Cohesin mediates Esco2-dependent transcriptional regulation in a zebrafish regenerating fin model of Roberts Syndrome

ABSTRACT Robert syndrome (RBS) and Cornelia de Lange syndrome (CdLS) are human developmental disorders characterized by craniofacial deformities, limb malformation and mental retardation. These birth defects are collectively termed cohesinopathies as both arise from mutations in cohesion genes. CdLS arises due to autosomal dominant mutations or haploinsufficiencies in cohesin subunits (SMC1A, SMC3 and RAD21) or cohesin auxiliary factors (NIPBL and HDAC8) that result in transcriptional dysregulation of developmental programs. RBS arises due to autosomal recessive mutations in cohesin auxiliary factor ESCO2, the gene that encodes an N-acetyltransferase which targets the SMC3 subunit of the cohesin complex. The mechanism that underlies RBS, however, remains unknown. A popular model states that RBS arises due to mitotic failure and loss of progenitor stem cells through apoptosis. Previous findings in the zebrafish regenerating fin, however, suggest that Esco2-knockdown results in transcription dysregulation, independent of apoptosis, similar to that observed in CdLS patients. Previously, we used the clinically relevant CX43 to demonstrate a transcriptional role for Esco2. CX43 is a gap junction gene conserved among all vertebrates that is required for direct cell-cell communication between adjacent cells such that cx43 mutations result in oculodentodigital dysplasia. Here, we show that morpholino-mediated knockdown of smc3 reduces cx43 expression and perturbs zebrafish bone and tissue regeneration similar to those previously reported for esco2 knockdown. Also similar to Esco2-dependent phenotypes, Smc3-dependent bone and tissue regeneration defects are rescued by transgenic Cx43 overexpression, suggesting that Smc3 and Esco2 cooperatively act to regulate cx43 transcription. In support of this model, chromatin immunoprecipitation assays reveal that Smc3 binds to a discrete region of the cx43 promoter, suggesting that Esco2 exerts transcriptional regulation of cx43 through modification of Smc3 bound to the cx43 promoter. These findings have the potential to unify RBS and CdLS as transcription-based mechanisms.


First person -Rajeswari Banerji
First Person is a series of interviews with the first authors of a selection of papers published in Biology Open, helping early-career researchers promote themselves alongside their papers. Rajeswari Banerji is first author on 'Cohesin mediates Esco2-dependent transcriptional regulation in zebrafish regenerating fin model of Roberts Syndrome', published in BiO. Rajeswari conducted the research in this article while a PhD student in the lab of M. Kathryn Iovine and Robert Skibbens at Lehigh University, PA, USA. She is currently a postdoctoral fellow in Manisha Patel's lab at the Skaggs School of Pharmacy, University of Colorado, investigating learning mechanisms of paediatric diseases in order to find pharmacological targets for proper treatment.

What is your scientific background and the general focus of your lab?
I recently completed my PhD in the Department of Cell and Molecular biology under the mentorships of Kathryn Iovine and Robert Skibbens. The Skibbens lab studies the role of cohesins and their associated factors using yeast as a model system. These factors such as ESCO2 are clinically significant as defects lead to various developmental disorders such as Robert's syndrome (RBS). The Iovine lab examines the role of the gap junction protein Cx43, which is also critical during skeletal regeneration. For instance, Cx43 mutations in humans results in oculodentodigital dysplasia (ODDD). My PhD research work was based on a collaboration between the two labs to develop a vertebrate model system to understand RBS and the mechanistic overlap with ODDD, using the zebrafish regenerating caudal fin as the model system.

What are the potential implications of these results for your field of research?
Our research identifies a novel transcription-based mechanism underlying RBS which is similar to CdLS. Thus, our study suggests that the underlying causation for both diseases are relateda link largely undeveloped in the field. The unified mechanism provides opportunities to find a single drug target from which new pharmaceutical treatments can be developed in the future.

How would you explain the main findings of your paper to non-scientific family and friends?
RBS is a severe type of human developmental disorder characterized by craniofacial deformities, limb malformation and mental retardation. Currently the treatment options are very limited and not very beneficial. Thus, it is important to understand the molecular mechanisms underlying RBS if we are to identify relevant pharmacological targets for more effective treatments. In a previous study we developed a zebrafish regenerating fin vertebrate model to Zebrafish caudal fin stained with calcein examine the skeletal defects of RBS. We established a genetic link between the gene mutated in RBS -esco2and the clinically relevant gap junction gene cx43, which suggested a transcriptional role for Esco2 in Cx43 regulation. This is the first study to provide evidence that unifies RBS and similar birth defect maladies such as Cornelia de Lange syndrome (CdLS).
What, in your opinion, are some of the greatest achievements in your field and how has this influenced your research?
One of the greatest achievements in our field is the use of various model systems such as zebrafish, Drosophila and yeast to study mechanisms underlying human diseases. In particular zebrafish research plays a valuable role in developmental studies and also drug discovery.
What has surprised you the most while conducting your research?
Both RBS and CdLS are severe birth defects with overlapping phenotypes, and arise from mutation of genes that function in a common pathway, but surprisingly the etiologies of these syndromes are believed to be distinct. Though it is widely accepted that CdLS arises from transcriptional deregulation of a set of genes, the mechanism underlying RBS remains unknown. A popular model states that RBS is caused by mitotic failure and cell death that is completely distinct from CdLS. I was surprised that similar developmental diseases such as RBS and CdLS could have such distinct mechanisms and believed that this area needed further investigation.

What changes do you think could improve the professional lives of early-career scientists?
I feel collaboration plays a beneficial role in any kind of research. I was fortunate to work in such an environment that gave me different perspectives and ideas that helped me progress in my work and shape my future. I highly recommend the kind of exposure one receives by pursuing collaborative research.
"I highly recommend the kind of exposure one receives by pursuing collaborative research." What's next for you?
Currently I am working as a postdoctoral researcher in the Department of Pharmacy. I strongly believe that zebrafish is an ideal model for paediatric research and high-throughput drug testing. At present I am working on a severe form of childhood epilepsy using zebrafish larvae and testing various drugs to decrease the severe seizures associated with this disease. In the long run I would like to continue with drug discovery and ultimately enter in the area of clinical trial studies.