Cristina Martín-Castellanos (Research Scientist, CSIC)
Luisa F. Bustamante Jaramillo (PhD Student)
María Álvarez Sánchez (Undergraduate Student)
Track record of the group
The World Health Organization (WHO) considers infertility as a disease (http://www.who.int/reproductivehealth/topics/infertility/multiple-definitions/en/) and estimates 1-2% of prevalence in couples in developed countries (3.6 million of infertile couples in high-income regions) (Mascarenhas et al., 2010). A key feature of meiosis, the cellular program that generates gametes, is the recombination between homologous chromosomes (maternal and paternal) that, in addition to generate genetic diversity, facilitates accurate chromosome segregation. Defects in chromosome segregation during meiosis cause miscarriages, infertility and genetic diseases, and reveal the important to understand the meiotic recombination process.
Our laboratory is interested in how this particular recombination is regulated, what determines in which chromosome regions takes place and how the activity of the topoisomerase-like protein that initiates the process is controlled. To this aim we are using the fission yeast Schizosaccharomyces pombe, since this organism is a proven model system for different eukaryotic conserved processes.
In particular we are studying how linear elements (LinEs) -dynamic chromosome structures similar to the synaptonemal complex of other eukaryotes-, and the conserved pre-recombination complexes (SFT and DSB-complexes) are regulated to generate the double-strand breaks (DSBs) required for the initiation of the recombination process. Our work in collaboration with Dr. Smith at the Fred Hutchinson Cancer Research Center (Seattle, USA) has identified several components of LinEs (Martín-Castellanos et al., 2005; Davis et al., 2008; Ma et al., 2017). LinE-components are specifically enriched at the recombination hotspots (genome sequences where DSB occurs), and their binding is proportional to the strength of the hotspot, indicating they function as determinants for DSB formation (Fowler et al., 2013; Martín-Castellanos et al., 2013). In addition, we have also established functional relationships among the different components of the complex and identified key protein residues involved in LinE-formation/nuclear import and complex organization (Ma et al., 2017). These structures serve as platforms where the pre-recombination complexes, which eventually will generate the breaks in the DNA, are loaded. Our work has also identified a component of these complexes, increasing our knowledge about the functional organization of the SFT-complex (Martín-Castellanos et al., 2005; Bonfils et al., 2011).
A strict regulation of DSB formation during meiosis is critical to maintain the genome stability during this physiological situation, in which self-inflicted DNA damage is used as a source of genetic recombination. Thus, DNA break formation has to be coordinated with meiotic progression and, indeed, the S-phase checkpoint blocks DSB formation during meiotic DNA replication both in fission and budding yeast. Apart from this coordination this aspect of meiosis is largely unknown in most species. An exception is budding yeast, where phosphorylation in key residues of one of the proteins of the pre-recombination complexes by kinases involved in cell cycle regulation is essential for DSB formation.
Legend to Figure. Approaches used for our research: cytological localization of proteins required for the initiation of meiotic recombination, genome localization, and physical analysis of DSB formation. Meiotic cohesins are required for LinE focus-formation (and hotspot-binding), structures where pre-recombination complexes are loaded. A correct organization of these structures and complexes is essential for the formation of DSBs at meiotic hotspots. Mutants in these proteins produced aberrant meiotic products with an abnormal DNA context due to an abnormal chromosome segregation
Most relevant publications
1) Functional organization of protein determinants of meiotic DNA break hotspots. Ma L, Fowler KR, Martín-Castellanos C, Smith GR. Sci Rep. 2017 May 3;7(1):1393. doi: 10.1038/s41598-017-00742-3. PMID: 28469148
2) Making chromosomes hot for breakage.Martín-Castellanos C*, Fowler KR, Smith GR*. Cell Cycle. 2013 May 1;12(9):1327-8. doi: 10.4161/cc.24576. Epub 2013 Apr 11. PMID: 23588069 *co-corresponding authors
3) Protein determinants of meiotic DNA break hot spots. Fowler KR, Gutiérrez-Velasco S, Martín-Castellanos C*, Smith GR*. Mol Cell. 2013 Mar 7;49(5):983-96. doi: 10.1016/j.molcel.2013.01.008. Epub 2013 Feb 7. PMID: 23395004 *co-corresponding authors
4) Functional interactions of Rec24, the fission yeast ortholog of mouse Mei4, with the meiotic recombination-initiation complex. Bonfils S, Rozalén AE, Smith GR, Moreno S, Martín-Castellanos C. J Cell Sci. 2011 Apr 15;124(Pt 8):1328-38. doi: 10.1242/jcs.079194. Epub 2011 Mar 23. PMID: 21429938
5) Rec25 and Rec27, novel linear-element components, link cohesin to meiotic DNA breakage and recombination. Davis L, Rozalén AE, Moreno S, Smith GR*, Martín-Castellanos C*. Curr Biol. 2008 Jun 3;18(11):849-54. doi: 10.1016/j.cub.2008.05.025. PMID: 18514516 *co-corresponding authors
6) Slk1 is a meiosis-specific Sid2-related kinase that coordinates meiotic nuclear division with growth of the forespore membrane. Pérez-Hidalgo L, Rozalén AE, Martín-Castellanos C, Moreno S. J Cell Sci. 2008 May 1;121(Pt 9):1383-92. doi: 10.1242/jcs.023812. Epub 2008 Apr 8. PMID: 18397994
7) Modified Cell Cycle Regulation in Meiosis. Pérez-Hidalgo, L., Moreno, S. and Martín-Castellanos, C. Genome Dynamics & Stability, Vol. 2: Recombination and Meiosis, 303-353. (Richard Egel and Dirk-Henner Lankenau eds.). Springer-Verlag. (2008). ISBN-13 978-3-540-75371-1
8) A large-scale screen in S. pombe identifies seven novel genes required for critical meiotic events. Martín-Castellanos C, Blanco M, Rozalén AE, Pérez-Hidalgo L, García AI, Conde F, Mata J, Ellermeier C, Davis L, San-Segundo P, Smith GR, Moreno S. Curr Biol. 2005 Nov 22;15(22):2056-62. PMID: 1630356
Aim of the research work
Our main current objective is to determine whether CDK (cyclin-dependent kinase) activity, which controls meiosis progression, is also required for DSB formation and recombination in fission yeast. We are using both genetic (recombination assays) and physical (DSB quantification) analyses in different experimental situations where CDK activity is down regulated.
Since proteins of LinEs and pre-recombination complexes harbor consensus phosphorylation sites by CDK, we are also addressing whether CDK regulates LinE formation and/or the loading of accessory proteins as possible mechanisms to control DSB formation. We are using both microscopy (protein localization) and cellular fractionation (chromatin binding) analyses to address possible defects in experimental situations with reduced CDK activity. This strategy is complemented with the analysis of the recombination proficiency of mutants (phospho-null) in several of these proteins.
Given that DSB formation is a key conserved feature of meiosis and the kinase under study is also conserved, we expect the results to be useful to understand the control of recombination in other organisms with sexual reproduction.
Research identification links
Researcher ID F-4502-2017