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Analysis of Meiotic Sister Chromatid Cohesion in Caenorhabditis elegans

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Cohesin and Condensin

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1515))

Abstract

In sexually reproducing organisms, the formation of healthy gametes (sperm and eggs) requires the proper establishment and release of meiotic sister chromatid cohesion (SCC). SCC tethers replicated sisters from their formation in premeiotic S phase until the stepwise removal of cohesion in anaphase of meiosis I and II allows the separation of homologs and then sisters. Defects in the establishment or release of meiotic cohesion cause chromosome segregation errors that lead to the formation of aneuploid gametes and inviable embryos. The nematode Caenorhabditis elegans is an attractive model for studies of meiotic sister chromatid cohesion due to its genetic tractability and the excellent cytological properties of the hermaphrodite gonad. Moreover, mutants defective in the establishment or maintenance of meiotic SCC nevertheless produce abundant gametes, allowing analysis of the pattern of chromosome segregation. Here I describe two approaches for analysis of meiotic cohesion in C. elegans. The first approach relies on cytology to detect and quantify defects in SCC. The second approach relies on PCR and restriction digests to identify embryos that inherited an incorrect complement of chromosomes due to aberrant meiotic chromosome segregation. Both approaches are sensitive enough to identify rare errors and precise enough to reveal distinctive phenotypes resulting from mutations that perturb meiotic SCC in different ways. The robust, quantitative nature of these assays should strengthen phenotypic comparisons of different meiotic mutants and enhance the reproducibility of data generated by different investigators.

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References

  1. Nasmyth K, Haering CH (2009) Cohesin: its roles and mechanisms. Annu Rev Genet 43:525–558. doi:10.1146/annurev-genet-102108-134233

    Article  CAS  PubMed  Google Scholar 

  2. Klein F, Mahr P, Galova M et al (1999) A central role for cohesins in sister chromatid cohesion, formation of axial elements, and recombination during yeast meiosis. Cell 98:91–103

    Article  CAS  PubMed  Google Scholar 

  3. Buonomo SB, Clyne RK, Fuchs J et al (2000) Disjunction of homologous chromosomes in meiosis I depends on proteolytic cleavage of the meiotic cohesin Rec8 by separin. Cell 103:387–398

    Article  CAS  PubMed  Google Scholar 

  4. Kitajima TS, Miyazaki Y, Yamamoto M, Watanabe Y (2003) Rec8 cleavage by separase is required for meiotic nuclear divisions in fission yeast. EMBO J 22:5643–5653

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Guacci V, Koshland D, Strunnikov A (1997) A direct link between sister chromatid cohesion and chromosome condensation revealed through the analysis of MCD1 in S. cerevisiae. Cell 91:47–57

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Michaelis C, Ciosk R, Nasmyth K (1997) Cohesins: chromosomal proteins that prevent premature separation of sister chromatids. Cell 91:35–45

    Article  CAS  PubMed  Google Scholar 

  7. Birkenbihl RP, Subramani S (1992) Cloning and characterization of rad21 an essential gene of Schizosaccharomyces pombe involved in DNA double-strand-break repair. Nucleic Acids Res 20:6605–6611

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Uhlmann F, Wernic D, Poupart MA et al (2000) Cleavage of cohesin by the CD clan protease separin triggers anaphase in yeast. Cell 103:375–386

    Article  CAS  PubMed  Google Scholar 

  9. Severson AF, Ling L, van Zuylen V, Meyer BJ (2009) The axial element protein HTP-3 promotes cohesin loading and meiotic axis assembly in C. elegans to implement the meiotic program of chromosome segregation. Genes Dev 23:1763–1778. doi:10.1101/gad.1808809

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Severson AF, Meyer BJ (2014) Divergent kleisin subunits of cohesin specify mechanisms to tether and release meiotic chromosomes. eLife 3, e03467. doi:10.7554/eLife.03467

    Article  PubMed  PubMed Central  Google Scholar 

  11. Lee J, Hirano T (2011) RAD21L, a novel cohesin subunit implicated in linking homologous chromosomes in mammalian meiosis. J Cell Biol 192:263–276. doi:10.1083/jcb.201008005

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Ishiguro K, Kim J, Fujiyama-Nakamura S et al (2011) A new meiosis-specific cohesin complex implicated in the cohesin code for homologous pairing. EMBO Rep 12:267–275. doi:10.1038/embor.2011.2

