Protein turnover downstream of the Nipbl/CRL4 axis contributes to abnormal development in zebrafish embryos.

Background: Mutations in cohesins cause cohesinopathies such as Cornelia de Lange Syndrome (CdLS) and Roberts Syndrome (RBS). Prior findings demonstrate that Esco2 (a cohesin activator) and Smc3 (a core cohesin subunit) regulate the CRL4 E3 ubiquitin ligase. SMC3 mutations, however, account for a small percentage of CdLS. Here, we test whether NIPBL, which when mutated is responsible for 65% of CdLS cases, also regulates CRL4.

Results: We report that Nipbl knockdown in zebrafish embryos produces developmental abnormalities and reduces the transcription of ddb1, which encodes a key component of CRL4 E3 ligase. The severity of phenotypes in Nipbl knockdown embryos is partially rescued by exogenous ddb1 mRNA, demonstrating that CRL4 ligase function is downstream of Nipbl. These findings suggest that aberrant accumulation of CRL4 ligase substrates contributes to developmental abnormalities. To test this model, we identified candidate CRL4 substrates in zebrafish embryos by LC-MS. The results reveal that elevated expression of one of these candidates, pparαa, is sufficient to produce developmental defects in zebrafish embryos.

Conclusions: Nipbl impacts CRL4 ligase activity via regulation of ddb1 expression. We provide evidence that the aberrant accumulation of substrates is sufficient to produce developmental abnormalities consistent with those observed in RBS and CdLS models.