Examining the role of cohesin subunit Rad21 in malignancy and development

Abstract

The cohesin complex is essential for cell survival, owing to its well-established roles in cell division, DNA repair and gene expression. Malignancy and developmental disorders termed the cohesinopathies can result when mutations are present in cohesin subunits, or in proteins that interact within cohesin.

In this thesis, I describe experiments using the rad21nz171 zebrafish mutant line, which carries a null allele for cohesin subunit Rad21, to address cohesin’s role in driving malignancy and normal development. The thesis is in two sections.

The first section examines the functionality of a germline RAD21 variant that may be responsible for the development of myelodysplastic syndrome in a cohort of patients. RAD21 functionality can be determined through the use of a zebrafish runx1 in situ hybridisation bioassay, and this research found that the familial variant produces a functional cohesin subunit. A novel RAD21 variant was designed and assayed alongside the familial variant, and found to be non-functional.

The second section focuses on the role of cohesin in regulating normal embryogenesis. High-throughput RNA-sequencing was performed on the elongating tailbuds of rad21nz171 zebrafish embryos compared to wildtype siblings, to elucidate which genes and biological pathways cohesin is fundamental in regulating during zebrafish embryogenesis. The tailbud was chosen for examination as the posterior of the zebrafish develops from a bipotent stem cell population in this structure, and a lack of cohesin-regulated gene expression is predicted to affect cell fate decisions of the stem cells. An abundance of metadata was generated from the RNA-sequencing analyses, providing numerous avenues for further exploration. The spatial expression of four genes identified in these analyses were examined via in situ hybridisation, providing a broader picture of cohesin-deficient development.

We discovered that in the absence of cohesin-regulation, the fate of bipotent stem cells in the developing zebrafish tailbud skews towards neural ectoderm over mesodermal progenitors, and theorise that this shift is due to dysregulation of wnt3a, a key regulator of zebrafish axial elongation. A role for cohesin in regulating ribosome biogenesis, and thus protein translation, was identified in these analyses. The involvement of cohesin in assisting in protein translation implicates cohesin as regulating gene expression not only at the gene, but protein level also – a role which has not been well explored in the literature to date.