Linker Histone Medicated Regulation of Mitotic Chromosome Compaction and Individualization

Mitotic chromosomes are scaled to the cell size to ensure effective chromosome segregation. Recent studies have shown how condensins and DNA topoisomerase II organize the mitotic chromosome. However, the regulation of these factors in maintaining proper chromosome size in different cell types remains a mystery. Here, I investigated the role of the linker histone variant H1.8 in regulating mitotic chromosome structure. I showed that H1.8 suppresses binding of condensins and topo II to mitotic chromatin in Xenopus egg extracts. Using an in vitro reconstitution system, I showed that H1.8 inhibits binding of purified condensins and topo II to nucleosome arrays. I also showed that condensin binding to nucleosome arrays is sensitive to magnesium dependent chromatin compaction. By using direct measurement of chromosome length, I then showed that H1.8 suppresses chromosome length solely through condensin I enrichment on chromatin. I then investigated the organization of Xenopus egg extract chromosomes using chromosome conformation capture technique Hi-C. Using Hi-C analysis, I showed that condensin I organizes both mitotic loops and loop layers of mitotic chromosomes and that H1.8 mediated suppression of condensin I increases both mitotic loop and layer sizes. This analysis also corroborates direct measurements of chromosome length. I also showed that nucleosome depletion results in further reduction in loop and layer sizes over H1.8 depletion. This suggests that chromosome length can be regulated by condensin I binding through competitive inhibition by both nucleosomes and linker histones. Mitotic chromosomes are organized in a rod to increase both physical rigidity of chromosomes and to ensure effective resolution. Using Hi-C data, I observed that both condensins play a role in maintaining chromosome rigidity and subsequently in maintaining chromosome individualization. I then showed that, like sister chromatid resolution, condensin activity drives topo II activity to continuously resolve interchromosomal links in mitosis. Since H1.8 suppresses both condensin and topo II, it suppresses chromosome individualization. I then go on to show that this suppression of chromosome individualization is necessary to maintain spindle integrity. Based on these data, I propose a model where mitotic chromosome length and individualization can be regulated by using linker histone stoichiometry on chromatin as a rheostat. As linker histones are a dynamic component of chromatin that have been shown to have extensive cell cycle dependent phosphorylation, I discuss the possibility that titrating linker histone stoichiometry on chromatin may be used as a mechanism to control the binding of DNA binding proteins in both interphase and mitosis and thus regulate cellular functions

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

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