The histone variant CENP-A is the epigenetic determinant for the centromere, where it is interspersed with canonical H3 to form a specialized chromatin structure that nucleates the kinetochore. How nucleosomes at the centromere arrange into higher order structures is unknown. Here we demonstrate that the human CENP-A-interacting protein CENP-N promotes the stacking of CENP-A-containing mononucleosomes and nucleosomal arrays through a previously undefined interaction between the α6 helix of CENP-N with the DNA of a neighboring nucleosome. We describe the cryo-EM structures and biophysical characterization of such CENP-N-mediated nucleosome stacks and nucleosomal arrays and demonstrate that this interaction is responsible for the formation of densely packed chromatin at the centromere in the cell. Our results provide first evidence that CENP-A, together with CENP-N, promotes specific chromatin higher order structure at the centromere.

组蛋白变体 CENP-A 是着丝粒的表观遗传决定因素，它与典型的 H3 散布在一起，形成一种特殊的染色质结构，使着丝粒成核。着丝粒的核小体如何排列成更高阶的结构尚不清楚。在这里，我们证明人类CENP-A相互作用蛋白CENP-N通过CENP-N的α6螺旋与邻近核小体的DNA之间先前未定义的相互作用促进含有CENP-A的单核小体和核小体阵列的堆叠。我们描述了这种 CENP-N 介导的核小体堆叠和核小体阵列的冷冻电镜结构和生物物理特征，并证明这种相互作用导致细胞着丝粒处密集染色质的形成。我们的结果提供了第一个证据，证明 CENP-A 与 CENP-N 一起促进着丝粒处特定的染色质高级结构。