Identification and characterization of histones in Physarum polycephalum evidence a phylogenetic vicinity of Mycetozoans to the animal kingdom.

Physarum polycephalum belongs to Mycetozoans, a phylogenetic clade apart from the animal, plant and fungus kingdoms. Histones are nuclear proteins involved in genome organization and regulation and are among the most evolutionary conserved proteins within eukaryotes. Therefore, this raises the question of their conservation in Physarum and the position of this organism within the eukaryotic phylogenic tree based on histone sequences. We carried out a comprehensive study of histones in Physarum polycephalum using genomic, transcriptomic and molecular data. Our results allowed to identify the different isoforms of the core histones H2A, H2B, H3 and H4 which exhibit strong conservation of amino acid residues previously identified as subject to post-translational modifications. Furthermore, we also identified the linker histone H1, the most divergent histone, and characterized a large number of its PTMs by mass spectrometry. We also performed an in-depth investigation of histone genes and transcript structures. Histone proteins are highly conserved in Physarum and their characterization will contribute to a better understanding of the polyphyletic Mycetozoan group. Our data reinforce that P. polycephalum is evolutionary closer to animals than plants and located at the crown of the eukaryotic tree. Our study provides new insights in the evolutionary history of Physarum and eukaryote lineages.

A nucleosome-free region locally abrogates histone H1-dependent restriction of linker DNA accessibility in chromatin.

Eukaryotic genomes are packaged into linker-oligonucleosome assemblies, providing compaction of genomic DNA and contributing to gene regulation and genome integrity. To define minimal requirements for initial steps in the transition of compact, closed chromatin to a transcriptionally active, open state, we developed a model in vitro system containing a single, unique, "target" nucleosome in the center of a 25-nucleosome array and evaluated the accessibility of the linker DNA adjacent to this target nucleosome. We found that condensation of H1-lacking chromatin results in ∼60-fold reduction in linker DNA accessibility and that mimics of acetylation within all four core histone tail domains of the target nucleosome synergize to increase accessibility ∼3-fold. Notably, stoichiometric binding of histone H1 caused >2 orders of magnitude reduction in accessibility that was marginally diminished by histone acetylation mimics. Remarkably, a nucleosome-free region (NFR) in place of the target nucleosome completely abrogated H1-dependent restriction of linker accessibility in the immediate vicinity of the NFR. Our results suggest that linker DNA is as inaccessible as DNA within the nucleosome core in fully condensed, H1-containing chromatin. They further imply that an unrecognized function of NFRs in gene promoter regions is to locally abrogate the severe restriction of linker DNA accessibility imposed by H1s.

Spermatid-specific linker histone HILS1 is a poor condenser of DNA and chromatin and preferentially associates with LINE-1 elements.

BACKGROUND: Linker histones establish and maintain higher-order chromatin structure. Eleven linker histone subtypes have been reported in mammals. HILS1 is a spermatid-specific linker histone, and its expression overlaps with the histone-protamine exchange process during mammalian spermiogenesis. However, the role of HILS1 in spermatid chromatin remodeling is largely unknown. RESULTS: In this study, we demonstrate using circular dichroism spectroscopy that HILS1 is a poor condenser of DNA and chromatin compared to somatic linker histone H1d. Genome-wide occupancy study in elongating/condensing spermatids revealed the preferential binding of HILS1 to the LINE-1 (L1) elements within the intergenic and intronic regions of rat spermatid genome. We observed specific enrichment of the histone PTMs like H3K9me3, H4K20me3 and H4 acetylation marks (H4K5ac and H4K12ac) in the HILS1-bound chromatin complex, whereas H3K4me3 and H3K27me3 marks were absent. CONCLUSIONS: HILS1 possesses significantly lower α-helicity compared to other linker histones such as H1t and H1d. Interestingly, in contrast to the somatic histone variant H1d, HILS1 is a poor condenser of chromatin which demonstrate the idea that this particular linker histone variant may have distinct role in histone to protamine replacement. Based on HILS1 ChIP-seq analysis of elongating/condensing spermatids, we speculate that HILS1 may provide a platform for the structural transitions and forms the higher-order chromatin structures encompassing LINE-1 elements during spermiogenesis.