Tatsuya Hirano

Condensins, Chromosome Condensation Protein Complexes Containing XCAP-C, XCAP-E and a Xenopus Homolog of the Drosophila Barren Protein

We report here purification and characterization of chromosome condensation protein complexes (termed condensins) containing XCAP-C and XCAP-E, two Xenopus members of the SMC family. Sucrose density gradient centrifugation reveals two major forms of condensins. The 8S form is a heterodimer of XCAP-C and XCAP-E, whereas the 13S form contains three additional subunits. One of them is identified as a homolog of the Drosophila Barren protein whose mutation shows a defect in chromosome segregation. Chromosomal targeting of condensins is mitosis-specific and is independent of topoisomerase IIalpha. 13S condensin is required for condensation, as demonstrated by immunodepletion and rescue experiments. Our results suggest that the condensin complexes represent the most abundant structural components of mitotic chromosomes and play a central role in driving chromosome condensation.

Differential Contributions of Condensin I and Condensin II to Mitotic Chromosome Architecture in Vertebrate Cells

The canonical condensin complex (henceforth condensin I) plays an essential role in mitotic chromosome assembly and segregation from yeast to humans. We report here the identification of a second condensin complex (condensin II) from vertebrate cells. Condensins I and II share the same pair of structural maintenance of chromosomes (SMC) subunits but contain different sets of non-SMC subunits. siRNA-mediated depletion of condensin I- or condensin II-specific subunits in HeLa cells produces a distinct, highly characteristic defect in chromosome morphology. Simultaneous depletion of both complexes causes the severest defect. In Xenopus egg extracts, condensin I function is predominant, but lack of condensin II results in the formation of irregularly shaped chromosomes. Condensins I and II show different distributions along the axis of chromosomes assembled in vivo and in vitro. We propose that the two condensin complexes make distinct mechanistic contributions to mitotic chromosome architecture in vertebrate cells.

ATP-Dependent Positive Supercoiling of DNA by 13S Condensin: A Biochemical Implication for Chromosome Condensation

13S condensin is a five-subunit protein complex that plays a central role in mitotic chromosome condensation in Xenopus egg extracts. Two core subunits of this complex, XCAP-C and XCAP-E, belong to an emerging family of putative ATPases, the SMC family. We report here that 13S condensin has a DNA-stimulated ATPase activity and exhibits a high affinity for structured DNAs such as cruciform DNA. 13S condensin is able to introduce positive supercoils into a closed circular DNA in the presence of bacterial or eukaryotic topoisomerase I. The supercoiling reaction is ATP-dependent. We propose that 13S condensin wraps DNA in a right-handed direction by utilizing the energy of ATP hydrolysis. This reaction may represent a key mechanism underlying the compaction of chromatin fibers during mitosis.

Condensin and cohesin display different arm conformations with characteristic hinge angles

Structural maintenance of chromosomes (SMC) proteins play central roles in higher-order chromosome dynamics from bacteria to humans. In eukaryotes, two different SMC protein complexes, condensin and cohesin, regulate chromosome condensation and sister chromatid cohesion, respectively. Each of the complexes consists of a heterodimeric pair of SMC subunits and two or three non-SMC subunits. Previous studies have shown that a bacterial SMC homodimer has a symmetrical structure in which two long coiled-coil arms are connected by a flexible hinge. A catalytic domain with DNA- and ATP-binding activities is located at the distal end of each arm. We report here the visualization of vertebrate condensin and cohesin by electron microscopy. Both complexes display the two-armed structure characteristic of SMC proteins, but their conformations are remarkably different. The hinge of condensin is closed and the coiled-coil arms are placed close together. In contrast, the hinge of cohesin is wide open and the coiled-coils are spread apart from each other. The non-SMC subunits of both condensin and cohesin form a globular complex bound to the catalytic domains of the SMC heterodimers. We propose that the “closed” conformation of condensin and the “open” conformation of cohesin are important structural properties that contribute to their specialized biochemical and physiological functions.

Human Wapl Is a Cohesin-Binding Protein that Promotes Sister-Chromatid Resolution in Mitotic Prophase

BACKGROUNDnThe linkage between duplicated chromosomes (sister chromatids) is established during S phase by the action of cohesin, a multisubunit complex conserved from yeast to humans. Most cohesin dissociates from chromosome arms when the cell enters mitotic prophase, leading to the formation of metaphase chromosomes with two cytologically discernible chromatids. This process is known as sister-chromatid resolution. Although two mitotic kinases have been implicated in this process, it remains unknown exactly how the cohesin-mediated linkage is destabilized at a mechanistic level.nnnRESULTSnThe wings apart-like (Wapl) protein was originally identified as a gene product that potentially regulates heterochromatin organization in Drosophila melanogaster. We show that the human ortholog of Wapl is a cohesin-binding protein that facilitates cohesins timely release from chromosome arms during prophase. Depletion of Wapl from HeLa cells causes transient accumulation of prometaphase-like cells with chromosomes that display poorly resolved sister chromatids with a high level of cohesin. Reduction of cohesin relieves the Wapl-depletion phenotype, and depletion of Wapl rescues premature sister separation observed in Sgo1-depleted or Esco2-depleted cells. Conversely, overexpression of Wapl causes premature separation of sister chromatids. Wapl physically associates with cohesin in HeLa-cell nuclear extracts. Remarkably, in vitro reconstitution experiments demonstrate that Wapl forms a stoichiometric, ternary complex with two regulatory subunits of cohesin, implicating its noncatalytic function in inactivating cohesins ability to interact with chromatin.nnnCONCLUSIONSnWapl is a new regulator of sister chromatid resolution and promotes release of cohesin from chromosomes by directly interacting with its regulatory subunits.

