Crystal structure of the human centromeric nucleosome containing CENP-A

Abstract

In eukaryotes, accurate chromosome segregation during mitosis and meiosis is coordinated by kinetochores, which are unique chromosomal sites for microtubule attachment. Centromeres specify the kinetochore formation sites on individual chromosomes, and are epigenetically marked by the assembly of nucleosomes containing the centromere-specific histone H3 variant, CENP-A. Although the underlying mechanism is unclear, centromere inheritance is probably dictated by the architecture of the centromeric nucleosome. Here we report the crystal structure of the human centromeric nucleosome containing CENP-A and its cognate α-satellite DNA derivative (147 base pairs). In the human CENP-A nucleosome, the DNA is wrapped around the histone octamer, consisting of two each of histones H2A, H2B, H4 and CENP-A, in a left-handed orientation. However, unlike the canonical H3 nucleosome, only the central 121 base pairs of the DNA are visible. The thirteen base pairs from both ends of the DNA are invisible in the crystal structure, and the αN helix of CENP-A is shorter than that of H3, which is known to be important for the orientation of the DNA ends in the canonical H3 nucleosome. A structural comparison of the CENP-A and H3 nucleosomes revealed that CENP-A contains two extra amino acid residues (Arg 80 and Gly 81) in the loop 1 region, which is completely exposed to the solvent. Mutations of the CENP-A loop 1 residues reduced CENP-A retention at the centromeres in human cells. Therefore, the CENP-A loop 1 may function in stabilizing the centromeric chromatin containing CENP-A, possibly by providing a binding site for trans-acting factors. The structure provides the first atomic-resolution picture of the centromere-specific nucleosome.

The Centromere: Epigenetic Control of Chromosome Segregation during Mitosis

“…The notion that CENP-A can function as a hemisome will not be corroborated until a hemisome is successfully isolated and characterized biochemically. In contrast to a CENP-A hemisome, the crystal structure of an octameric CENP-A nucleosome has been resolved, revealing only subtle differences to canonical octameric H3 nucleosomes (Tachiwana et al 2011). Octameric CENP-A nucleosomes wrap slightly less DNA than H3 nucleosomes ( 120 bp vs. 147 bp), resulting in different DNA entry/exit angles from the nucleosome, and several exposed residues exist in CENP-A's extended loop 1 region that are important for CENP-A nucleosome stability (Tachiwana et al 2011).…”

“…In contrast to a CENP-A hemisome, the crystal structure of an octameric CENP-A nucleosome has been resolved, revealing only subtle differences to canonical octameric H3 nucleosomes (Tachiwana et al 2011). Octameric CENP-A nucleosomes wrap slightly less DNA than H3 nucleosomes ( 120 bp vs. 147 bp), resulting in different DNA entry/exit angles from the nucleosome, and several exposed residues exist in CENP-A's extended loop 1 region that are important for CENP-A nucleosome stability (Tachiwana et al 2011). In support of the octameric CENP-A nucleosome model, ChIP-Seq data revealed that CENP-A mononucleosomes wrap more DNA in cells than hemisomes could wrap (Hasson et al 2013), immunoprecipitation of Drosophila CENP-C CID mononucleosomes revealed the presence of CENP-A CID dimers (Zhang et al 2012), and CAL1-assembled CENP-A CID nucleosomes in Drosophila are octameric .…”

“…The amino-terminal 40 amino acids of CENP-A, the most divergent domain from histone H3, undergoes specific posttranslational modifications that are thought to influence CENP-A nucleosome structure and function, although their significance remains largely unclear (Zeitlin et al 2001;Kunitoku et al 2003;Bailey et al 2013;Boeckmann et al 2013). The most amino-terminal a helix in CENP-A (residues 48 -56) is three residues shorter than that observed in histone H3 (residues 45 -56), yet this small difference results in a loss of a DNA interaction that causes different entry/exit angles of DNA from the CENP-A nucleosome, protects less DNA in classic nucleosome micrococcal nuclease digestion assays, and most likely has a key influence of overall centromeric chromatin structure (Conde e Silva et al 2007;Panchenko et al 2011;Tachiwana et al 2011). In the histone fold domain of CENP-A, a two-residue insertion (R80 and G81) in a loop domain is exposed in the CENP-A nucleosome and influences CENP-A nucleosome stability (Sekulic et al 2010;Tachiwana et al 2011).…”

“…The most amino-terminal a helix in CENP-A (residues 48 -56) is three residues shorter than that observed in histone H3 (residues 45 -56), yet this small difference results in a loss of a DNA interaction that causes different entry/exit angles of DNA from the CENP-A nucleosome, protects less DNA in classic nucleosome micrococcal nuclease digestion assays, and most likely has a key influence of overall centromeric chromatin structure (Conde e Silva et al 2007;Panchenko et al 2011;Tachiwana et al 2011). In the histone fold domain of CENP-A, a two-residue insertion (R80 and G81) in a loop domain is exposed in the CENP-A nucleosome and influences CENP-A nucleosome stability (Sekulic et al 2010;Tachiwana et al 2011). Importantly, a central region (residues 75 -114 in humans) constitutes the CENP-A targeting domain (CATD) (Black et al 2004) that is essential for numerous aspects of CENP-A function and structure, including binding of the CENP-A chaperone Holliday junction recognition protein (HJURP) and the centromere protein CENP-N (discussed below).…”

Principles and practice of nucleosome positioningin vitro

“…A large majority of nucleosome crystal structures are based on a palindromic 73 bp region from the human alpha satellite repeat (Harp et al 1996). However, the functionally relevant nucleosome positioning is unclear and complicated by active debate over the repeating chromatin unit size, the wrapping topology, and whether relevant centromeric chromatin is wrapped as either H3 and CENP-A containing nucleosomes or both (Tachiwana et al 2011), or possibly even other histone fold structures of centromeric proteins (Nishino et al 2012).…”