Disturbance in function and expression of condensin affects chromosome compaction in HeLa cells

Condensin, a major non-histone protein complex on chromosomes, is responsible for the formation of rod-shaped chromosome in mitosis. A heterodimer composed of SMC2 (structural maintenance of chromosomes) and SMC4 subunits constitutes the core part of condensin. Although extensive studies have been done in yeast, fruit fly and Xenopus to uncover the mechanisms and molecular nature of SMC proteins, little is known about the complex in mammalian cells. We have conducted a series of experiments to unveil the nature of condensin complex in human chromosome formation. The results show that overexpression of the C-terminal domain of SMC subunits disturbs chromosome condensation, leading to formation of swollen chromosomes, while knockdown of SMC subunits severely disturbs mitotic chromosome formation, resulting in chromatin bridges between daughter cells and multiple nuclei in single cells. The salt extraction assay indicates that a fraction of the condensin complex is bound to chromatin in interphase, but most of the condensin bind to chromatin at the onset of mitosis. Thus, disturbance in condensin function or expression affects chromosome condensation and influences mitotic progression.     

Condensin and Repo-Man-PP1 co-operate in the regulation of chromosome architecture during mitosis

The reversible condensation of chromosomes during cell division remains a classic problem in cell biology. Condensation requires the condensin complex in certain experimental systems, but not in many others. Anaphase chromosome segregation almost always fails in condensin-depleted cells, leading to the formation of prominent chromatin bridges and cytokinesis failure. Here, live-cell analysis of chicken DT40 cells bearing a conditional knockout of condensin subunit SMC2 revealed that condensin-depleted chromosomes abruptly lose their compact architecture during anaphase and form massive chromatin bridges. The compact chromosome structure can be preserved and anaphase chromosome segregation rescued by preventing the targeting subunit Repo-Man from recruiting protein phosphatase 1 (PP1) to chromatin at anaphase onset. This study identifies an activity critical for mitotic chromosome structure that is inactivated by Repo-Man-PP1 during anaphase. This activity, provisionally termed 'regulator of chromosome architecture' (RCA), cooperates with condensin to preserve the characteristic chromosome architecture during mitosis.     

Functional characterization of individual genomic condensin-binding sites in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> using minichromosome mitotic segregation stability assay

Proper chromatin compaction in mitosis (condensation) is required for equal chromosome distribution and the precise inheritance of genetic information. A protein complex called condensin is responsible for mitotic chromosome condensation, chromosome individualization, the timely separation of sister chromatids in mitosis, and proper tension in the mitotic spindle. The mitotic function of condensin depends on the recognition of specific binding sites in the chromosome. The mechanism for binding condensin to individual sites of mitotic chromosomes, as well as the molecular anatomy of these sites, remains to be elucidated. Even less is known about the process that translates condensin binding to individual sites into the segregation of chromosomes in anaphase. In the present work, by using minichromosome assay, we analyze seven individual condensin-binding sites in S. cerevisiae identified in the whole-genome ChIP-on-chip screening. This approach allowed us to estimate the individual contribution of condensin-binding sites to the segregation fidelity of minichromosomes.     

Contribution of hCAP-D2, a non-SMC subunit of condensin I, to chromosome and chromosomal protein dynamics during mitosis

Condensins are heteropentameric complexes that were first identified as structural components of mitotic chromosomes. They are composed of two SMC (structural maintenance of chromosomes) and three non-SMC subunits. Condensins play a role in the resolution and segregation of sister chromatids during mitosis, as well as in some aspects of mitotic chromosome assembly. Two distinct condensin complexes, condensin I and condensin II, which differ only in their non-SMC subunits, exist. Here, we used an RNA interference approach to deplete hCAP-D2, a non-SMC subunit of condensin I, in HeLa cells. We found that the association of hCAP-H, another non-SMC subunit of condensin I, with mitotic chromosomes depends on the presence of hCAP-D2. Moreover, chromatid axes, as defined by topoisomerase II and hCAP-E localization, are disorganized in the absence of hCAP-D2, and the resolution and segregation of sister chromatids are impaired. In addition, hCAP-D2 depletion affects chromosome alignment in metaphase and delays entry into anaphase. This suggests that condensin I is involved in the correct attachment between chromosome kinetochores and microtubules of the mitotic spindle. These results are discussed relative to the effects of depleting both condensin complexes.     

