Cohesin mediates Esco2-dependent transcriptional regulation in 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.

Esco2 and Cohesin Regulate CRL4 Ubiquitin Ligaseddb1Expression and Thalidomide Teratogenicity

“…A significant reduction of Esco2 and Smc3 protein levels in 24 hpf embryos were obtained with esco2-ATG MO and smc3-ATG MO respectively (Fig S1 A, B). These findings extend the previously validated target specificity and KD efficacy of both MOs in fin regeneration to embryonic development (13,21,22). Here, we assessed the role of Esco2 KD and Smc3 KD in developing zebrafish embryos.…”

“…In the former case, cohesins redistribute to over 18,000 new loci during zebrafish development (25), suggesting that regulating CRL4 subunit expression is only one example of many regulatory circuits through which Esco2 and cohesin function. A second example is CX43, a gap junction gene involved in skeletal development that is similarly under control of the Esco2-cohesin axis (21,22). Mutations in CX43 cause oculodentodigital dysplasia (ODDD) in humans and defects in bone segment regrowth in zebrafish (6,26).…”

Simplet-dependent regulation of β-catenin signaling influences skeletal patterning downstream of Cx43

“…We use the percentage similarity method to determine whether the gene-targeting MO has an effect (i.e. Banerji et al, 2017;Govindan et al, 2016). With this method, we take the ratio of the injected side over the uninjected side and multiply by 100, and we compare these values for the Smp-MOand for the STD-MO-treated fins (values close to 100% indicate little effect of the MO; values with low similarity indicate that the MO had an effect).…”

“…ImageJ software was used to measure the band intensities. Relative pixel densities of gel bands were measured using a gel analysis tool in ImageJ software as described (Banerji et al, 2017). The density of each band was obtained as the area under the curve.…”

Condensins and cohesins – one of these things is not like the other!

“…In fact, multiple studies document cohesin-dependent genespecific transcription regulation (Rollins et al, 2004;Stedman et al, 2008;Rhodes et al, 2010;Gimigliano et al, 2012;Yan et al, 2013;Banerji et al, 2016Banerji et al, , 2017bTsai et al, 2018). In post-ZGA zebrafish, however, only half of the genes that exhibit altered mRNA levels upon cohesin depletion corresponded to gene loci that were otherwise normally bound by cohesin (Meier et al, 2018).…”