The N-terminal tail of CENP-A is highly divergent from other H3 variants. Canonical histone N-termini are hotspots of conserved post-translational modification; however, no broadly conserved modifications of the vertebrate CENP-A tail have been previously observed. Our lab has identified novel post-translational modifications on human CENP-A N-termini using high-resolution MS. These include the trimethylation of Gly1 at the alpha-amino position and side-chain phosphorylation of Ser16 and Ser18. CENP-A is subjected to constitutive initiating methionine removal, similar to other H3 variants. The nascent N-terminal residue Gly1 becomes trimethylated on the α-amino group. We identified the methyltransferase NRMT as the enzyme responsible for modifying the CENP-A amino terminus. Methylation occurs in the pre-nucleosomal form and marks the majority of CENP-A nucleosomes. Serine 16 and 18 become phosphorylated in pre-nucleosomal CENP-A and are phosphorylated on asynchronous and mitotic nucleosomal CENP-A and is important for chromosome segregation during mitosis. The double phosphorylation motif forms a salt-bridged secondary structure and causes CENP-A N-terminal tails to form intramolecular associations. Analytical ultracentrifugation of phospho-mimetic CENP-A nucleosome arrays demonstrates that phosphorylation results in greater intranucleosome associations and counteracts the hyper-oligomerized state exhibited by unmodified CENP-A nucleosome arrays.
Our studies have revealed that the major modifications on the N-terminal tail of CENP-A alter the physical properties of the chromatin fiber at the centromere. We are presently working to understand the function of amino-terminal methylation in CENP-A function.