Project description:Nucleosomes with histone H3 replaced by CENP-A direct kinetochore assembly. CENP-A nucleosomes from human and Drosophila have been reported to have reduced heights as compared to canonical octameric H3 nucleosomes, thus suggesting a unique tetrameric hemisomal composition. We demonstrate that octameric CENP-A nucleosomes assembled in vitro exhibit reduced heights, indicating that they are physically distinct from H3 nucleosomes and negating the need to invoke the presence of hemisomes.

| S-EPMC3676451 | biostudies-literature

Reduction in Step Height Variation and Correcting Contrast Inversion in Dynamic AFM of WS2 Monolayers.

Project description:A model has been developed to account for and prevent the anomalies encountered in topographic images of transition metal dichalcogenide monolayers using dynamic atomic force microscopy (dAFM). The height of WS2 monolayers measured using dAFM appeared to be increased or decreased, resulting from the interactions between the tip and the surface. The hydrophilic SiO2 substrate appeared higher than the weakly hydrophilic WS2 when the tip amplitude was low or at a high set point (high force). Large amplitudes and low set points corrected the step height inversion, but did not recover the true step height. Removing water from the sample resulted in an order of magnitude reduced variation in step height, but the WS2 appeared inverted except at low amplitudes and high set points. Our model explains the varying step heights in dAFM of TMDs as a result of varying tip-sample interactions between the sample and substrate, in the presence or absence of capillaries. To eliminate contrast inversion, high amplitudes can be used to reduce the effect of capillary forces. However, when capillaries are not present, low amplitudes and high set points produce images with proper contrast due to tool operation in the repulsive regime on both materials.

| S-EPMC5736643 | biostudies-literature

Decoding the centromeric nucleosome through CENP-N.

Project description: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.

| S-EPMC5777823 | biostudies-literature

Cryo-EM structure of the CENP-A nucleosome in complex with phosphorylated CENP-C.

Project description:The CENP-A nucleosome is a key structure for kinetochore assembly. Once the CENP-A nucleosome is established in the centromere, additional proteins recognize the CENP-A nucleosome to form a kinetochore. CENP-C and CENP-N are CENP-A binding proteins. We previously demonstrated that vertebrate CENP-C binding to the CENP-A nucleosome is regulated by CDK1-mediated CENP-C phosphorylation. However, it is still unknown how the phosphorylation of CENP-C regulates its binding to CENP-A. It is also not completely understood how and whether CENP-C and CENP-N act together on the CENP-A nucleosome. Here, using cryo-electron microscopy (cryo-EM) in combination with biochemical approaches, we reveal a stable CENP-A nucleosome-binding mode of CENP-C through unique regions. The chicken CENP-C structure bound to the CENP-A nucleosome is stabilized by an intramolecular link through the phosphorylated CENP-C residue. The stable CENP-A-CENP-C complex excludes CENP-N from the CENP-A nucleosome. These findings provide mechanistic insights into the dynamic kinetochore assembly regulated by CDK1-mediated CENP-C phosphorylation.

| S-EPMC7917552 | biostudies-literature

Molecular basis for CENP-N recognition of CENP-A nucleosome on the human kinetochore.

Project description: Not available

| S-EPMC5835772 | biostudies-literature

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

Project description:Centromeres are defined epigenetically by nucleosomes containing the histone H3 variant CENP-A, upon which the constitutive centromere-associated network of proteins (CCAN) is built. CENP-C is considered to be a central organizer of the CCAN. We provide new molecular insights into the structure of human CENP-A nucleosomes, in isolation and in complex with the CENP-C central region (CENP-CCR ), the main CENP-A binding module of human CENP-C. We establish that the short αN helix of CENP-A promotes DNA flexibility at the nucleosome ends, independently of the sequence it wraps. Furthermore, we show that, in vitro, two regions of human CENP-C (CENP-CCR and CENP-Cmotif ) both bind exclusively to the CENP-A nucleosome. We find CENP-CCR to bind with high affinity due to an extended hydrophobic area made up of CENP-AV 532 and CENP-AV 533 . Importantly, we identify two key conformational changes within the CENP-A nucleosome upon CENP-C binding. First, the loose DNA wrapping of CENP-A nucleosomes is further exacerbated, through destabilization of the H2A C-terminal tail. Second, CENP-CCR rigidifies the N-terminal tail of H4 in the conformation favoring H4K20 monomethylation, essential for a functional centromere.

| S-EPMC6776904 | biostudies-literature

Molecular basis of CENP-C association with the CENP-A nucleosome at yeast centromeres.

Project description:Histone CENP-A-containing nucleosomes play an important role in nucleating kinetochores at centromeres for chromosome segregation. However, the molecular mechanisms by which CENP-A nucleosomes engage with kinetochore proteins are not well understood. Here, we report the finding of a new function for the budding yeast Cse4/CENP-A histone-fold domain interacting with inner kinetochore protein Mif2/CENP-C. Strikingly, we also discovered that AT-rich centromere DNA has an important role for Mif2 recruitment. Mif2 contacts one side of the nucleosome dyad, engaging with both Cse4 residues and AT-rich nucleosomal DNA. Both interactions are directed by a contiguous DNA- and histone-binding domain (DHBD) harboring the conserved CENP-C motif, an AT hook, and RK clusters (clusters enriched for arginine-lysine residues). Human CENP-C has two related DHBDs that bind preferentially to DNA sequences of higher AT content. Our findings suggest that a DNA composition-based mechanism together with residues characteristic for the CENP-A histone variant contribute to the specification of centromere identity.

| S-EPMC5710141 | biostudies-literature

The cryo-EM structure of the CENP-A nucleosome in complex with the phosphorylated CENP-C:: CENP-A nucleosome in complex with phosphorylated CENP-C C-terminal domain(601-864)

Project description: Not available

| EMPIAR-10569 | biostudies-other

The cryo-EM structure of the CENP-A nucleosome in complex with the phosphorylated CENP-C:: CENP-A nucleosome in complex with CENP-C motif (655-675) and CENP-N N-terminal domain (1-211)

Project description: Not available

| EMPIAR-10571 | biostudies-other