The condensin I subunit Barren/CAP-H is essential for the structural integrity of centromeric heterochromatin during mitosis

During cell division, chromatin undergoes structural changes essential to ensure faithful segregation of the genome. Condensins, abundant components of mitotic chromosomes, are known to form two different complexes, condensins I and II. To further examine the role of condensin I in chromosome structure and in particular in centromere organization, we depleted from S2 cells the Drosophila CAP-H homologue Barren, a subunit exclusively associated with condensin I. In the absence of Barren/CAP-H the condensin core subunits DmSMC4/2 still associate with chromatin, while the other condensin I non-structural maintenance of chromosomes family proteins do not. Immunofluorescence and in vivo analysis of Barren/CAP-H-depleted cells showed that mitotic chromosomes are able to condense but fail to resolve sister chromatids. Additionally, Barren/CAP-H-depleted cells show chromosome congression defects that do not appear to be due to abnormal kinetochore-microtubule interaction. Instead, the centromeric and pericentromeric heterochromatin of Barren/CAP-H-depleted chromosomes shows structural problems. After bipolar attachment, the centromeric heterochromatin organized in the absence of Barren/CAP-H cannot withstand the forces exerted by the mitotic spindle and undergoes irreversible distortion. Taken together, our data suggest that the condensin I complex is required not only to promote sister chromatid resolution but also to maintain the structural integrity of centromeric heterochromatin during mitosis.     

Mutations in the Drosophila condensin subunit dCAP-G: defining the role of condensin for chromosome condensation in mitosis and gene expression in interphase

Chromosomes are dynamic structures that are reorganized during the cell cycle to optimize them for distinct functions. SMC and non-SMC condensin proteins associate into complexes that have been implicated in the process of chromosome condensation. The roles of the individual non-SMC subunits of the complex are poorly understood, and mutations in the CAP-G subunit have not been described in metazoans. Here we elucidate a role for dCAP-G in chromosome condensation and cohesion in Drosophila. We illustrate the requirement of dCAP-G for condensation during prophase and prometaphase; however, we find that alternate mechanisms ensure that replicated chromosomes are condensed prior to metaphase. In contrast, dCAP-G is essential for chromosome condensation in metaphase of single, unreplicated sister chromatids, suggesting that there is an interplay between replicated chromatids and the condensin complex. In the dcap-g mutants, defects in sister-chromatid separation are also observed. Chromatid arms fail to resolve in prophase and are unable to separate at anaphase, whereas sister centromeres show aberrant separation in metaphase and successfully move to spindle poles at anaphase. We also identified a role for dCAP-G during interphase in regulating heterochromatic gene expression.     

Cell cycle-dependent phosphorylation, nuclear localization, and activation of human condensin

Condensin, one of the most abundant components of mitotic chromosomes, is a conserved protein complex composed of two structural maintenance of chromosomes (SMC) subunits (SMC2- and SMC4-type) and three non-SMC subunits, and it plays an essential role in mitotic chromosome condensation. Purified condensin reconfigures DNA structure using energy provided by ATP hydrolysis. To know the regulation of condensin in somatic cells, the expression level, subcellular localization, and phosphorylation status of human condensin were examined during the cell cycle. The levels of condensin subunits were almost constant throughout the cell cycle, and the three non-SMC subunits were phosphorylated at specific sites in mitosis and dephosphorylated upon the completion of mitosis. Subcellular fractionation studies revealed that a proportion of condensin was tightly bound to mitotic chromosomes and that this form was phosphorylated at specific sites. Condensin purified from mitotic cells had much stronger supercoiling activity than that purified from interphase cells. These results suggest that condensin functions in somatic cells are regulated by phosphorylation in two ways during the cell cycle; the phosphorylation of specific sites correlates with the chromosomal targeting of condensin, and its biochemical activity is stimulated by phosphorylation.     

Functional characteristics of the individual genomic condensin binding sites of Saccharomyces cerevisiae using minichromosome mitotic segregation stability model

Proper chromatin compaction in mitosis (condensation) is required for equal chromosome distribution and precise genetic information inheritance. Protein complex named condensin is responsible for the mitotic condensation, it also individualizes chromosomes, and ensures chromatin separation between sister chromatids in mitosis as well as proper mitotic spindle tension. Mitotic condensin function depends on recognition of the specific binding sites on the chromosome. Mechanism of condensin binding on the individual sites of the mitotic chromosomes, as well as molecular anatomy of these sites remains to be unclear. Even less known is how condensin binding on the individual sites helps separating chromosomes in anaphase. In current paper using minichromosome test, we analyze seven individual condensin binding sites in Saccharomyces cerevisiae found in previous all-genome CHIP on CHIP screening in our lab. This approach allowed us to find out what was the individual contribution of condensin binding sites in securing mitotic stability of the minichromosomes.     

