Meet the CCB lab

 

Research

The CCB laboratory is interested in meiosis, the cellular program that generates reproductive cells. Between each generation, organisms shuffle the chromosomes that they inherited from their parents in order to produce reproductive cells with unique combinations of alleles. This is a central goal of sexual reproduction because it promotes genetic diversity, thereby driving evolution. The chromosomal exchanges, called crossovers, are formed by a mechanism of DNA recombination that is initiated by the programmed introduction of DNA double-strand breaks (Figure 1).

The laboratory uses biochemistry and molecular genetics approaches with the model organism Saccharomyces cerevisiae to gain insights into the fundamental mechanisms that lead to the formation of meiotic DNA double-strand breaks and their repair by recombination.

 

Figure 1: (A) Meiotic recombination occurs during prophase I and allows the accurate segregation of homologous chromosomes at the first meiotic division. (B) Recombination is initiated by Spo11-induced double strand breaks and leads to the formation of crossovers.

 

Meiotic DSBs are catalyzed by Spo11, a transesterase that evolved from an archaeal type II topoisomerase (Topo VI). Two Spo11 subunits work in concert to make a DSB using an active-site tyrosine residue to attack the DNA backbone and generate a covalent 5′-phosphotyrosyl link.

Breaking DNA is intrinsically dangerous. Indeed, DSBs are normally undesirable DNA damages that severely compromise cell physiology and survival. Yet meiotic cells induce high levels of breaks. Break formation is therefore controlled in terms of their number, timing and distribution, which involve multi-layered control mechanisms. How these control mechanisms work at the molecular level and are integrated within the cell is not well understood.

Spo11 activity depends on at least nine co-factors that can be classified into three sub-complexes (Figure 2): The core complex that contains Spo11, the RMM complex, and the MRX complex. A central goal of our research is to understand how these proteins collaborate to catalyze DSB formation. We have isolated recombinant DSB proteins and are using in vitro methods to characterize their biochemical properties. We seek to gain insights into their molecular structure, to address how they interact with one another and with DNA, and to understand how the co-factors assist Spo11 in cleaving DNA.

 

Figure 2: (A) Upon double-strand break formation, Spo11 remains covalently attached to the 5’-ends of DNA. (B) The S. cerevisiae meiotic DSB proteins form three sub-complexes and also depend on two regulatory kinases. The Core complex can be considered the DSB enzyme that is related to Topo VI. The biochemical properties of the DSB proteins and how they collaborate in DSB formation is unclear. (B) The purified yeast DSB machinery.

 

Meiotic chromosomes are arranged as a series of DNA loop anchored along a nucleoprotein axis (Figure 3). DSB proteins are mostly located along the chromosome axis, but DNA cleavage happens preferentially within the loops. The tethered loop-axis model proposes that a loop becomes tethered to the axis, allowing axis-bound Spo11 to cleave within the loop. However, the underlying molecular assemblies remain poorly understood.

We have recently proposed that this assembly relies on the condensation of RMM along the chromosome. Our current model is that RMM condensates provide a platform that recruit the core complex and MRX (Figure 5C). The connection between the loop and the axis is then mediated by Spp1, that binds H3K4me3 methylation marks along the loop and axis-bound Mer2 proteins.

We aim to gain a better understanding of the relationship between DSB proteins and the chromosome axis. How do the DSB proteins become enriched along the axis? What are the molecular interactions that involved in the assembly of the DSB machinery?

 

Figure 3: (A) The loop-axis organization of meiotic chromosomes. (B) The tethered loop-axis model for DSB formation. (C) Model of assembly of the DSB machinery. RMM is recruited to the chromosome axis, composed of Hop1 (yellow ovals) and Red1 (red squares). RMM forms macromolecular assemblies along the chromosome. These RMM condensates recruit the core complex and MRX, Spp1 links H3K4me3 marks within DNA loops to axis-bound Mer2 proteins, allowing Spo11 to cleave the loop.

 

The team

 

Corentin Claeys Bouuaert (PI).

Corentin earned his PhD in 2011 from the University of Nottingham, where he studied the mechanism of DNA transposition under the supervision of Dr. Ronald Chalmers. He then moved to New York to join the laboratory of Dr. Scott Keeney at the Memorial Sloan Kettering Cancer Center, where he initiated his research on meiosis. He returned to his alma mater UCLouvain to open his laboratory within the LIBST institute in 2019.

