Within canonical eukaryotic nuclei, DNA is packaged with highly conserved histone proteins into nucleosomes, which facilitate DNA condensation and contribute to genomic regulation. Yet the dinoflagellates, a group of unicellular algae, are a striking exception to this otherwise universal feature as they have largely abandoned histones and acquired apparently viral-derived substitutes termed DVNPs (dinoflagellate-viral-nucleoproteins). Despite the magnitude of this transition, its evolutionary drivers remain unknown. Here, using Saccharomyces cerevisiae as a model, we show that DVNP impairs growth and antagonizes chromatin by localizing to histone binding sites, displacing nucleosomes, and impairing transcription. Furthermore, DVNP toxicity can be relieved through histone depletion and cells diminish their histones in response to DVNP expression suggesting that histone reduction could have been an adaptive response to these viral proteins. These findings provide insights into eukaryotic chromatin evolution and highlight the potential for horizontal gene transfer to drive the divergence of cellular systems.

We employ targeted mass spectrometry to compare the levels of H2AX S139 phosphorylation (γH2AX) and Y142 phosphorylation. We use synthetic peptides to facilitate MS optimization and estimate relative detection efficiencies for the different modifications. Despite phosphopeptide enrichment from large amounts of starting material, we are unable to detect endogenous H2AX Y142 phosphorylation, indicating that it is present at low abundance (<1%). We also calculate the relative levels of H2AX compared to other H2A isoforms and quantify the proportion of H2AX that is phosphorylated on S139 (γH2AX) after ionizing radiation. Introduction S139 phosphorylationThe histone H2A family variant, H2AX, is distinguished from canonical H2A family members through a 22 amino acid C-terminal tail [1]. Phosphorylation of the C-terminal domain of H2AX at position 139 (γH2AX) is a rapid response to DNA double-strand breaks (DSB). S139 is phosphorylated by ATM, ATR and DNA-PK, which are phosphatidylinositol 3-kinase-related kinases. γH2AX foci are widely used as diagnostic markers of DSB. The utility of γH2AX as a marker stems from the rapid (<1 min) and extensive nature of this modification. Rogakou et al. observed that approximately 1% of total H2AX becomes phosphorylated per gray of ionizing radiation (IR), and extrapolated from H2AX relative abundance that each DSB results in γH2AX covering on average 2 million bp [2]. The biological function of such large γH2AX domains is not clear, and the H2AX histone is not essential for DSB repair, however H2AX -/-mice show increased ionizing radiation sensitivity, as well as increases in chromatid breaks and dicentric chromosomes [3]. Y142 phosphorylationThe H2AX C-terminal domain can also be phosphorylated on tyrosine 142 by the WSTF remodelling factor kinase [4][5][6]. Cook et al. show that dephosphorylation of Y142 upon DNA damage avoids apoptosis. Using synthetic phosphopeptides, they demonstrate binding of pro-apoptotic factors to S139 Y142 doubly-phosphorylated peptides: the implication is that Y142 phosphorylation is abundant, and will be located in proximity to DNA damage. While kinases and phosphatases responsible for creating and removing this modification have been identified, the basal level of Y142 phosphorylation is unknown, although our earlier intact histone MS analysis indicates that in HeLa cells it is not greater than~10% [7]. Scully et al. expressed epitope-tagged H2AX in H2AX -/-mouse ES cells and identified a number of H2AX modifications by mass spectrometry, including S139 and T101 phosphorylation, however Y142 phosphorylation was not detected [8]. The role of Y142ph in the DNA damage response is of great interest, with the identification of putative interacting proteins that recognise the doubly phosphorylated C-terminal tail [9]. Mutation of these residues has been carried out in the chicken DT40 cell line and revealed that Y142A IR sensitivity is rescued by co-mutation of S139A [10]. H2AX levels across cell lines and in the genomeGiven the role of H2AX phospho...

Many animals achieve sperm chromatin compaction and stabilisation by replacing canonical histones with sperm nuclear basic proteins (SNBPs) such as protamines during spermatogenesis. Hydrozoan cnidarians and echinoid sea urchins lack protamines and have evolved a distinctive family of sperm-specific histone H2Bs (spH2Bs) with extended N termini rich in SPK(K/R) motifs. Echinoid sperm packaging is regulated by spH2Bs. Their sperm is negatively buoyant and fertilises on the sea floor. Hydroid cnidarians undertake broadcast spawning but their sperm properties are poorly characterised. We show that Hydractinia echinata and H. symbiolongicarpus sperm chromatin possesses higher stability than somatic chromatin, with reduced accessibility to transposase Tn5 integration and to endonucleases in vitro. In contrast, nuclear dimensions are only moderately reduced in mature Hydractinia sperm. Ectopic expression of spH2B in the background of H2B.1 knockdown results in downregulation of global transcription and cell cycle arrest in embryos, without altering their nuclear density. Taken together, SPKK-containing spH2B variants act to stabilise chromatin and silence transcription in Hydractinia sperm with only limited chromatin compaction. We suggest that spH2Bs could contribute to sperm buoyancy as a reproductive adaptation.

Many animals achieve sperm chromatin compaction and stabilisation during spermatogenesis by replacing canonical histones with sperm nuclear basic proteins (SNBPs) such as protamines. A number of animals including hydrozoan cnidarians and echinoid sea urchins lack protamines and have instead evolved a distinctive family of sperm-specific histone H2Bs (spH2Bs) with extended N-termini rich in SPKK-related motifs. Sperm packaging in echinoids such as sea urchins is regulated by spH2Bs and their sperm is negatively buoyant for fertilization on the sea floor. Hydroid cnidarians also package sperm with spH2Bs but undertake broadcast spawning and their sperm properties are poorly characterised. We show that sperm chromatin from the hydroid Hydractinia possesses higher stability than its somatic equivalent, with reduced accessibility of sperm chromatin to transposase Tn5 integration in vivo and to endonucleases in vitro. However, nuclear dimensions are only moderately reduced in mature Hydractinia sperm compared to other cell types. Ectopic expression of spH2B in the background of H2B knockdown resulted in downregulation of global transcription and cell cycle arrest in embryos without altering their nuclear density. Taken together, spH2B variants containing SPKK-related motifs act to stabilise chromatin and silence transcription in Hydractinia sperm without significant chromatin compaction. This is consistent with a contribution of spH2B to sperm buoyancy as a reproductive adaptation.