Decoding the centromeric nucleosome through CENP-N

Abstract

Centromere protein (CENP) A, a histone H3 variant, is a key epigenetic determinant of chromosome domains known as centromeres. Centromeres nucleate kinetochores, multi-subunit complexes that capture spindle microtubules to promote chromosome segregation during mitosis. Two kinetochore proteins, CENP-C and CENP-N, recognize CENP-A in the context of a rare CENP-A nucleosome. Here, we reveal the structural basis for the exquisite selectivity of CENP-N for centromeres. CENP-N uses charge and space complementarity to decode the L1 loop that is unique to CENP-A. It also engages in extensive interactions with a 15-base pair segment of the distorted nucleosomal DNA double helix, in a position predicted to exclude chromatin remodelling enzymes. Besides CENP-A, stable centromere recruitment of CENP-N requires a coincident interaction with a newly identified binding motif on nucleosome-bound CENP-C. Collectively, our studies clarify how CENP-N and CENP-C decode and stabilize the non-canonical CENP-A nucleosome to enforce epigenetic centromere specification and kinetochore assembly.

The COMA complex is required for positioning Ipl1 activity proximal to Cse4 nucleosomes in budding yeast

“…Interestingly, our EMSA and XLMS data suggest that in contrast to the human ortholog, CENP-N/L, the Chl4/Iml3 heterodimer does not directly interact with Cse4-NCP, but is linked to Ame1/Okp1 and Mif2 (Figure 1A, C). Recent electron microscopy reconstructions of human CENP-A nucleosomes in complex with CENP-N/L identified the RG loop in the CATD (Zhou et al, 2011) to be required for CENP-N interacting with CENP-A (Chittori et al, 2018, Pentakota et al, 2017, Tian et al, 2018. The lack of this RG loop in the budding yeast Cse4 CATD is consistent with our finding of Chl4/Iml3 being positioned distal from Cse4 nucleosomes ( Figure 1A, C).…”

“…In vertebrates two CCAN proteins, CENP-N and CENP-C, were shown to directly and selectively interact with CENP-A. CENP-C binds divergent hydrophobic residues of the CENP-A C-terminal tail, whereas CENP-N associates with the CENP-A CATD (Carroll et al, 2009, Carroll et al, 2010, Guse et al, 2011, Kato et al, 2013, Weir et al, 2016, Pentakota et al, 2017.…”

“…Across different species the CENP-C signature motif interacts with divergent hydrophobic residues of the CENP-A C-terminal tail (Musacchio and Desai, 2017). Electron microscopy studies have recently resolved the interaction of CENP-N with the CENP-A centromere-targeting domain (CATD) of CENP-A-NCP in vertebrates (Carroll et al, 2009, Guse et al, 2011, Pentakota et al, 2017, Chittori et al, 2018, Tian et al, 2018. For budding yeast Cse4 a direct interaction has so far only been demonstrated with Mif2 (Westermann et al, 2003, Xiao et al, 2017.…”

CENP-C unwraps the CENP-A nucleosome through the H2A C-terminal tail

“…Crystal structure of CENP-A nucleosome (Tachiwana et al, 2011), in vivo MNase experiments (Hasson et al, 2013) and initial cryoEM studies (Roulland et al, 2016) Next most intriguing question is how CENP-A nucleosome directly binds 2 CCAN components, CENP-N and CENP-C, and how is their binding changing the nucleosome. Recent work (Chittori et al, 2018;Pentakota et al, 2017;Tian et al, 2018) provides the atomic resolution view at CENP-A/CENP-N interaction, which involves recognition of the solvent-exposed L1 loop on CENP-A and interaction with DNA, but structure of CENP-A nucleosome in complex with CENP-C is still missing. Currently, 2 different parts of CENP-C are proposed to bind CENP-A nucleosome: CENP-C CR and CENP-C motif .…”

“…Initial clues came from the crystal structure of the CENP-A nucleosome (Tachiwana et al, 2011) that implied octameric histone core, similar to canonical nucleosome, with unwrapped DNA ends. Recently, a cryoEM structure of the human CENP-A nucleosome in complex with human CENP-N has been reported by several groups (Chittori et al, 2018;Pentakota et al, 2017;Tian et al, 2018) revealing high-resolution molecular determinants for the CENP-A/CENP-N interaction. However, high-resolution structure of human CENP-A nucleosome in complex with CENP-C is still missing.…”