract Marine picoeukaryotes of the order Mamiellales possess some of the most compact genomes among free-living eukaryotes, yet each species maintains two “outlier chromosomes” containing regions of markedly lower GC content. These regions have been recognised since the first Mamiellales genome was sequenced nearly two decades ago, but whether they occupy a distinct spatial compartment within the nucleus has remained unknown. Here, we report the first chromosome conformation capture (Hi-C) analysis of Ostreococcus tauri , the smallest known free-living eukaryote, and show that its outlier regions form topologically associating domain (TAD)-like structures with sharp boundaries that spatially insulate them from the rest of the genome. Comparative analysis of available transcriptomic, methylation, and nucleosome positioning data across Mamiellales reveals that outlier regions share a conserved chromatin profile characterised by hypomethylation, transcriptional hyperactivity, and altered nucleosome spacing. The two outlier chromosomes differ in this respect: transcription is elevated in the big outlier region (BOC1) in every species examined, whereas transcription in the small outlier region (SOC1) varies between species. Outlier regions are enriched in transposable elements (TEs) in most Mamiellales species, whereas Bathycoccus prasinos and O. lucimarinus lack substantial TE enrichment but retain the characteristic sequence composition and elevated BOC1 transcription. This decoupling identifies spatial insulation as a property of outlier chromosomes that TE content does not predict, adding a three-dimensional criterion to the sequence features by which these chromosomes have been defined. Analysis of published Hi-C data from Mantoniella tinhauana and the distantly related stramenopile Pelagomonas calceolata reveals consistent patterns of insulation, suggesting that compartmentalisation of outlier regions may be a recurrent feature of compact picoeukaryotic genomes. Our findings establish outlier chromosomes as spatially insulated compartments whose organisation tracks chromatin state rather than TE content alone.