Chromosome Cohesion - Dr Nobuaki Kudo
In recent years it has become more commonplace for women to consider pregnancy at around the age of 35 or older, even though it is at this time that the risk of pregnancy loss, as well as innate diseases such as Down syndrome, sharply increases. Aneuploidy (i.e. an abnormal number of chromosomes in the cells) is one of the leading causes of such incidents, and is also associated with cancer in somatic cells. Most early embryonic aneuploidy is generated by the fertilization of aneuploid eggs or sperm that have originated from chromosome mis-segregation during a specialized type of cell division, called meiosis. We wish to understand, at the molecular level, what causes aneuploidy and why it increases in a maternal age-dependent manner, thereby allowing us to devise methods or treatments to avoid it.
Figure 1. A full set of chromosomes (blue) from a mouse oocyte, at metaphase I stage, spread on a glass slide and stained with antibodies detecting cohesin Rec8 (magenta) and kinetochores (green). Nineteen homologous paternal and maternal chromosome pairs are connected with crossovers and cohesin distal to crossover sites, however one pair has been precociously segregated (circles). This situation is thought to be the most common causes for embryonic aneuploidy leading to infertility, spontaneous abortion and Down syndrome in humans.
Following a mitotic cell division, the chromosome number is the same in both the mother and daughter cells. In contrast, meiotic cell division, which takes place in the germ line, produces cells with half the number of chromosomes (see Fig. 2). This is the result of two sequential rounds of chromosome separation without an intervening round of DNA duplication. As in mitotic cells, meiotic cells also use the tension from microtubules from opposite poles of the cell to align and segregate chromosomes. However, the requirement of homologous chromosome segregation (meiosis I) in addition to sister chromatid segregation (meiosis II) adds a few functional modifications: firstly, pairs of homologous chromosomes must be linked to establish tension between them. Secondly, unlike in mitosis, sister chromatids must go to the same spindle pole at the first meiotic division. Thirdly, sister chromatids have to remain connected to facilitate their bi-orientation in meiosis II. These changes are achieved by three meiosis I-specific mechanisms: (i) synapsis and crossing-over between homologous chromosomes, (ii) mono-polar attachment of sister kinetochores to spindle microtubules, (iii) chromosome arm-specific loss of sister chromatid cohesion. Malfunction in any of the above mechanisms can cause chromosome mis-segregation and thus aneuploidy, and therefore it is important to understand their precise molecular mechanisms.
Figure 2. Chromosome segregation patterns in meiosis and mitosis. See main text for details. Enlarge Figure 2
Cohesion between sister chromatids is generated during DNA replication and maintained until their segregation into two daughter cells. It is mediated by an evolutionally-conserved hetero-tetrameric protein complex, called cohesin; cohesion is essential for faithful chromosome segregation in mitosis as well as meiosis. In meiosis, DNA replication is followed by recombination of sister chromatid strands between homologous paternal and maternal chromosomes, which creates crossovers. Crossovers are physical linkages between homologous chromosomes, and are in fact supported by sister chromatid cohesion distal to crossover sites. Loss of cohesion between chromosome arms allows segregation of homologous chromosomes during meiosis I while loss of cohesion between sister centromeres allows segregation of sister chromatids in meiosis II. Therefore, accurate regulation of the cohesin complex is one of the most important aspects of faithful chromosome segregation.
In addition to cohesin's canonical role in sister chromatid cohesion, recent studies provide evidence that cohesin regulates gene expression. Human developmental diseases such as Cornelia de Lange and Roberts syndromes were shown to be caused by mutations in genes encoding cohesin or its regulators. Latest investigations in mammalian mitotic cells show that many cohesin binding sites on chromosome arms are co-occupied with an insulator element binding protein called CTCF and they co-operate to regulate expression of a subset of genes. One of the genes regulated by these is the human H19/Igf2 locus, whose expression is controlled by parental origin-dependent DNA methylation of the H19 imprinting control region (ICR). Therefore, a certain fraction of cohesin, together with CTCF, is thought to be required for maintaining expression and repression of imprinted genes by defining chromosomal boundaries, possibly by affecting higher order chromatin structure, in somatic cells.
Studies in various model organisms have revealed molecular mechanisms of cohesin regulation and chromosome segregation; however, much less is understood in mammalian germ cells. Studying mammalian meiotic chromosome segregation with a special focus on cohesin is particularly interesting and clinically important, since cohesion must be maintained for up to 40 years in human oocytes - in cellular terms, an exceedingly long period of time. The significance of cohesin regulation for mammalian fertility has been illustrated by recent studies involving Smc1β and Separase knockout mice. Furthermore, the new discovery of a potential role for cohesin in gene regulation has prompted the hypothesis that deterioration of cohesion in germ cells might cause an aberrant gene expression pattern and thus physiological aging of eggs. Therefore, we are actively studying the mechanisms of chromosome cohesion and segregation in mammalian germ cell development.
Figure 3. Chromosomal development during meiotic prophase (leptotene, zygotene, pachytene and diplotene) and 2 rounds of meiotic cell division in mouse spermatogenesis. Interkinesis is the secondary spermatocyte in interphase between the first and second meiotic division. Chromosome spreads were prepared from the testis of a transgenic mouse expressing Rec8-myc and stained with antibodies against the myc epitope (green) and a synaptonemal complex protein Sycp3 (magenta). Transgene-derived Rec8-myc is perfectly functional and is a useful tool to study cohesin function during meiotic chromosomal development. Enlarge Figure 3
Where does cohesin bind on meiotic chromosomes?