 

Pascaline Liloku (Research Technician).

Pascaline completed a BS in Biochemistry at the Haute Ecole de la Province de Liège in 2018. She joined UCLouvain in September 2019 and became the first member of the CCB lab. In addition to making sure that the lab runs smoothly, Pascaline exploits her biochemistry skills to tackle questions regarding the connection between the meiotic DSB machinery and chromatin.

 

Oger (Postdoc).

Cédric has a long-standing interest in the molecular mechanisms of DNA-processing enzymes. After completing his Master degree in Biochemistry, Cellular, and Molecular Biology at UCLouvain in 2011, Cedric secured a FRIA fellowship to fund his PhD training under the supervision of Dr. Bernard Hallet. During his thesis, Cédric studied the mechanism of the Tn3-family transposon, Tn4430. He graduated in 2018 and joined the CCB lab in 2019. Cédric really wants to know what triggers Spo11’s DNA cleavage activity.

 

Hajar Aït Bella (PhD student).

Hajar obtained a Master in Molecular Engineering from the University of Lorraine (Nancy, France) in 2017. After a couple of internships at the I2BC Paris-Sarclay, Hajar joined the CCB lab in 2019 to investigate the dynamics of the interaction between Spo11 and DNA.

 

Dima Daccache (PhD student).

Dima did a Master in Genomics and Functional Proteomics at Saint Joseph University in Beirut (Lebanon). She joined the CCB lab in October 2019 and secured an Aspirant fellowship from the FNRS to investigate the mechanism of DNA-dependent condensation by the RMM proteins.

 

Chi Wai Wong (PhD student).

Chi Wai earned a Master in Biomedical Sciences at the Chinese University of Hong Kong. After three years as a research assistant in Hong Kong, Chi Wai brought his laboratory skills to Belgium to start his PhD in 2019. Chi Wai wants to understand the relationships between the meiotic DNA double-strand break machinery and the chromosomes axis.

 

Marita Haddad (PhD student).

Marita completed a Masters in Genomics and Biomedical Sciences at the Lebanese University in 2020. She developed an interest in meiosis during her internship in the laboratory of Dr. Thomas Robert in Montpellier (France), then joined the CCB lab in March 2021. Marita is exploring the connection between RMM condensation, chromatin folding and DSB formation.

 

Karen Mechleb (PhD student).

Karen did a Master in Biochemistry and Molecular Genetics at the American University of Beirut (Lebanon). She joined the CCB lab in March 2021 and is developing innovative approaches based on atomic force microscopy to characterize the interactions between meiotic DNA double-strand break proteins.

 

Priyanka Priyadarshini (PhD student).

Priyanka earned a Masters in Biotechnology at the Pondicherry Central University (India). In October 2021, Priyanka secured an Aspirant fellowship from the FNRS to study the role of the MRX complex in meiotic DSB formation.

 

Alice Chanteau (Master student).

Alice is a Master 2 student in Health Biology from Institut Polytechnique of Paris.

 

Christelle Danielle Ossogo Mballa (Master student).

Christelle is doing a Master in Molecular and Cellular Biology at UCLouvain.

 

 

Selected publications

Yadav V.K., Claeys Bouuaert C. Mechanism and control of meiotic DNA double-strand break formation in S. cerevisiae. Frontiers in Cell and Developmental Biology (2021).

Claeys Bouuaert, C., Pu S., Wang J., Oger C., Daccache D., Xie W., Patel D.J., Keeney S. DNA-driven condensation assembles the meiotic DNA break machinery. Nature (2021).

Johnson D., Crawford M., Cooper T., Claeys Bouuaert C., Keeney S., Llorente B., Garcia V., Neale M.J. Concerted cutting by Spo11 illuminates meiotic DNA break mechanics. Nature (2021).

Claeys Bouuaert C., Tischfield S.E., Pu S., Mimitou E.P., Arias-Palomo E., Berger J.M., Keeney S. Structural and functional characterization of the Spo11 core complex. Nature Structural & Molecular Biology (2021) 28, 92-102.