CE12 - Génétique, génomique et ARN

Functions of condensin: from a structural role to the control of genomic stability – CONDENSin3R

Functions of Condensin: from a structural role to the control of genomic stability

The SMC-condensin complexes are recognized as the “universal organizers of chromosomes”, with a role in anaphase/metaphase chromosome compaction, essential for their segregation. Recent evidences indicate additional functions for condensin in DNA repair processes involving homologous recombination (HR). However, this condensin’s function remains poorly studied. HR is a faithful DNA repair process, which relies on the formation of specific DNA structures to properly repair DNA lesions. In particular, HR dependent replication fork restart following replication stress or chromosome structure dependent meiotic HR, are model situations of this interplay between chromosome structure and HR regulation. Understand whether condensin has a conserved function in repair processes either by modulating the activity of DNA repair factors or by controlling the formation of DNA structures required for DNA repair is a central question.

In search of a comprehensive view of the role of condensin in DNA repair processes

The objective of the CONDENSin3R project is to characterize the role of the SMC-condensin complexes in the regulation of HR-associated processes. Three work packages are developed (WP). The WP1 aims at clarifying whether condensins associate with DNA during repair and what triggers this association. The WP2 and 3 aim at deciphering the molecular mechanisms underlying the role of condensin in DNA repair. A direct regulatory role for condensin on HR proteins is investigated in WP2, while WP3 focuses on characterizing condensin as a chromosomal organizer of DNA repair. The two repair model situations of blocked replication fork in yeast and human cells and meiotic HR in the mouse are studied in parallel. <br /><br />Based on the development of a multi-organism approach, coupled with a multi-system approach, the long-term goal of the CONDENSin3R project is to get a unified view of the SMC-condensin role in the control of DNA repair processes, and genomic stability.

The CONDENSin3R project is designed based on a multi-organisms and multi-techniques approaches to decipher the role of condensins in DNA repair processes, and in particular in homologous recombination (HR) related repair processes.
The project relies on the use of three different model organisms: the yeast Saccharomyces cerevisiae, the human cell lines, and the mouse. These different organisms, considered as model systems to study DNA repair processes, were chosen to uncover different aspects of the condensin’s role in HR related DNA repair processes (i.e. replication fork restart, meiotic recombination control).
In the three different systems, genetic characterization of specific condensin mutants’ phenotypes is proposed, based on the use of model-specific tools, with the objective to get a unified view of the role of condensins.
More precisely, P1 proposes the use of yeast and human cells, taking advantage of the combine approaches of genomic (ChiP-Seq), iPOND (isolation of proteins on Nascent DNA wich relies on the labelling of nascent DNA with EdU), SIRF/PLA technique, molecular biology approaches (protein/DNA binding assays…), electron microscopy, to asses the function of condensin in the control of replication fork restarts after DNA damage.
P2 on the other hand proposes to develop biochemical approaches (protein purification, biophysical and structural characterization), genomic, proteomic and cytological approaches in the mouse, to decipher the role of condensin in the control of chromosome structure and homologous recombination during the first meiotic division.
Altogether, this unprecedented multi-organisms and multi-approaches project will be essential to decipher the role of condensin in different DNA repair situations, to pinpoint differences as well as common point and to propose a unified view of the role of condensins in DNA repair.

WP1. Condensin & DNA repair: When? Where? & Who? WP1 aims at identifying repair situations that involve condensin. First, P1 and P2 investigated the dynamics of condensin association to DNA during DNA replication and meiotic prophase I. P1 analysed the pattern of condensin binding to chromatin during the cell cycle. To this purpose, P1 has used the ChiP-Seq and the ChEC-Seq techniques. Maps obtained (i) in metaphase show a strong enrichment at centromeres and at the RFB of rDNA repeats, and (ii) in S phase challenged by damaging agents, at replication sites. In this context, P1 has found that the chromatin remodeler Chd1 supports condensin binding. P2 characterized the pattern of condensin localization in prophase I meiotic nucleus and showed a peak of recruitment during HR initiation steps and a colocalization with chromatin. Preliminary results indicate that condensin I interacts with the meiotic axes and co-localize with recombination proteins.

WP2. Condensin, a direct regulator of repair proteins? P1 and P2 investigated whether condensin acts as a direct regulator of DNA repair proteins. P2 initiated the molecular characterization of the NCAPD2/TOPOVIBL interaction. Soluble NCAPD2 and TOPOVIBL truncated constructs were purified and the biophysical analysis of this construct reveals dimerization and DNA interaction properties. P2 initiated the phenotypic characterization of the Ncapd2cKO mutant strain to get functional insights and found a miss-regulation of the meiotic HR process in the mutant. P1 investigated whether condensin contributes to the recruitment of stalled replication forks processing and restarting factors. Although nascent DNA resection is defective at stalled replication forks in the absence of condensin, P1 found that the major nuclease is well recruited, suggesting that condensin enables its activity rather than its recruitment.

WP3. Condensin as a chromosomal organizer for DNA repair? The objective of WP3 is to determine whether condensin reorganize DNA structure to favor DNA repair. Condensation has been linked to the resolution of chromosomal intertwines upon mitosis entry. To test whether this function is conserved in S phase, P1 analysed fork processing in the absence of topoisomerases in yeast and human cells. Clearly, Top2 depletion recapitulates the absence of CAPG2 and their functions are epistatic. This suggests a universal role for condensin, allowing DNA strands supercoiling to regulate the formation of DNA structures and the dynamic association of chromosomal components. The role of condensin in meiotic chromosome reorganization is currently analyzed by P2 by cytological analysis in the WT and Ncapd2KOc mutant. Preliminary experiments revealed that condensin I is required for a normal recruitment of different structural components of the meiotic chromosome.

