A “Tail” of Unusual Histone H2A Variants in Bdelloid Rotifers

Abstract

In eukaryotes, the nucleosome is comprised of an octamer of four canonical core histones (two copies each of H2A, H2B, H3, and H4) around which ~147bp of DNA is wrapped. Histone variants (non‐allelic versions of the core histones) are also present in chromatin, and these proteins have diverse functions in DNA repair, meiotic recombination, chromosome segregation and transcription regulation. Bdelloid rotifers are aquatic microinvertebrates that possess H2A variants with C‐terminal tails ~20–40 residues longer than in other eukaryotes. While the genome project for the bdelloid Adineta vaga annotated three distinct histone H2A variants (H2Abd, H2Abd1 and H2Abd2), our search of the genome revealed a gene encoding a novel fourth H2A variant (hereafter called “H2A‐HDX”). The predicted translation of H2A‐HDX is 256 amino acids; this makes it unusual in that its C‐terminal tail is estimated to be over 120 residues longer than canonical H2A. The objective of this project was to characterize the sequence and function of histone H2A variants, including the newly discovered H2A‐HDX variant, found in bdelloid rotifers using phylogenetic, gene expression and mass spectrometry analyses. First, a phylogenetic analysis including H2A variants was done. We searched databases of protein and expressed sequence tag sequences for H2A variants from diverse eukaryotes. The analysis revealed that bdelloid H2A variants are present in a clade of other eukaryotic H2A homologs. In addition, H2A‐HDX represents an independent lineage of H2A variants distinct from other animals. Interestingly, bdelloids have the ability to effectively repair DNA double‐strand breaks (DSBs) induced by high doses of ionizing radiation. Since certain H2A variants (such as H2AX) function in DNA repair in other eukaryotes, we wanted to determine if H2A variants in bdelloid rotifers performed a similar role. We exposed the rotifers to 280 Gray of ionizing radiation to induce DNA damage, and used real‐time PCR to quantify expression levels of H2A variants. We found the expression of H2A variants in irradiated samples was not elevated relative to non‐irradiated samples, suggesting that upregulation is not associated with DNA repair. Next, mass spectrometry was used to test whether the long C‐terminal tails of the H2A variants are the targets of post‐translational modifications (PTMs) during DNA repair. An acid extraction protocol was used to isolate histones from irradiated and non‐irradiated rotifers. High‐resolution mass spectrometric analysis done using a Thermo Velos Orbitrap mass spectrometer identified all four bdelloid‐specific H2A variants, and validated the long C‐terminal tails of H2Abd, H2Abd1 and H2Abd2. H2A‐HDX was present in a lower abundance relative to other H2A variants, and only the first 194 of the predicted 256 amino acids were confirmed. However, reverse transcription PCR showed that the H2A‐HDX gene transcribes the full‐length C‐terminal tail. We also identified PTMs specific to irradiated samples in the N‐ and C‐terminal tails of the H2A variants, suggesting a potential role for epigenetics and histone H2A variants during DNA repair. Future pulldown assays using proteins and N‐ and C‐terminal tail peptides (+/− PTMs) will identify binding partners to H2A‐HDX and shed light on the role of histone H2A variants in DNA repair or other cellular processes in bdelloid rotifers.Support or Funding InformationThis work was supported by grants from the National Center for Research Resources (5P20RR016460) & National Institute of General Medical Sciences (8P20GM103429) from the NIH.