The involvement of histones and post‐translational modifications during DNA repair in bdelloid rotifers

Abstract

Histones are architectural proteins that package eukaryotic DNA into nucleosomes and regulate the expression and replication of the genome. Four canonical histones (H2A, H2B, H3, and H4) are present in duplicates to form an octamer within a nucleosome. Histone N‐and C‐terminal tails are often targets for post‐translational modifications (PTMs). For example, C‐terminal phosphorylation of the histone variant H2AX serves as an epigenetic marker for DNA damage and links damaged chromatin to DNA repair proteins. Bdelloid rotifers are aquatic microinvertebrates that have the ability to effectively repair DNA double‐strand breaks (DSBs) induced by high doses (>1000 Gray) of ionizing radiation. The bdelloid Adineta vaga possesses five histone H2A variants, three of which (H2Abd, H2Abd1, H2Abd2) have C‐terminal tails that are significantly longer than those in other eukaryotes. These tails have the potential to act as targets for PTMs. The objective of this project was to investigate the role of bdelloid H2A variants in DNA repair by characterizing PTMs associated with these proteins and to identify their binding partners. We induced DSBs by irradiating three species of bdelloids with 280 Gray of ionizing radiation. Histones were then isolated at different time points post‐irradiation and high‐resolution mass spectrometry was used to characterize the histones present. We identified each of the five bdelloid‐specific H2A variants along with histones H2B, H3 and H4. Several PTMs (acetylations, methylations and phosphorylations) were identified on the N‐and C‐terminal tails of all histones. Of the identified PTMs, many were only present during DNA repair; among these, several were located in the long C‐terminal tails of H2A variants. To determine the significance of the H2A variants and associated PTMS in bdelloids, we want to identify binding partners to these proteins during DNA repair. To accomplish this, we produced 6×histidine‐tagged proteins representing each of the five variants in A. vaga. Western blots with custom polyclonal antibodies specific for each variant were used to verify the identity of the tagged proteins. Recombinant proteins and peptides (+/− PTMs) will be used in pulldown assays to identify binding partners of histones during DNA repair. In sum, we have identified several novel histone PTMs associated with DNA repair in bdelloid rotifers. These findings will improve our understanding of the role of histones and epigenetics in the specialized DNA repair system in bdelloids.Support or Funding InformationThis work was supported by grants from the NCRR (5P20RR016460) & NIGMS (8P20GM103429) from the NIH and from the Arkansas Science and Technology Authority.