Identification of the Post-translational Modifications Present in Centromeric Chromatin

Abstract

The centromere is the locus on the chromosome that acts as the essential connection point between the chromosome and the mitotic spindle. A histone H3 variant, CENP-A, defines the location of the centromere, but centromeric chromatin consists of a mixture of both CENP-A-containing and H3-containing nucleosomes. We report a surprisingly uniform pattern of primarily monomethylation on lysine 20 of histone H4 present in short polynucleosomes mixtures of CENP-A and H3 nucleosomes isolated from functional centromeres. Canonical H3 is not a component of CENP-A-containing nucleosomes at centromeres, so the H3 we copurify from these preparations comes exclusively from adjacent nucleosomes. We find that CENP-A-proximal H3 nucleosomes are not uniformly modified but contain a complex set of PTMs. Dually modified K9me2-K27me2 H3 nucleosomes are observed at the centromere. Side-chain acetylation of both histone H3 and histone H4 is low at the centromere. Prior to assembly at centromeres, newly expressed CENP-A is sequestered for a large portion of the cell cycle (late S-phase, G2, and most of mitosis) in a complex that contains its partner, H4, and its chaperone, HJURP. In contrast to chromatin associated centromeric histone H4, we show that prenucleosomal CENP-A-associated histone H4 lacks K20 methylation and contains side-chain and ␣-amino acetylation. We show HJURP displays a complex set of serine phosphorylation that may potentially regulate the deposition of CENP-A.

Acetylation of histone H4 lysine 5 and 12 is required for CENP-A deposition into centromeres

“…2e,f), although a greater proportion of H4K12ac was present in human CENP-A chromatin compared with chicken. This result is consistent with a recent proteomics analysis18.…”

A Dual Inhibitory Mechanism Sufficient to Maintain Cell-Cycle-Restricted CENP-A Assembly

“…Key proteins necessary for the process of CENP-A deposition include the Mis18 complex and the CENP-A chaperone HJURP which bears CENP-A-specific nucleosome assembly activity (Dunleavy et al, 2009; Foltz et al, 2009; Fujita et al, 2007). HJURP and M18BP1 (also known as HsKNL2), a member of the Mis18 complex, are phosphoproteins (Bailey et al, 2016; Dephoure et al, 2008; Kato et al, 2007; McKinley and Cheeseman, 2014; Müller et al, 2014; Silva et al, 2012; Wang et al, 2014) and localize to centromeres in a cell cycle controlled manner, in early G1 phase (Dunleavy et al, 2009; Foltz et al, 2009; Fujita et al, 2007; Maddox et al, 2007), indicating they are putative targets for Cdk regulation. In addition, recent work has identified the mitotic kinase Plk1 as a critical component to drive CENP-A assembly (McKinley and Cheeseman, 2014).…”

“…However, while Plk1 is itself a cell cycle controlled kinase, it does not restrict CENP-A assembly to G1 phase as it is required for both canonical assembly in G1 phase as well as for premature assembly upon Cdk inhibition. In addition, several residues on CENP-A itself are phosphorylated (Bailey et al, 2016; Yu et al, 2015; Zeitlin et al, 2001). One of these, serine 68, is proposed to phosphorylated by mitotic Cdk activity (Yu et al, 2015) but the relevance of this is being disputed (Fachinetti et al, 2017) and mutation of this residue does not lead to a change in the timing of CENP-A deposition.…”

CENP-A Modifications on Ser68 and Lys124 Are Dispensable for Establishment, Maintenance, and Long-Term Function of Human Centromeres

“…Nascent CENP-A is expressed across the cell cycle but with elevated synthesis in late S-phase and G2 (Shelby et al, 1997), incorporated into a complex containing its partner histone H4 and a dedicated histone chaperone named HJURP (Dunleavy et al, 2009; Foltz et al, 2009), but only assembled into centromeric chromatin at mitotic exit (Jansen et al, 2007; Schuh et al, 2007) in a process that requires the centromere targeting of Mis18 and its partner molecules (comprising the ‘Mis18 complex’) (Barnhart et al, 2011; Fujita et al, 2007; Moree et al, 2011; Silva et al, 2012). The sub-cellular/sub-chromosomal targeting and cell cycle regulation of CENP-A and its chromatin assembly components may be controlled by post-translational modifications, and recent reports have indeed described candidate post-translational modifications of CENP-A (Bade et al, 2014; Bailey et al, 2013; Bui et al, 2012; Niikura et al, 2015; Yu et al, 2015; Zeitlin et al, 2001), HJURP (Bailey et al, 2016; Kato et al, 2007; Müller et al, 2014; Wang et al, 2014), and the Mis18 complex (McKinley and Cheeseman, 2014; Silva et al, 2012). …”