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

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. Importantly, Smc3-dependent bone and tissue regeneration defects are rescued by transgenic Cx43 overexpression, providing evidence that Smc3 directly contributes to RBS-type phenotypes (i.e. skeletal defects) in the regenerating fin model. Moreover, chromatin immunoprecipitation (ChIP) 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.