Saturday, April 26, 2025
4/26/2025
Novel targets of CRL4 ligase within Cohesinopathy pathways - Every year, approximately ~120,000 infants are born with birth defects that require special care and interventions to survive. In fact, birth defects are the leading cause of infant death. Thus, developmental maladies represent a significant emotional and financial burden on both families and the health care system. Transcription dysregulation, or gene mutations affecting developmental programs, account for the majority of birth defects. While numerous gene mutations that underlie developmental maladies have been identified, this often does not provide insight into treatments or symptom amelioration through pharmacological approaches. Here, we leverage our expertise on two related genetic syndromes, Roberts Syndrome (RBS) and Cornelia de Lange Syndrome (CdLS). RBS and CdLS patients exhibit a range of severe phenotypes that include craniofacial defects (microcephaly, eye defects, hearing loss), reduced limb size (phocomelia), abnormalities of the heart, GI and respiratory tracts, and intellectual disabilities. The genetic basis of RBS and CdLS are known - both arise through mutation of a cohesin-based pathway that regulates gene transcription and ensures genome integrity. We recently discovered that the cohesin pathway regulates the transcription of ddb1 - encoding a key component of the Cullin4 Ring Ligase (CRL4) ubiquitination complex. We hypothesize that RBS (esco2 mutated) and CdLS (smc3 mutated), and likely other developmental maladies, arise in large part through reduced CRL4 activity. In support of this hypothesis, exogenous expression of ddb1 reduces the severity of developmental defects that otherwise arise in smc3 knockdown zebrafish embryos. We performed liquid chromatography–mass spectrometry on embryos knocked down for esco2 (RBS), smc3 (CdLS) and ddb1. We obtained a prioritized list of candidates, common across all treatments, that we predict are downstream of CRL4 activity and involved in RBS/CdLS phenotypes. In Specific Aim 1 of this proposal, we validate the LC-MS data and further test the extent to which knockdown of these targets impact RBS/CdLS-type developmental defects in zebrafish embryos simultaneously reduced in either esco2 or smc3 expression. In Specific Aim 2, we test our hypothesis that exogenous expression of these candidates is teratogenic. In combination, these studies will reveal new targets through which birth defect severity can be reduced.
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