Cohesin and its regulators: from chromosome dynamics and nuclear architecture to human diseases
Lead Research Organisation:
Plasticell Ltd
Department Name: Therapeutics
Abstract
Cohesin is an evolutionarily conserved multi-protein complex that belongs to the structural maintenance of chromosomes protein family. It can topologically entrap DNA molecules mediating sister chromatid cohesion, a function important for accurate chromosome segregation and DNA replication/repair. It has been recently discovered that cohesin can generate and maintain DNA loops by an ATPase-dependent in cis DNA tethering activity, called loop-extrusion, critical for organising chromatin architecture and regulating gene transcription, and as such thought to be important for cell differentiation and development. However, the molecular mechanisms by which the cohesin complex achieve these key cellular functions remain to be elucidated. Mutations in genes coding for cohesin and its regulators lead to a class of developmental disorders collectively called "cohesinopathies" and have been found in several types of cancer. Nonetheless, the pathogenesis of these devastating human diseases is poorly understood. CohesiNet aims to answer outstanding questions in the field of cohesin biology by a highly innovative research programme. This consortium will investigate fundamental mechanisms on how cohesin acts during chromosomal cohesion and loop extrusion, identify regulatory modules of cohesin functions and get insights into the molecular bases of cohesin-related diseases. These ambitious goals will be achieved using multi-disciplinary hypothesis-driven and exploratory approaches while promoting a culture of communication and cooperation among academic and private institutions across the European Union. A team of ten PhD students will be trained by the CohesiNet consortium to address these scientific questions and be empowered with skills that will enhance their career perspectives, while experiencing first-hand the value of Open Science and the importance of inclusion, transparency, accessibility and integrity in scientific research.
People |
ORCID iD |
| Marina Tarunina (Principal Investigator) |
| Title | Establishment of differentiation protocols for stem cell-based models of cohesinopathies |
| Description | To study how the cohesinopathy disease-relevant mutations affect the phenotype and behaviour of differentiating cells we first established the protocols for differentiation of parental (unedited) iPS cell lines into NCC. Two published methods were compared: STEMdiff Neural Crest (NCC) differentiation kit supplied by Stemcell technologies and published NCC differentiation protocol. Efficiency of NCC differentiation was evaluated by FACS analysis focusing on expression of NCC specific cell surface markers. |
| Type Of Material | Cell line |
| Year Produced | 2025 |
| Provided To Others? | No |
| Impact | Both methods used for differentiation of iPSCs into NCCs resulted in sizeable population of Neural Crest Cells. Expression of NCC-specific cell surface markers as well as NCC-specific intracellular markers in 2 iPSC cell lines was confirmed by immunocytochemistry. To analyse ability of iPSC derived NCC for further differentiation we conducted initial experiments demonstrating their ability to differentiate into mesenchymal stem cells (MSC). |
| Title | Establishment of stem cell-based models of cohesinopathies |
| Description | Used CRISPR/Cas9 technique to target cohesin related genes that are mutated in patients suffering from cohesinopathies. Generated a set of mutated iPSC lines that can be used to study cellular consequences of cohesinopathy-related mutations using various functional assays and find out which pathways are impaired in the different cell lines. hiPSCs were electroporated with CRISPR-Cas9/gRNA RNP complex using Neon Transfection System (Invitrogen) following manufacturers protocol with modification that led to improvement of transfection efficiency. Following electroporation cells were plated in vitronectin coated tissue culture-treated 24 well plates. Kock-in and knock-out efficiency in each bulk iPSC was assessed with Sanger sequencing. Isolation of single cell clones carrying CRISPR/Cas9-induced mutations was performed using limiting dilution cloning in vitronectin coated tissue culture-treated 96 well plates. Wells with positive clones were identified and the cells were harvested to identify pure clonal populations by Sanger sequencing. |
| Type Of Material | Technology assay or reagent |
| Year Produced | 2024 |
| Provided To Others? | No |
