Visualization of the three-dimensional structure of the human centromere in mitotic chromosomes by super-resolution microscopy

The human centromere comprises large arrays of repetitive alpha-satellite DNA at the primary constriction of mitotic chromosomes. In addition, centromeres are epigenetically specified by the centromere-specific histone H3 variant CENP-A that supports kinetochore assembly to enable chromosome segregation. Since CENP-A is bound to only a fraction of the alpha-satellite elements within the megabase-sized centromere DNA, correlating the three-dimensional (3D) organization of alpha-satellite DNA and CENP-A remains elusive. To visualize centromere organization within a single chromatid, we used a combination of the Centromere Chromosome Orientation Fluorescent In Situ Hybridization (Cen-CO-FISH) technique together with Structured Illumination Microscopy (SIM). Cen-CO-FISH allows the differential labeling of the sister chromatids without the denaturation step used in conventional FISH that may affect DNA structure. Our data indicate that alpha-satellite DNA is arranged in a ring-like organization within prometaphase chromosomes, in presence or absence of spindle's microtubules. Using expansion microscopy (ExM), we found that CENP-A organization within mitotic chromosomes follows a rounded pattern similar to that of alpha-satellite DNA, often visible as a ring thicker at the outer surface oriented towards the kinetochore-microtubules interface. Collectively, our data provide a 3D reconstruction of alpha-satellite DNA along with CENP-A clusters that outline the overall architecture of the mitotic centromere. [Media: see text] [Media: see text] [Media: see text] [Media: see text

The Chromosomal Passenger Complex Is Required for Chromatin-Induced Microtubule Stabilization and Spindle Assembly

AbstractIn cells lacking centrosomes, such as those found in female meiosis, chromosomes must nucleate and stabilize microtubules in order to form a bipolar spindle. Here we report the identification of Dasra A and Dasra B, two new components of the vertebrate chromosomal passenger complex containing Incenp, Survivin, and the kinase Aurora B, and demonstrate that this complex is required for chromatin-induced microtubule stabilization and spindle formation. The failure of microtubule stabilization caused by depletion of the chromosomal passenger complex was rescued by codepletion of the microtubule-depolymerizing kinesin MCAK, whose activity is negatively regulated by Aurora B. By contrast, we present evidence that the Ran-GTP pathway of chromatin-induced microtubule nucleation does not require the chromosomal passenger complex, indicating that the mechanisms of microtubule assembly by these two pathways are distinct. We propose that the chromosomal passenger complex regulates local MCAK activity to permit spindle formation via stabilization of chromatin-associated microtubules

Linker histone H1.8 inhibits chromatin-binding of condensins and DNA topoisomerase II to tune chromosome compaction and individualization [preprint]

DNA loop extrusion by condensins and decatenation by DNA topoisomerase II (topo II) drive mitotic chromosome compaction and individualization. Here, we reveal that the linker histone H1.8 regulates chromatin levels of condensins and topo II. In vitro chromatin reconstitution experiments demonstrate that H1.8 inhibits binding of condensins and topo II to nucleosome arrays. Accordingly, H1.8 depletion in Xenopus egg extracts increased condensins and topo II levels on mitotic chromatin. Chromosome morphology and Hi-C analyses suggest that H1.8 depletion makes chromosomes thinner and longer likely through shortening the average loop size and reducing DNA amount in each layer of mitotic loops. Furthermore, H1.8-mediated suppression of condensins and topo II binding to chromatin limits chromosome individualization by preventing resolution of interchromosomal linkages. While linker histones locally compact DNA by clustering nucleosomes, we propose that H1.8 controls chromosome morphology and topological organization through restricting the loading of condensins and topo II on chromatin

Linker histone H1.8 inhibits chromatin binding of condensins and DNA topoisomerase II to tune chromosome length and individualization

DNA loop extrusion by condensins and decatenation by DNA topoisomerase II (topo II) are thought to drive mitotic chromosome compaction and individualization. Here, we reveal that the linker histone H1.8 antagonizes condensins and topo II to shape mitotic chromosome organization. In vitro chromatin reconstitution experiments demonstrate that H1.8 inhibits binding of condensins and topo II to nucleosome arrays. Accordingly, H1.8 depletion in Xenopus egg extracts increased condensins and topo II levels on mitotic chromatin. Chromosome morphology and Hi-C analyses suggest that H1.8 depletion makes chromosomes thinner and longer through shortening the average loop size and reducing the DNA amount in each layer of mitotic loops. Furthermore, excess loading of condensins and topo II to chromosomes by H1.8 depletion causes hyper-chromosome individualization and dispersion. We propose that condensins and topo II are essential for chromosome individualization, but their functions are tuned by the linker histone to keep chromosomes together until anaphase

Ku80 removal from DNA through double strand break–induced ubiquitylation

The Ku70/Ku80 heterodimer, or Ku, is the central component of the nonhomologous end joining (NHEJ) pathway of double strand break (DSB) repair. Because Ku forms a ring through which the DSB threads, it likely becomes topologically attached to DNA during repair. The mechanism for its removal was unknown. Using a method to identify proteins recruited to DSBs in Xenopus laevis egg extract, we show that DSB-containing DNAs accumulate members of the Skp1–Cul1–F-box complex and K48-linked polyubiquitylated proteins in addition to known repair proteins. We demonstrate that Ku80 is degraded in response to DSBs in a ubiquitin-mediated manner. Strikingly, K48-linked polyubiquitylation, but not proteasomal degradation, is required for the efficient removal of Ku80 from DNA. This removal is DNA length dependent, as Ku80 is retained on duplex oligonucleotides. Finally, NHEJ completion and removal of Ku80 from DNA are independent from one another. We propose that DSB-induced ubiquitylation of Ku80 provides a mechanism to efficiently eliminate Ku from DNA for pre- and postrepair processes

Two Birds with One Stone— Dealing with Nuclear Transport and Spindle Assembly

Spindle assembly and nuclear transport both utilize the same simple device: Ran-GTP-sensitive interaction of importin β and its cargo proteins. In this issue of Cell, Blower et al. (2005) report that one of these cargos required for spindle assembly turns out to be Rae1, previously known as an mRNA export protein. This study reveals the importance of RNAs in spindle structure

Cell cycle-dependent specific positioning and clustering of centromeres and telomeres in fission yeast

Abstract. Fluorescence in situ hybridization (FISH) shows that fission yeast centromeres and telomeres make up specific spatial arrangements in the nucleus. Their positioning and clustering are cell cycle regulated. In G2, centromeres cluster adjacent to the spindle pole body (SPB), while in mitosis, their association with each other and with the SPB is disrupted. Similarly, telomeres cluster at the nuclear periphery in G2 and their associations are disrupted in mitosis. Mitotic centromeres interact with the spindle. They remain undivided until the spindle reaches a critical length, then separate and move towards the poles. This demonstrated, for the first time, that anaphase A occurs in fission yeast. The mode of anaphase A and B is similar to that of higher eukaryotes. In nda3 an