Fig. 2. Structural basis for CENP-N-dependent nucleosome-nucleosome interaction.
a, The CENP-N α6 helix interacts with nucleosomal DNA of a second nucleosome without contacting histones (nonspecific nucleosome). Top, overview of interactions. Lower left, the interface between CENP-N α6 and DNA is highlighted. Lower middle, a surface charge representation (unit, kT e−1) in the same orientation. Lower right, the specific interaction on the other side of CENP-N with the CENP-A RG loop. b, Single mutations on CENP-N α6 affect nucleosome-nucleosome interactions, as shown by AUC. c, FRET analysis of the interaction between CENP-A mononucleosome (CA MN) and H3 mononucleosome (H3 MN) in the absence or presence of CENP-N. The donor is CENP-A mononucleosomes containing Alexa 488-labeled H2B; the acceptor is a H3 mononucleosome containing Atto N 647-labeled H2B (250 nM donor and acceptor nucleosome were used); FRET intensity changes in dependence of [CENP-N]. Error bars are from five independent measurements of two biological replicates. Data are presented as mean values ± s.d. d, The H4 N-terminal tail is essential for CENP-N-mediated nucleosome stacking (van Holde–Weischet plots of sedimentation). ∆19 indicates H4 tail deletion (amino acids 1–18). e, Comparison of different modes of nucleosome stacking. ‘Nuc1’ represents the reference nucleosome that interacts specifically with the indicated factor. ‘Nuc2’ is the neighboring nucleosome which interacts with Nuc1 or its binding factors non-specifically. Top, models for stacked mononucleosomes. 1AOI is the PDB code for a previously published nucleosome structure. Bottom, ‘superhelix locations’ (SHLs) (1-6) and the nucleosomal dyad axis (SHL 0; ɸ) of nuc2 (brown color, DNA only), are indicated, with nuc1 shown in a dotted circle (gray color), depicts the relative orientations of nuc1 and nuc2 and CENP-N or cGAS, respectively. Only half of the nucleosomal DNA is shown for clarity.