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

Rajeswari Banerji,M Kathryn Iovine,Robert V Skibbens

doi:10.1242/bio.026013

Abstract

ABSTRACTRobert 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.

Highlights

Expression of smc3 in the regenerating fin Esco2 is a critical regulator of fin skeletal and tissue regeneration that is required for expression of the developmental signaling factor cx43 (Banerji et al, 2016)
While Esco2 is essential for modifying the cohesin subunit Smc3 to produce sister chromatid tethering and high fidelity chromosome segregation, a role for Smc3 in mediating Esco2dependent Roberts syndrome (RBS)-like skeletal and tissue defects remains unknown
The results reveal that smc3 mRNA is strongly expressed at 3 dpa, similar to esco2 expression (Fig. 1A). smc3 expression decreased by 5 dpa and was negligible by 8 dpa (Fig. 1A)

Summary

Introduction

Roberts syndrome (RBS) is a multi-spectrum developmental disorder characterized by severe skeletal deformities resulting in craniofacial. Cornelia de Lange Syndrome (CdLS) patients exhibit phenotypes similar to RBS patients, including severe longbone growth defects, missing digits, craniofacial abnormalities, organ defects and severe mental retardation (Tonkin et al, 2004; Krantz et al, 2004; Gillis et al, 2004; Musio et al, 2006). At least a subset of cohesin subunits form rings that appear to topologically entrap individual DNA segments (Guacci et al, 1997; Michaelis et al, 1997; Toth et al, 1999; Hartman et al, 2000; Panizza et al, 2000; Haering et al, 2002; Gruber et al, 2003; Arumugam et al, 2003; Tong and Skibbens, 2014; Eng et al, 2015; Stigler et al, 2016)

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Results

Conclusion