| Impact | Generation of isogenic iPS cell lines that mimic genetic defects associated with cohesinopathies provides a unique tool for studying the physiological consequences of the related mutations. Differentiation of pluripotent stem cells into different lineages and distinctive cell types (neural, heart, skeletal muscle, blood) recapitulates early developmental events and can help to understand the molecular mechanisms by which cohesin and its regulators impact organ development. To study the role of Nipped-B-like (NIPBL) protein during cellular differentiation, we mutated NIPBL gene in human iPSCs rendering a non-functional NIPBL protein. Since NIPBL has an essential role in cohesin function, such severe mutations in patients lead to haploinsufficiency of the NIPBL protein. iPSC clones with successfully edited NIPBL gene were isolated using limiting dilution and analyzed with Sanger sequencing. The ICE data analysis revealed 3 clones with ?90% purity. Generation of iPSC lines with mutated NIPBL gene will allow us to study how the introduction of these mutations will effect differentiation pathways that control differentiation of iPSCs into Neural Crest cells and terminally differentiated cell types. For other cohesinopathies associated mutations we either generated knock-in clones that carry homozygous mutations or performed knock-out of these genes in the hiPSCs. We are currently assessing the knock-in efficiency for DDX11 and SGOL1 genes by analyzing Sanger sequencing results using ICE (Interference of CRISPR Edits) analysis tool. |
| Description | Construction of a cohesinopathy-mutant cell panel. This is a collaboration with Dr Job de Lange laboratory at Amsterdam University Medical Center. |
| Organisation | Amsterdam Medical Center |
| Country | Netherlands |
| Sector | Hospitals |
| PI Contribution | Generation of isogenic iPS cell lines that mimic genetic defects associated with cohesinopathies provides a unique tool for studying the physiological consequences of the related mutations. We aim to determine the extent to which deregulation of these diverse cellular processes contributes to the etiology of the different pathologies. In collaboration with Dr Job de Lange laboratory at AUMC we plan to create a panel of isogenic human cell lines harbouring different cohesinopathy mutations. Working at Dr de Lange lab Plasticell's PhD student Ivana Milas targeted NIPBL (Cornelia de Lange Syndrome, CdLS), SGOL1 (Chronic Atrial and Intestinal Dysrithmia, CAID), ESCO2 (mutated in Roberts Syndrome, RBS) and DDX11 (Warsaw breakage syndrome, WABS) genes in iPSCs that will allow us to establish cellular model cohesinopathy in vitro. In this collaboration Plasticell is responsible for differentiation of gene modified pluripotent stem cells into different lineages and study distinctive cell types (neural, heart, skeletal muscle, blood) that will recapitulate early developmental events and can help to understand the molecular mechanisms by which cohesin and its regulators impact organ development. |
| Collaborator Contribution | Dr Job de Lange lab previously used CRISPR/Cas9 to target ESCO2 (mutated in RBS) and DDX11 (WABS) in RPE1 cells and constructed mutations in BUB1. In the current project they further expanded this panel by constructing disease-mimicking mutations in NIPBL (CdLS), SGOL1 (CAID) and STAG2 and introducing them into two iPS lines EhiPS and KOLF2.1J. All the gene editing work took place at Dr de Lange lab. The created cell panel will be instrumental to study cohesin-related features, including (but not limited to) sister chromatid cohesion and global DNA architecture. |
| Impact | This collaboration is multi-disciplinary. Dr Job de Lange's lab specialises in editing techniques that introduce gene modifications to create isogenic mutant cell lines or correct mutations in patient-derived cells. Plasticell is specialising in finding optimal conditions for differentiation of pluripotent stem cells into defined lineages. Combining these two approaches will allow to model cohesinopathies in vitro and to study disease related abnormalities at different stages of cell differentiation. |
| Start Year | 2024 |
| Description | Horizon Europe MSCA 2021 - Doctoral Network CohesiNet Consortium meeting in Amsterdam University Medical Center |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Postgraduate students |
| Results and Impact | 12 PhD students and their supervisors attended the meeting. PhD students had the opportunity to discuss their work and set up future collaborations |
| Year(s) Of Engagement Activity | 2024 |
| Description | Horizon Europe MSCA 2021 - Doctoral Network CohesiNet Consortium meeting in Vienna BioCenter |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Postgraduate students |
| Results and Impact | The members of consortium used this opportunity to discuss the progress of the project and set up future collaborations |
| Year(s) Of Engagement Activity | 2025 |