Novel targets of CRL4 ligase within Cohesinopathy pathways

每年约有120，000名婴儿出生时患有先天缺陷， 需要特殊的护理和干预才能生存。事实上，出生缺陷是 婴儿死亡的原因。因此，发展性疾病代表了一个重要的情感问题， 给家庭和医疗保健系统带来经济负担。转录 失调，或影响发育程序的基因突变，解释了 大多数出生缺陷。虽然许多基因突变导致了发育 疾病已经确定，这往往不能提供深入的治疗或 通过药理学方法改善症状。 在这里，我们利用我们的专业知识对两个相关的遗传综合征，罗伯茨 综合征（RBS）和科尔内利亚德兰格综合征（CdLS）。RBS和CdLS患者 表现出包括颅面缺陷（小头畸形， 眼睛缺陷，听力损失），肢体尺寸减小（短肢畸形），心脏异常， 消化道和呼吸道，以及智力残疾。RBS的遗传基础和 CdLS是已知的-两者都是通过基于粘着蛋白的途径的突变产生的， 调节基因转录并确保基因组的完整性。我们最近发现 粘附素途径调节ddb 1的转录，ddb 1编码一个关键组分， Cullin 4环连接酶（CRL 4）泛素化复合物。我们假设RBS （esco 2突变）和CdLS（smc 3突变），以及可能的其他发育疾病， 这在很大程度上是通过降低CRL 4活性而产生的。为了支持这一假设， DDB 1的外源性表达降低了发育缺陷的严重性， 否则在SMC 3敲低的斑马鱼胚胎中出现。 我们用液相色谱-质谱联用技术分析了 对于esco 2（RBS）、smc 3（CdLS）和ddb 1下降。我们得到了一份 所有治疗中常见的候选者，我们预测是CRL 4的下游 活性并参与RBS/CdLS表型。在本建议的具体目标1中，我们 验证LC-MS数据，并进一步测试这些靶标的敲低程度 同时影响斑马鱼胚胎RBS/CdLS型发育缺陷 降低ESCO 2或SMC 3表达。在具体目标2中，我们测试了我们的假设 这些候选物的外源表达是致畸的。结合起来，这些 研究将揭示降低出生缺陷严重程度的新目标。

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.