Recently numerous research groups mapped multiple cohesin binding sites on chromosome arms in mammalian mitotic cells and found that cohesin preferentially binds to DNase I hypersensitive sites with co-localization of the insulator protein CTCF. In mitotic division, sister chromatid cohesion at chromosome arms is essentially dispensable for bi-orientation of sister kinetochores and accurate sister chromatid segregation. In fact, most cohesin complexes are released from arms before all chromatid pairs are aligned on the cell equator plane. In striking contrast, chromatid arm cohesion is essential for bi-orientating homologous chromosomes during meiosis I. Therefore it is very interesting to explore the distribution of cohesin during meiosis and to determine whether or not mitotic and meiotic cohesins share the same sites. We are performing chromatin immunoprecipitation (ChIP) assays for the meiotic cohesin subunit Rec8 in spermatocytes. Our long-term aims include genome-wide mapping of meiotic cohesin sites, comparison between young and aged oocytes and the relationship of cohesin positions and reprogramming of the sex-specific genome imprinting pattern that is also a unique feature of germ cell development.
How are sister kinetochores mono-orientated during meiosis I?
One of the modifications that distinguishes meiosis I chromosome segregation from that in meiosis II and mitosis is kinetochore mono-orientation. Molecular mechanisms accomplishing this have been studied in other eukaryotic model organisms; however, it is assumed that they are not applicable to mammalian germ cells. One reason for this is that the centromeric DNA structure, on which the kinetochore is assembled, exhibits a large degree of diversity between species; another is that the molecules which have been found to play a role in mono-orientation, show very low conservation. Therefore the mechanism that determines kinetochore orientation in mammalian meiosis I remains totally unclear. We are taking a candidate approach, as well as interaction-based screening, to identify the relevant molecules. We are also testing whether the bi-orientated sister kinetochore configuration in mitotic cells can be converted to a mono-orientated arrangement.
How is cohesin regulated during meiotic prophase?
Though cohesin is required for crossover formation in all model organisms so far tested, its precise role in the process is not understood. Even when crossovers are successfully created, sister chromatid cohesion distal to the crossover sites remains essential for the maintenance of such crossovers. This is particularly important in human oogenesis, because female germ cells undergo homologous recombination during fetal development before birth and homologous chromosome segregation after puberty, indicating that crossovers must be maintained for approximately 10-40 years. Interestingly, cohesin is not replenished after DNA replication in an unchallenged yeast cell cycle. Is this also true for mammalian oogenesis? In addition, how oocytes maintain the cell cycle arrest for such a long time is also an interesting question. We are studying genetically modified mice to address these questions.
Human meiotic problems and germ line stem cells
In close collaboration with Dr. Carol Readhead and Dr. Sheba Jarvis in the IRDB and the IVF unit at Hammersmith Hospital, we are studying meiotic chromosomal abnormalities in non-obstructive azoospermic patients. We are also characterizing mammalian spermatogonial stem cells with the aim of identifying future application in regenerative medicine.
Petronczki M, Siomos MF, Nasmyth K. (2003) Un ménage à quatre: the molecular biology of chromosome segregation in meiosis. Cell. 112: 423-40.
Hauf S, Watanabe Y. (2004) Kinetochore orientation in mitosis and meiosis. Cell. 119: 317-27.
McNairn AJ, Gerton JL. (2008) The chromosome glue gets a little stickier. Trends Genet. 24: 382-9.
Hassold T, Hunt P. (2001) To err (meiotically) is human: the genesis of human aneuploidy. Nat Rev Genet. 2: 280-91.
Kudo NR, Anger M, Peters AH, Stemmann O, Theussl HC, Helmhart W, Kudo, H, Heyting C, Nasmyth K.
(2009) Role of cleavage by separase of the Rec8 kleisin subunit of cohesion during mammalian meiosis I. J Cell Sci. 122: 2686-98.
McGuinness BE, Anger M, Kouznetosva A, Gil-Bernabe AM, Helmhart W, Kudo NR, Wuensche A, Taylor S, Hoog C, Novak B, Nasmyth K. (2009) Regulation of APC/C activity in oocytes by a Bub1-dependant spindle assembly checkpoint. Curr Biol. 19: 369-80.
Kudo NR, Wassmann K, Anger M, Schuh M, Wirth KG, Xu H, Helmhart W, Kudo H, McKay M, Maro B, Ellenberg J, de Boer P, Nasmyth K. (2006) Resolution of chiasmata in oocytes requires separase-mediated proteolysis. Cell. 126: 135-146. doi
Wirth KG, Wutz G, Kudo NR, Desdouets C, Zetterberg A, Taghybeeglu S, Seznec J, Ducos GM, Ricci R, Firnberg N, Peters JM, Nasmyth K. (2006) Separase: a universal trigger for sister chromatid disjunction but not chromosome cycle progression. J Cell Biol. 172: 847-860.
McGuinness BE, Hirota T, Kudo NR, Peters JM, Nasmyth K. (2005) Shugoshin prevents dissociation of cohesin from centromeres during mitosis in vertebrate cells. PLoS Biol. 3: 433-449. doi
Wirth KG, Ricci R, Giménez-Abián JF, Taghybeeglu S, Kudo NR, Jochum W, Vasseur-Cognet M, Nasmyth K. (2004) Loss of the anaphase-promoting complex in quiescent cells causes unscheduled hepatocyte proliferation. Genes Dev. 18: 88-98. doi
The Royal Society
Medical Research Council (MRC) - New Investigator Award
Institiute of Obstetrics & Gynaecology (IOGT)
Nobuaki Kudo, PhD (Head of Group, Non-clinical Lecturer)
Xiangwei Fu, PhD (Postdoc)
Ben Rogers, PhD (Postdoc)
Kasia Kuleszewicz, MSc (PhD student)
James Crichton, BSc (MSc student)
Joao Pedro Sousa Martins, MSc
Giulia Grimaldi, BSc
Emily Waiyaiya, BSc