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Herran Y, Gutierrez-Caballero C, Sanchez-Martin M et al (2011) The cohesin subunit RAD21L functions in meiotic synapsis and exhibits sexual dimorphism in fertility. EMBO J 30:3091–3105. doi:10.1038/emboj.2011.222

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Llano E, Herran Y, Garcia-Tunon I et al (2012) Meiotic cohesin complexes are essential for the formation of the axial element in mice. J Cell Biol 197:877–885. doi:10.1083/jcb.201201100

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Yuan L, Yang X, Ellis JL et al (2012) The Arabidopsis SYN3 cohesin protein is important for early meiotic events. Plant J 71:147–160. doi:10.1111/j.1365-313X.2012.04979.x

    Article  CAS  PubMed  Google Scholar 

  16. Ishiguro K-I, Kim J, Shibuya H et al (2014) Meiosis-specific cohesin mediates homolog recognition in mouse spermatocytes. Genes Dev 28:594–607. doi:10.1101/gad.237313.113

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Belmont AS (2001) Visualizing chromosome dynamics with GFP. Trends Cell Biol 11:250–257

    Article  CAS  PubMed  Google Scholar 

  18. Belmont AS, Straight AF (1998) In vivo visualization of chromosomes using lac operator-repressor binding. Trends Cell Biol 8:121–124

    Article  CAS  PubMed  Google Scholar 

  19. Robinett CC, Straight A, Li G et al (1996) In vivo localization of DNA sequences and visualization of large-scale chromatin organization using lac operator/repressor recognition. J Cell Biol 135:1685–1700

    Article  CAS  PubMed  Google Scholar 

  20. Straight AF, Belmont AS, Robinett CC, Murray AW (1996) GFP tagging of budding yeast chromosomes reveals that protein-protein interactions can mediate sister chromatid cohesion. Curr Biol 6:1599–1608

    Article  CAS  PubMed  Google Scholar 

  21. Askjaer P, Ercan S, Meister P (2014) Modern techniques for the analysis of chromatin and nuclear organization in C. elegans. WormBook 2:1–35. doi:10.1895/wormbook.1.169.1

    Article  Google Scholar 

  22. Yuen KWY, Nabeshima K, Oegema K, Desai A (2011) Rapid de novo centromere formation occurs independently of heterochromatin protein 1 in C. elegans embryos. Curr Biol 21:1800–1807. doi:10.1016/j.cub.2011.09.016

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Bilgir C, Dombecki CR, Chen PF et al (2013) Assembly of the synaptonemal complex is a highly temperature-sensitive process that is supported by PGL-1 during Caenorhabditis elegans meiosis. G3 (Bethesda) pii:g3.112.005165v1. doi:10.1534/g3.112.005165

    Google Scholar 

  24. Gonzalez-Serricchio A, Sternberg P (2006) Visualization of C. elegans transgenic arrays by GFP. BMC Genet 7:36–44

    Article  PubMed  PubMed Central  Google Scholar 

  25. Monen J, Maddox PS, Hyndman F et al (2005) Differential role of CENP-A in the segregation of holocentric C. elegans chromosomes during meiosis and mitosis. Nat Cell Biol 7:1148–1155. doi:10.1038/ncb1331

    Article  CAS  Google Scholar 

  26. Martinez-Perez E, Villeneuve AM (2005) HTP-1-dependent constraints coordinate homolog pairing and synapsis and promote chiasma formation during C. elegans meiosis. Genes Dev 19:2727–2743

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Reddy KC, Villeneuve AM (2004) C. elegans HIM-17 links chromatin modification and competence for initiation of meiotic recombination. Cell 118:439–452

    Article  CAS  PubMed  Google Scholar 

  28. Nabeshima K, Villeneuve AM, Hillers KJ (2004) Chromosome-wide regulation of meiotic crossover formation in Caenorhabditis elegans requires properly assembled chromosome axes. Genetics 168:1275–1292

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Schvarzstein M, Pattabiraman D, Libuda DE et al (2014) DNA helicase HIM-6/BLM both promotes MutSγ-dependent crossovers and antagonizes MutSγ-independent interhomolog associations during Caenorhabditis elegans meiosis. Genetics 198:193–207. doi:10.1534/genetics.114.161513

