Chromosome Dynamics Laboratory

Outline

The long-term goal in our laboratory is to understand the molecular mechanisms of chromosome assembly and segregation during mitosis and meiosis. Central to this process are two multiprotein complexes, known as condensin and cohesin, which regulate chromosome condensation and cohesion, respectively. The two complexes are structurally related to each other and contain members of a large family of chromosomal ATPases, known as SMC (structural maintenance of chromosomes) proteins. Mutations in the subunits of condensin and cohesin cause various defects in chromosome segregation, leading to genome instability in many model organisms. Furthermore, emerging lines of evidence suggest that functional perturbation of condensin and cohesin is tightly associated with several developmental diseases in humans. Our laboratory takes multidisciplinary approaches to understanding how condensin, cohesin and SMC proteins might work at a mechanistic level both in vivo and in vitro.

Recent Research Topic

Chromosome shaping by condensins and cohesion

Fig. 1 Bivalent chromosomes in a mouse oocyte

Bivalent chromosomes were prepared from a mouse oocyte at metaphase I and immunofluorescently labeled with antibodies against condensin II (red) and centromeres (green). DNA was stained with DAPI (blue).

The shape of chromosomes is known to vary widely among different organisms and different developmental stages in a single organism. Most eukaryotic species possess two different condensin complexes, known as condensins I and II, that play central roles in chromosome assembly during mitosis. To understand how various shapes of chromosomes are determined by the concerted actions of condensins I and II (and cohesin), we have devised a series of sophisticated protocols in which their levels can be precisely manipulated in Xenopus egg extracts. When the relative ratio of condensin I to II is forced to be smaller, embryonic chromosomes become shorter and thicker, being reminiscent of somatic chromosomes. Cohesin helps juxtapose sister chromatid arms by collaborating with condensin I and counteracting condensin II. These results demonstrate that chromosome shaping is achieved by an exquisite balance among condensin I, condensin II and cohesin [Shintomi and Hirano, 2011].

Regulation of condensin II by MCPH1

Mutations in human MCPH1 (hMCPH1) cause primary microcephaly, characterized by marked reduction of brain size. Interestingly, hMCPH1-mutant patient cells display unique cellular phenotypes including premature chromosome condensation (PCC) in G2 phase. To test whether hMCPH1 directly regulates the action of condensins, we have developed a cell-free assay using Xenopus egg extracts. Our results demonstrate that an N-terminal domain of hMCPH1 specifically inhibits the action of condensin II (but not of condensin I) by competing for its chromosomal binding sites in vitro. Importantly, this activity is greatly reduced by mutations that cause primary microcephaly in vivo. A complementation assay using patient cells revealed that, whereas the N-terminal domain of hMCPH1 is sufficient to rescue the PCC phenotype, its central domain plays an auxiliary role in shaping metaphase chromosomes by physically interacting with condensin II. Thus, hMCPH1 acts as a composite modulator of condensin II to regulate chromosome condensation and shaping [Yamashita, et al. 2011].

Dynamics of condensins in mammalian meiosis

To understand how condensins I and II contribute to meiotic chromosome dynamics and segregation, we have investigated their spatiotemporal distributions in mouse oocytes. At the germinal vesicle stage (prophase I), condensin I is present in the cytoplasm while condensin II is localized within the nucleus. After germinal vesicle breakdown, condensin II starts to associate with chromosomes and becomes concentrated onto chromatid axes of bivalent chromosomes by metaphase I. REC8 “glues” chromosome arms along their lengths. In striking contrast to condensin II, condensin I localizes primarily around centromeric regions at metaphase I, and starts to associate stably with chromosome arms only after anaphase I. Antibody injection experiments show that condensin functions are required for many aspects of meiotic chromosome dynamics. We suggest that condensin II plays a primary role in resolving sister chromatid axes, whereas condensin I contributes to monopolar attachment of sister kinetochores, possibly by assembling a unique centromeric structure underneath [Lee, et al. 2011].

Fig. 2 Chromosome assembly and segregation

Replicated chromatids (sister chromatids) are held together by the action of cohesin. At the onset of mitosis, bulk cohesin dissociates from chromosome arms whereas condensin associates with them to induce condensation. These processes lead to the formation of metaphase chromosomes in which sister chromatids are microscopically distinguishable from each other (resolution). In late mitosis, residual cohesin is cleaved, thereby promoting irreversible separation of sister chromatids.

Fig. 3 Subunit composition of condensin and cohesin

Condensin is composed of a heterodimeric pair of SMC2-SMC4 and three non-SMC regulatory subunits. Cohesin is composed of a pair of SMC1-SMC3 and two regulatory subunits. The SMC dimers are unique V-shaped molecules, each containing two long coiled-coil arms.