The onset and regulation of mitosis is dependent on phosphorylation of

The onset and regulation of mitosis is dependent on phosphorylation of a wide array of proteins. acid a phosphomimetic or to alanine an unmodifiable residue result in an increase in cells in prophase and an increase in anaphase/telophase bridges respectively. SILAC-coupled mass spectrometry shows that phosphorylated H3T80 (H3T80ph) preferentially interacts with histones H2A and H4 relative to non-phosphorylated H3T80 and this result is usually supported by increased binding of H3T80ph to histone octamers in vitro. These findings support a model where H3T80ph protruding from your nucleosome surface promotes interactions between adjacent nucleosomes to promote chromatin compaction during mitosis in metazoan cells. phosphorylation of H3S10 is essential for both proper chromosome condensation and segregation. 20 However in mammals H3S10 is usually dispensable for both of Roflumilast these functions.21 22 This leaves open the possibility that another histone modification at a different site may be important for chromosome condensation in metazoans. Unlike the N-terminal phosphorylation sites phosphorylation sites within the globular domain name of H3 are poorly characterized. These sites include H3T45 H3T80 and H3T118. Unlike H3T45 and H3T118 which are located at the DNA-histone interface H3T80 projects from the surface of the nucleosome. This position of H3T80ph makes it less likely to influence DNA-histone interactions but more likely to be accessible to one or more reader proteins. Much like H3S10 and H3S28 H3T80 is located adjacent to a lysine residue (H3K79) that is known to be mono- di- and tri-methylated.23-25 Despite the identification of H3T80 phosphorylation (H3T80ph) in mammalian cells in 4 mass spectrometry studies it has never been observed in the presence of H3K79 methylation (H3K79me).9-12 Nonetheless Martinez et al. used a commercial H3K79me3T80ph antibody to study histone H3K79me3T80ph in vivo.26 However our data shows that the H3K79me3T80ph antibody used previously actually recognizes H3K9me3S10ph calling into question not only the existence of the H3K79me3H3T80ph dual modification but also its dependence on the Aurora B kinase and the proposed link between this dual modification and cancer. Furthermore it highlights that H3T80ph shown to exist via mass spectrometry studies has not yet been characterized in vivo. Here we demonstrate using an antibody specific to H3T80ph that it is enriched during mitosis but unlike other mitotic H3 phsophorylation marks such as H3S10ph and H3S28ph 14 H3T80ph is not dependent on the Aurora B kinase and does not initiate at pericentric heterochromatin. Furthermore Roflumilast mutations to prevent or mimic H3T80 phosphorylation result in mitotic defects. Functionally phosphorylated H3T80 binds to other core histones leading us to propose that H3T80ph may promote chromatin condensation during mitosis. Results A commercially available H3K79me3T80ph antibody recognizes H3K9me3S10ph Like most histone H3 residues both H3T80 and the adjacent H3K79 are highly conserved (Fig.?1A). Spatially H3T80 and H3K79 are located around the nucleosome surface with H3T80 positioned in such a way Roflumilast that it protrudes from the surface (Fig.?1B).1 Previous work using a commercially available antibody raised against a H3K79me3T80ph peptide concluded that H3K79me3T80ph serves as a mitotic indicator in melanoma samples.26 However the staining pattern yielded by this GPR44 antibody within the cell and the ablation of this transmission by inhibitors of Aurora B kinase suggested to us the possibility that this putative H3K79me3T80ph antibody might recognize the highly abundant dual modification H3K9me3S10ph.5 14 Therefore we tested the specificity of this antibody (MC491) in 3 ways: dot blots with modified and unmodified H3 peptides; immunoblot analyses using lysates from cells lacking the H3K79 methyltransferase Dot1L;27 and indirect Roflumilast immunofluorescence in cells reduced in their capacity to phosphorylate H3S10. On dot blots and consistent with earlier work 26 the MC491 antibody did not recognize H3S10ph alone. However when the dual H3K9me3S10ph peptide was used the MC491 antibody acknowledged both H3K9me3S10ph and H3K79me3T80ph peptides to comparable extents (Fig.?1C). Immunoblot analyses revealed that this MC491 antibody acknowledged epitopes in both.