    Article  PubMed  PubMed Central  Google Scholar 

  30. Frøkjær-Jensen C, Davis MW, Sarov M et al (2014) Random and targeted transgene insertion in Caenorhabditis elegans using a modified Mos1 transposon. Nat Methods 11:529–534. doi:10.1038/nmeth.2889

    Article  PubMed  PubMed Central  Google Scholar 

  31. Checchi PM, Lawrence KS, Van MV et al (2014) Pseudosynapsis and decreased stringency of meiotic repair pathway choice on the hemizygous sex chromosome of Caenorhabditis elegans males. Genetics 197:543–560. doi:10.1534/genetics.114.164152

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Cortes DB, McNally KL, Mains PE, McNally FJ (2015) The asymmetry of female meiosis reduces the frequency of inheritance of unpaired chromosomes. eLife 4:e06056. doi:10.7554/eLife.06056

    Article  PubMed  PubMed Central  Google Scholar 

  33. Darby RAJ, Hine AV (2005) LacI-mediated sequence-specific affinity purification of plasmid DNA for therapeutic applications. FASEB J 19:801–803. doi:10.1096/fj.04-2812fje

    CAS  PubMed  Google Scholar 

  34. Sambrook J, Russel DW (2001) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY

    Google Scholar 

  35. Skop AR, White JG (1998) The dynactin complex is required for cleavage plane specification in early Caenorhabditis elegans embryos. Curr Biol 8:1110–1116

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Schindelin J, Arganda-Carreras I, Frise E et al (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9:676–682. doi:10.1038/nmeth.2019

    Article  CAS  PubMed  Google Scholar 

  37. Schneider CA, Rasband WS, Eliceiri KW (2012) NIH Image to ImageJ: 25 years of image analysis. Nat Methods 9:671–675

    Article  CAS  PubMed  Google Scholar 

  38. Davis MW, Hammarlund M, Harrach T et al (2005) Rapid single nucleotide polymorphism mapping in C. elegans. BMC Genomics 6:118. doi:10.1186/1471-2164-6-118

    Article  PubMed  PubMed Central  Google Scholar 

  39. Phillips CM, McDonald KL, Dernburg AF (2009) Cytological analysis of meiosis in Caenorhabditis elegans. Methods Mol Biol 558:171–195. doi:10.1007/978-1-60761-103-5_11

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Edgar LG (1995) Blastomere culture and analysis. Methods Cell Biol 48:303–321

    Article  CAS  PubMed  Google Scholar 

  41. Nabeshima K (2011) Chromosome structure and homologous chromosome association during meiotic prophase in Caenorhabditis elegans. Methods Mol Biol 745:549–562. doi:10.1007/978-1-61779-129-1_32

    Article  CAS  PubMed  Google Scholar 

  42. Greenwald I, Horvitz HR (1986) A visible allele of the muscle gene sup-10X of C. elegans. Genetics 113:63–72

    CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

I would like to acknowledge Barbara Meyer for her support and guidance during my postdoctoral research, when I developed many of the techniques described here. Strains used during the development of these methods were provided by the CGC, which is funded by the NIH Office of Research Infrastructure Programs (P40 OD010440), and the National Bioresource Project. This work was supported through NIH grant R15GM117548, startup funds provided by Cleveland State University and the Center for Gene Regulation in Health and Disease, and a Graduate Faculty Travel Award administered through the CSU Office of Research.

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Authors and Affiliations

  1. Department of Biological, Geological, and Environmental Sciences, Center for Gene Regulation in Health and Disease, Cleveland State University, 2121 Euclid Avenue SI 219, Cleveland, OH, 44115-2214, USA

    Aaron F. Severson

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  1. Aaron F. Severson
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Correspondence to Aaron F. Severson .

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Editors and Affiliations

  1. Department of Biological Chemistry, School of Medicine University of California— Irvine, Irvine, California, USA

    Kyoko Yokomori

  2. Laboratory of Genome Structure and Function Research Center for Epigenetic Disease, Institute of Molecular and Cellular Biosciences The University of Tokyo, Tokyo, Japan

    Katsuhiko Shirahige

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Severson, A.F. (2017). Analysis of Meiotic Sister Chromatid Cohesion in Caenorhabditis elegans . In: Yokomori, K., Shirahige, K. (eds) Cohesin and Condensin. Methods in Molecular Biology, vol 1515. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6545-8_5

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  • DOI: https://doi.org/10.1007/978-1-4939-6545-8_5

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6543-4

  • Online ISBN: 978-1-4939-6545-8

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