Centromeres are maintained by fastening CENP-A to DNA and directing an arginine anchor-dependent nucleosome transition

Lucie Y Guo,Kara L Mckinley,Nikolina Sekulic,Daniele Fachinetti,Ben E Black,Glennis A Logsdon,Praveen Kumar Allu,Levani Zandarashvili,Jennine M Dawicki-Mckenna,Ryan M Jamiolkowski,Don W Cleveland,Iain M Cheeseman

doi:10.1038/ncomms15775

Abstract

Maintaining centromere identity relies upon the persistence of the epigenetic mark provided by the histone H3 variant, centromere protein A (CENP-A), but the molecular mechanisms that underlie its remarkable stability remain unclear. Here, we define the contributions of each of the three candidate CENP-A nucleosome-binding domains (two on CENP-C and one on CENP-N) to CENP-A stability using gene replacement and rapid protein degradation. Surprisingly, the most conserved domain, the CENP-C motif, is dispensable. Instead, the stability is conferred by the unfolded central domain of CENP-C and the folded N-terminal domain of CENP-N that becomes rigidified 1,000-fold upon crossbridging CENP-A and its adjacent nucleosomal DNA. Disrupting the ‘arginine anchor’ on CENP-C for the nucleosomal acidic patch disrupts the CENP-A nucleosome structural transition and removes CENP-A nucleosomes from centromeres. CENP-A nucleosome retention at centromeres requires a core centromeric nucleosome complex where CENP-C clamps down a stable nucleosome conformation and CENP-N fastens CENP-A to the DNA.

Highlights

Maintaining centromere identity relies upon the persistence of the epigenetic mark provided by the histone H3 variant, centromere protein A (CENP-A), but the molecular mechanisms that underlie its remarkable stability remain unclear
Since all constructs are expressed at roughly equal levels as the auxin-inducible degron (AID)-EYFP-tagged version before indole-3-acetic acid (IAA) treatment, there is an expected drop in the amount that is detectable at centromeres after IAA treatment even with the wild-type full-length version [CENP-C(FL); Fig. 1d,e and Supplementary Fig. 1e,f]
Our physical studies of CENP-A nucleosome complexes combined with gene replacement and rapid depletion of the non-histone centromereassociated network (CCAN) proteins, CENP-C and CENP-N, provide the molecular basis for the extraordinary stability of CENP-A nucleosomes that is at the heart of the epigenetic mechanism that maintains the identity of centromere location on every chromosome

Summary

Introduction

Maintaining centromere identity relies upon the persistence of the epigenetic mark provided by the histone H3 variant, centromere protein A (CENP-A), but the molecular mechanisms that underlie its remarkable stability remain unclear. A model for the epigenetic specification of centromere identity has emerged wherein pre-existing nucleosomes with a histone H3 variant named centromere protein A (CENP-A)[3,4] direct the local assembly of newly synthesized CENP-A5,6, with CENP-A deposition occurring once per cell cycle following completion of mitosis[7,8]. This model relies on the stable maintenance of CENP-A nucleosomes at a single site on each chromosome throughout the remainder of the cell cycle. An arginine anchor is the shared feature of a diverse set of nucleosome-binding proteins studied to date[21,22,23,24,25], establishing an emerging paradigm for nucleosome recognition[26]

Methods

Results

Conclusion