The condensin complex is required for proper spindle assembly and chromosome segregation in Xenopus egg extracts

Chromosome condensation is required for the physical resolution and segregation of sister chromatids during cell division, but the precise role of higher order chromatin structure in mitotic chromosome functions is unclear. Here, we address the role of the major condensation machinery, the condensin complex, in spindle assembly and function in Xenopus laevis egg extracts. Immunodepletion of condensin inhibited microtubule growth and organization around chromosomes, reducing the percentage of sperm nuclei capable of forming spindles, and causing dramatic defects in anaphase chromosome segregation. Although the motor CENP-E was recruited to kinetochores pulled poleward during anaphase, the disorganized chromosome mass was not resolved. Inhibition of condensin function during anaphase also inhibited chromosome segregation, indicating its continuous requirement. Spindle assembly around DNA-coated beads in the absence of kinetochores was also impaired upon condensin inhibition. These results support an important role for condensin in establishing chromosomal architecture necessary for proper spindle assembly and chromosome segregation.     

Chromosome condensation factor Brn1p is required for chromatid separation in mitosis

This work describes BRN1, the budding yeast homologue of Drosophila Barren and Xenopus condensin subunit XCAP-H. The Drosophila protein is required for proper chromosome segregation in mitosis, and Xenopus protein functions in mitotic chromosome condensation. Mutant brn1 cells show a defect in mitotic chromosome condensation and sister chromatid separation and segregation in anaphase. Chromatid cohesion before anaphase is properly maintained in the mutants. Some brn1 mutant cells apparently arrest in S-phase, pointing to a possible function for Brn1p at this stage of the cell cycle. Brn1p is a nuclear protein with a nonuniform distribution pattern, and its level is up-regulated at mitosis. Temperature-sensitive mutations of BRN1 can be suppressed by overexpression of a novel gene YCG1, which is homologous to another Xenopus condensin subunit, XCAP-G. Overexpression of SMC2, a gene necessary for chromosome condensation, and a homologue of the XCAP-E condensin, does not suppress brn1, pointing to functional specialization of components of the condensin complex.     

A human condensin complex containing hCAP-C-hCAP-E and CNAP1, a homolog of Xenopus XCAP-D2, colocalizes with phosphorylated histone H3 during the early stage of mitotic chromosome condensation

Structural maintenance of chromosomes (SMC) family proteins play critical roles in structural changes of chromosomes. Previously, we identified two human SMC family proteins, hCAP-C and hCAP-E, which form a heterodimeric complex (hCAP-C-hCAP-E) in the cell. Based on the sequence conservation and mitotic chromosome localization, hCAP-C-hCAP-E was determined to be the human ortholog of the Xenopus SMC complex, XCAP-C-XCAP-E. XCAP-C-XCAP-E is a component of the multiprotein complex termed condensin, required for mitotic chromosome condensation in vitro. However, presence of such a complex has not been demonstrated in mammalian cells. Coimmunoprecipitation of the endogenous hCAP-C-hCAP-E complex from HeLa extracts identified a 155-kDa protein interacting with hCAP-C-hCAP-E, termed condensation-related SMC-associated protein 1 (CNAP1). CNAP1 associates with mitotic chromosomes and is homologous to Xenopus condensin component XCAP-D2, indicating the presence of a condensin complex in human cells. Chromosome association of human condensin is mitosis specific, and the majority of condensin dissociates from chromosomes and is sequestered in the cytoplasm throughout interphase. However, a subpopulation of the complex was found to remain on chromosomes as foci in the interphase nucleus. During late G(2)/early prophase, the larger nuclear condensin foci colocalize with phosphorylated histone H3 clusters on partially condensed regions of chromosomes. These results suggest that mitosis-specific function of human condensin may be regulated by cell cycle-specific subcellular localization of the complex, and the nuclear condensin that associates with interphase chromosomes is involved in the reinitiation of mitotic chromosome condensation in conjunction with phosphorylation of histone H3.     

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.     

Expression and functional dynamics of the XCAP-D2 condensin subunit in Xenopus laevis oocytes

The 13 S condensin complex plays a crucial role in the condensation and segregation of the two sets of chromosomes during mitosis in vivo as well as in cell-free extracts. This complex, conserved from yeast to human, contains a heterodimer of structural maintenance of chromosome (SMC) family proteins and three additional non-SMC subunits. We have investigated the expression of the non-SMC condensin component XCAP-D2 in Xenopus laevis oocytes. When studied during meiotic maturation, XCAP-D2 starts to accumulate at the time of germinal vesicle breakdown and reaches its maximal amount in metaphase II oocytes. This accumulation is specifically blocked by injection of antisense oligonucleotides. XCAP-D2 antisense-injected oocytes progress normally through meiosis until metaphase II. At this stage, however, chromosomes exhibit architecture defaults, and resolution of sister chromatids is impaired. Surprisingly, in mitotic extracts made from XCAP-D2 knocked-down oocytes, sperm chromatin normally condenses into compacted chromosomes, whereas the amounts of both free and chromosome-bound XCAP-D2 are markedly reduced. This apparent discrepancy is discussed in light of current knowledge on chromosome dynamics.     