13S Condensin Actively Reconfigures DNA by Introducing Global Positive Writhe: Implications for Chromosome Condensation

Xenopus 13S condensin converts interphase chromatin into mitotic-like chromosomes, and, in the presence of ATP and a type I topoisomerase, introduces (+) supercoils into DNA. The specific production of (+) trefoil knots in the presence of condensin and a type II topoisomerase shows that condensin reconfigures DNA by introducing an ordered, global, (+) writhe. Knotting required ATP hydrolysis and cell cycle-specific phosphorylation of condensin. Condensin bound preferentially to (+) supercoiled DNA in the presence of ATP but not in its absence. Our results suggest a mechanism for the compaction of chromatin by condensin during mitosis.

The making of the mitotic chromosome: modern insights into classical questions.

The condensation of mitotic chromosomes is essential for the faithful segregation of sister chromatids in anaphase. An emerging view is that chromosome assembly is an active and dynamic process of chromatin reorganization in which two ATP hydrolyzing enzymes, topoisomerase II and the condensin complex, play central roles. In this review, we discuss recent work that sheds new light on the molecular and structural dynamics of mitotic chromosomes.

Condensins: Organizing and Segregating the Genome

Condensins are multi-subunit protein complexes that play a central role in mitotic chromosome assembly and segregation. The complexes contain structural maintenance of chromosomes (SMC) ATPase subunits, and induce DNA supercoiling and looping in an ATP-hydrolysis-dependent manner in vitro. Vertebrate cells have two different condensin complexes, condensins I and II, each containing a unique set of regulatory subunits. Condensin II participates in an early stage of chromosome condensation within the prophase nucleus. Condensin I gains access to chromosomes only after the nuclear envelope breaks down, and collaborates with condensin II to assemble metaphase chromosomes with fully resolved sister chromatids. The complexes also play critical roles in meiotic chromosome segregation and in interphase processes such as gene repression and checkpoint responses. In bacterial cells, ancestral forms of condensins control chromosome dynamics. Dissecting the diverse functions of condensins is likely to be central to our understanding of genome organization, stability and evolution.

Visualization of early chromosome condensation: a hierarchical folding, axial glue model of chromosome structure

Current models of mitotic chromosome structure are based largely on the examination of maximally condensed metaphase chromosomes. Here, we test these models by correlating the distribution of two scaffold components with the appearance of prophase chromosome folding intermediates. We confirm an axial distribution of topoisomerase IIα and the condensin subunit, structural maintenance of chromosomes 2 (SMC2), in unextracted metaphase chromosomes, with SMC2 localizing to a 150–200-nm-diameter central core. In contrast to predictions of radial loop/scaffold models, this axial distribution does not appear until late prophase, after formation of uniformly condensed middle prophase chromosomes. Instead, SMC2 associates throughout early and middle prophase chromatids, frequently forming foci over the chromosome exterior. Early prophase condensation occurs through folding of large-scale chromatin fibers into condensed masses. These resolve into linear, 200–300-nm-diameter middle prophase chromatids that double in diameter by late prophase. We propose a unified model of chromosome structure in which hierarchical levels of chromatin folding are stabilized late in mitosis by an axial “glue.”

Scc2 Couples Replication Licensing to Sister Chromatid Cohesion in Xenopus Egg Extracts

The cohesin complex is a central player in sister chromatid cohesion, a process that ensures the faithful segregation of chromosomes in mitosis and meiosis. Previous genetic studies in yeast show that Scc2/Mis4, a HEAT-repeat-containing protein, is required for the loading of cohesin onto chromatin. In this study, we have identified two isoforms of Scc2 in humans and Xenopus (termed Scc2A and Scc2B), which are encoded by a single gene but have different carboxyl termini created by alternative splicing. Both Scc2A and Scc2B bind to chromatin concomitant with cohesin during DNA replication in Xenopus egg extracts. Simultaneous immunodepletion of Scc2A and Scc2B from the extracts impairs the association of cohesin with chromatin, leading to severe defects in sister chromatid pairing in the subsequent mitosis. The loading of Scc2 onto chromatin is inhibited in extracts treated with geminin but not with p21(CIP1), suggesting that this step depends on replication licensing but not on the initiation of DNA replication. Upon mitotic entry, Scc2 is removed from chromatin through a mechanism that requires cdc2 but not aurora B or polo-like kinase. Our results suggest that vertebrate Scc2 couples replication licensing to sister chromatid cohesion by facilitating the loading of cohesin onto chromatin.