Failures to repair DNA lesions lead to mutations causing genomic disorders, with profound impact on physiological processes such as development, tissue homeostasis, neurological disorders and aging. DNA repair mechanisms are then essential to preserve genome integrity. Homologous recombination (HR) is a faithful conserved DNA repair process which relies on the tight regulation of specific DNA topological structures. Whereas the different HR proteins are well known, the topological environment in which they proceed remains to be understand. The aim of this project is to exploit exogenous and endogenous DNA threats to characterize the function of a structural component of chromosomes, Condensin, in the regulation of DNA repair. This complex belongs to the structural maintenance of chromosomes (SMC) protein family which regulates nearly all aspects of chromosome biology and are critical for genomic stability. In addition to improve our academic knowledge, this project should also have important long-term repercussions for human health. Indeed, Condensin-deficient patients present neuronal disorders such as microcephaly, characterized with frequent anaphase chromatin bridges leading to micronuclei formation. “Condensinopathies” belong to microcephalic disorders. Importantly, P1 team has recently made a breakthrough discovery showing that the release of ssDNA fragments during the processing of stalled RF results in a microcephaly syndrome, through a process also involving the formation of micronuclei. For those reasons, studying the processing of stalled forks in Condensin deficient cells in this project may reveal the origin of microcephaly in Condensinopathies. One of the objectives of the Condensin3R project is also to study the role of Condensin during the specialized meiotic cell division. Genetic rearrangements in meiosis due to improper HR are the cause of various genetic disorders, gross chromosomal rearrangements and fertility issues. Therefore, multiple layers of control of meiotic HR are needed, and among those, the control by the chromosome structure is essential. The identification of Condensin as an interactor of a protein required for meiotic DSB formation, offers the opportunity to study the role of these proteins in establishing the meiotic chromosome higher-order structure as well as in regulating HR. Understanding the different regulatory mechanisms of meiotic HR acquired in the course of evolution is thus a key point to unravel the cause of various genomic diseases and reproduction problems. Overall, this proposal tackles an important fundamental question which is to decipher the impacts of Condensin deficiency onto DNA repair. The answers to this question will have broad impacts on different fields, from the understanding of microcephaly up to fertility issues in mammals.

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DNA replication and segregation are the pivotal events of cell division. All along these processes, the maintenance of a proper chromosome structure is essential for the transmission of an intact genetic material. Amongst the different factors that control DNA structure, the SMC related complex condensin is recognized as the “universal organizer of chromosomes”, with a well characterized anaphase/metaphase chromosome compaction role, essential for segregation. Interestingly, recent studies suggest additional functions for condensin, in particular in DNA repair processes involving homologous recombination (HR). However, this potential crucial role of condensin in the control of genomic stability remains poorly studied.
HR is a faithful DNA repair process, which relies on the tight regulation of specific DNA structures. During replication, replication forks (RF) encounter obstacles such as DNA lesions. The subsequent slowing or stalling of RF defines replication stress (RS), which is a major source of genomic instability and different studies point toward an HR-mediated fork restart process. In meiosis, the formation of DNA double strand breaks (DSB), repaired by HR, is essential for the segregation of the homologous chromosomes, and the production of haploid gametes. In both cases, HR is associated to an important chromosomal reorganization, essential for a proper repair. Namely, blocked RF undergoes an extensive remodeling that enables the subsequent HR proteins recruitment and fork restart. And, in meiosis, DSB formation and repair take place in the context of a complete structural reorganization of the chromosomes. However, despite their key role, the factors that mediate these structural reorganization events and coordinate them with HR are poorly understood.
The objective of the CONDENSin3R project is to characterize the role of the SMC-condensin complex, as a chromosome organizer, in the regulation of HR-associated DNA repair process.
To assess in depth the role of condensin in DNA repair, we propose to develop three research axes. The first one aims at clarifying whether condensin indeed associate with DNA and what trigger this association during repair. The molecular mechanisms underlying the role of condensin in DNA repair will be studied in the axes 2 and 3. The goal of axe 2 will be to investigate an unexpected direct regulatory role of condensin on HR proteins, while axe 3 will focus on characterizing condensin as a chromosomal organizer for DNA repair. Importantly, two repair model situations will be studied in parallel, blocked RF in yeast and human cells and meiotic HR in the mouse, to shed light on a conserved role of condensin during HR repair.
Altogether, the CONDENSin3R project, based on the development of a multi-organism approach (yeast, human cells and mouse), coupled with a multi-system approach (HR-mediated fork restart, meiotic HR), will allow to tackle common questions: Are condensin involved in DNA repair processes? Is there a direct link between condensin and HR repair proteins? What is the role of condensin in the control of the DNA repair mediated chromosome structural reorganization? Answering to these questions will be of great importance to get a unified view of the SMC-Condensin role in the control of DNA repair processes, and genomic stability. Moreover, it will update and enlarge our vision of the canonical role of condensin, the “universal organizer of chromosome”.

Project coordination

Armelle Lengronne (Institut de Génétique Humaine)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

CBS Centre de biochimie structurale
IGH Institut de Génétique Humaine

Help of the ANR 584,814 euros
Beginning and duration of the scientific project: December 2020 - 48 Months

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