Posttranslational modification of CENP-A influences the conformation of centromeric chromatin

Abstract

Centromeres are chromosomal loci required for accurate segregation of sister chromatids during mitosis. The location of the centromere on the chromosome is not dependent on DNA sequence, but rather it is epigenetically specified by the histone H3 variant centromere protein A (CENP-A). The N-terminal tail of CENP-A is highly divergent from other H3 variants. Canonical histone N termini are hotspots of conserved posttranslational modification; however, no broadly conserved modifications of the vertebrate CENP-A tail have been previously observed. Here, we report three posttranslational modifications on human CENP-A N termini using high-resolution MS: trimethylation of Gly1 and phosphorylation of Ser16 and Ser18. Our results demonstrate that 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 demonstrate that the N-terminal RCC1 methyltransferase is capable of modifying the CENP-A N terminus. Methylation occurs in the prenucleosomal form and marks the majority of CENP-A nucleosomes. Serine 16 and 18 become phosphorylated in prenucleosomal CENP-A and are phosphorylated on asynchronous and mitotic nucleosomal CENP-A and are 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 hyperoligomerized 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.epigenetics | kinetochore | mass spectrometry

Structural basis for substrate recognition by the human N-terminal methyltransferase 1

“…This study of NTMT1 in complex with its cofactor (SAH) and physiological substrate (RCC1) provides a structural foundation on which the field of α-N methylation can expand. Given that retinoblastoma protein RB1 and SET (Tooley et al 2010), DDB2 (Cai et al 2014), centromere H3 variants CENP-A/B (Bailey et al 2013;Dai et al 2015), Drosophila H2B (Villar-Garea et al 2012), and poly(ADP-ribose) polymerase 3 (Dai et al 2015) have been recently reported to be modified through α-N-terminal methylation and that there exists potentially >300 putative substrates harboring the NTMT1/2's consensus sequence (Tooley and Schaner Tooley 2014), the biological functions of α-Nterminal methylation are likely pleiotropic and may not be limited to only the modulation of DNA-binding abilities of the modified proteins. Thus, our proposed model of the recognition of a consensus XPK substrate motif by NTMT1 may shed light on the understanding of NTMT1's substrate specificity and catalytic mechanism in general.…”

“…In addition to RCC1, several other proteins are subject to α-N-terminal methylation (Webb et al 2010), including retinoblastoma protein RB1 and SET (Tooley et al 2010), damaged DNA-binding protein 2 (DDB2) (Cai et al 2014), centromere H3 variants CENP-A/B (Bailey et al 2013;Dai et al 2015), Drosophila H2B (Villar-Garea et al 2012), and poly(ADP-ribose) polymerase 3 (Dai et al 2015), while data bank analysis of NTMT1/2's consensus sequence predicts the existence of potentially >300 targets for α-N-terminal methylation (Tooley and Schaner Tooley 2014). NTMT1 is responsible for the α-N-terminal methylation of DDB2 in response to the generation of UV-induced cyclobutane pyrimidine dimers (Cai et al 2014).…”

Accessible DNA and Relative Depletion of H3K9me2 at Maize Loci Undergoing RNA-Directed DNA Methylation

“…CentC displayed lower levels of H3K9me2, H3K27me2, and CHG methylation than knob180 and TR-1 (Figures 3C and 3F; Supplemental Figures 4C, 4E, and 4F). This makes sense because, in centromeres, a subset of histone H3 is replaced by the histone H3 variant cenH3/CENP-A, which lacks the Lys-9 and Lys-27 residues (Bailey et al, 2013). Besides CentC, maize centromeres (defined as regions of cenH3 enrichment) contain a complex mixture of other DNA elements, including abundant and polymorphic retrotransposons that allow for the mapping of unique reads (excluding most CentC and other sequences with near-perfect copies).…”

Identification of the Post-translational Modifications Present in Centromeric Chromatin

“…To increase resolution using advanced chromatin-optimized proteomics, we undertook an unbiased approach to identify the histone PTMs that are closely associated with CENP-A, likely within the CENP-A nucleosome or present in neighboring nucleosomes. Previously, we used affinity purification and mass spectrometry to show that CENP-A molecules are phosphorylated at serines 16 and 18, and amino-terminally trimethylated (31). Here, using this biochemical system to purify specific populations of CENP-A, we identify and measure the levels of the PTMs on the proximal binding partner proteins, histone H3, H4, and HJURP.…”