Cohesin and Regulation of Gene Expression During Zebrafish Development

Abstract

Made up of four subunits, Structural Maintenance of Chromosomes (SMC) proteins, the SMC1 and SMC3 subunits, Rad21 and stromalin antigens (SA1/2), the ring- shaped protein cohesin is an essential part of both the mitotic and meiotic cell cycle. In addition to maintaining cohesion between sister chromatids at the anaphase stage of the cell cycle, cohesin also plays a role in regulating gene expression during development. Mutations in the cohesin subunits and the related cohesin network are often found in somatically derived cancers such as Acute Myeloid Leukemia (AML), Chronic Myelomonocytic Leukemia (CMML), Myeloproliferative Neoplasms (MPNs) and Myelodysplastic Syndrome (MDS), as well as a distinct group of inherited developmental disorders called Cohesinopathies, Roberts syndrome (RBS) and Cornelia de Lange Syndrome (CdLS).

The zebrafish (Danio rerio) animal model, rad21nz171, with a nonsense mutation in the rad21 gene, has been utilized as an animal model to study the effect of depletion of cohesin. Genes indentified to bind cohesin and be affected in cohesin depleted embryos were studied in this project and include the following, ankrd9, foxo3b, has2, pax2a, sema3ab, s1pr1, sox9b, wnt3a and wnt7ba. Whole-mount in situ hybridization was carried out using riboprobes for these genes on embryos produced by the incross of adult heterozygous rad21nz171 zebrafish. Half of the embryos were expected to be heterozygous for the mutant rad21 allele, while the rest would be homozygous for the mutant allele and wild type siblings. Heterozygotes were also expected to have lower levels of Rad21 compared to wild type embryos, but more than their homozygous siblings. Previous experiments using this same model have found cohesin to regulate expression of the transcription factor, runx1 in a tissue-specific manner. It was thus hypothesized that cohesin could also regulate other genes in a similar manner.

Overall, heterozygotes showed similar expression patterns as the wild type control while homozygotes lacked expression for most genes. It was found that cohesin regulates the expression of has2, pax2a and wnt3a in a tissue-specific, dosage-sensitive manner and decrease in cohesin levels brings about ectopic expression of the tailbud region in heterozygote rad21nz171 embryos for these genes. Cohesin could also indirectly regulate wnt3a and wnt7ba expression possibly by directly regulating foxo3b expression. Cohesin also regulated the tissue-specific expression of sox9b in the tailbud region. This positions cohesin as an important regulator of gene expression during development in a dosage-dependent, tissue-specific manner and can help shed light on the range of phenotypes observed in patients suffering from cohesinopathies.