The condensin complexes play distinct roles to ensure normal chromosome morphogenesis during meiotic division in Arabidopsis

Meiosis is a specialized cell division essential for sexual reproduction. During meiosis the chromosomes are highly organized, and correct chromosome architecture is required for faithful segregation of chromosomes at anaphase I and II. Condensin is involved in chromosome organization during meiotic and mitotic cell divisions. Three condensin subunits, AtSMC4 and the condensin I and II specific subunits AtCAP‐D2 and AtCAP‐D3, respectively, have been studied for their role in meiosis. This has revealed that both the condensin I and condensin II complexes are required to maintain normal structural integrity of the meiotic chromosomes during the two nuclear divisions. Their roles appear functionally distinct in that condensin I is required to maintain normal compaction of the centromeric repeats and 45S rDNA, whereas loss of condensin II was associated with extensive interchromosome connections at metaphase I. Depletion of condensin is also associated with a slight reduction in crossover formation, suggesting a role during meiotic prophase I.     

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.     

Diverse mitotic and interphase functions of condensins in Drosophila

The condensin complex has been implicated in the higher-order organization of mitotic chromosomes in a host of model eukaryotes from yeasts to flies and vertebrates. Although chromosomes paradoxically appear to condense in condensin mutants, chromatids are not properly resolved, resulting in chromosome segregation defects during anaphase. We have examined the role of different condensin complex components in interphase chromatin function by examining the effects of various condensin mutations on position-effect variegation in Drosophila melanogaster. Surprisingly, most mutations affecting condensin proteins were often found to result in strong enhancement of variegation in contrast to what might be expected for proteins believed to compact the genome. This suggests either that the role of condensin proteins in interphase differs from their expected role in mitosis or that the way we envision condensin's activity needs to be modified to accommodate alternative possibilities.     

Building and breaking bridges between sister chromatids

Eukaryotic chromosomes undergo dramatic changes and movements during mitosis. These include the individualization and compaction of the two copies of replicated chromosomes (the sister chromatids) and their subsequent segregation to the daughter cells. Two multisubunit protein complexes termed 'cohesin' and 'condensin', both composed of SMC (Structural Maintenance of Chromosomes) and kleisin subunits, have emerged as crucial players in these processes. Cohesin is required for holding sister chromatids together whereas condensin, together with topoisomerase II, has an important role in organizing individual axes of sister chromatids prior to their segregation during anaphase. SMC and kleisin complexes also regulate the compaction and segregation of bacterial nucleoids. New research suggests that these ancient regulators of chromosome structure might function as topological devices that trap chromosomal DNA between 50 nm long coiled coils.     

Chromosome morphogenesis: condensin-dependent cohesin removal during meiosis

During meiosis, segregation of homologous chromosomes necessitates the coordination of sister chromatid cohesion, chromosome condensation, and recombination. Cohesion and condensation require the SMC complexes, cohesin and condensin, respectively. Here we use budding yeast Saccharomyces cerevisiae to show that condensin and Cdc5, a Polo-like kinase, facilitate the removal of cohesin from chromosomes prior to the onset of anaphase I when homologs segregate. This cohesin removal is critical for homolog segregation because it helps dissolve the recombination-dependent links between homologs that form during prophase I. Condensin enhances the association of Cdc5 with chromosomes and its phosphorylation of cohesin, which in turn likely stimulates cohesin removal. Condensin/Cdc5-dependent removal of cohesin underscores the potential importance of crosstalk between chromosome structural components in chromosome morphogenesis and provides a mechanism to couple chromosome morphogenesis with other meiotic events.     

Spatial and temporal regulation of Condensins I and II in mitotic chromosome assembly in human cells

Two different condensin complexes make distinct contributions to metaphase chromosome architecture in vertebrate cells. We show here that the spatial and temporal distributions of condensins I and II are differentially regulated during the cell cycle in HeLa cells. Condensin II is predominantly nuclear during interphase and contributes to early stages of chromosome assembly in prophase. In contrast, condensin I is sequestered in the cytoplasm from interphase through prophase and gains access to chromosomes only after the nuclear envelope breaks down in prometaphase. The two complexes alternate along the axis of metaphase chromatids, but they are arranged into a unique geometry at the centromere/kinetochore region, with condensin II enriched near the inner kinetochore plate. This region-specific distribution of condensins I and II is severely disrupted upon depletion of Aurora B, although their association with the chromosome arm is not. Depletion of condensin subunits causes defects in kinetochore structure and function, leading to aberrant chromosome alignment and segregation. Our results suggest that the two condensin complexes act sequentially to initiate the assembly of mitotic chromosomes and that their specialized distribution at the centromere/kinetochore region may play a crucial role in placing sister kinetochores into the back-to-back orientation.