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CENP-N promotes the compaction of centromeric chromatin.

ABSTRACT: 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.

SUBMITTER: Zhou K

PROVIDER: S-EPMC9010303 | biostudies-literature |

REPOSITORIES: biostudies-literature

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CENP-C facilitates the recruitment of M18BP1 to centromeric chromatin.

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Assembly of CENP-A into centromeric chromatin requires a cooperative array of nucleosomal DNA contact sites.

Project description:We investigated the requirements for targeting the centromeric histone H3 homologue CENP-A for assembly at centromeres in human cells by transfection of epitope-tagged CENP-A derivatives into HeLa cells. Centromeric targeting is driven solely by the conserved histone fold domain of CENP-A. Using the crystal structure of histone H3 as a guide, a series of CENP-A/histone H3 chimeras was constructed to test the role of discrete structural elements of the histone fold domain. Three elements were identified that are necessary for efficient targeting to centromeres. Two correspond to contact sites between histone H3 and nucleosomal DNA. The third maps to a homotypic H3-H3 interaction site important for assembly of the (H3/H4)2 heterotetramer. Immunoprecipitation confirms that CENP-A self-associates in vivo. In addition, targeting requires that CENP-A expression is uncoupled from histone H3 synthesis during S phase. CENP-A mRNA accumulates later in the cell cycle than histone H3, peaking in G2. Isolation of the gene for human CENP-A revealed a regulatory motif in the promoter region that directs the late S/G2 expression of other cell cycle-dependent transcripts such as cdc2, cdc25C, and cyclin A. Our data suggest a mechanism for molecular recognition of centromeric DNA at the nucleosomal level mediated by a cooperative series of differentiated CENP-A-DNA contact sites arrayed across the surface of a CENP-A nucleosome and a distinctive assembly pathway occurring late in the cell cycle.

| S-EPMC2134286 | biostudies-literature

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