Novel targets of CRL4 ligase within Cohesinopathy pathways

每年大约有120,000名婴儿出生时就有缺陷 需要特殊的护理和干预才能生存。事实上，出生缺陷是导致 婴儿死亡原因。因此，发育障碍代表了一种重要的情感 以及家庭和医疗保健系统的经济负担。转录 调节失调，或影响发育计划的基因突变，解释了 大多数先天缺陷。虽然作为发育基础的大量基因突变 疾病已被识别，这通常不能提供对治疗或 通过药理学方法改善症状。 在这里，我们利用我们在两种相关基因综合征上的专业知识，罗伯茨 RBS综合征(RBS)和Cornelia de Lange综合征(CDLS)。RBS和CDLS患者 表现出一系列严重的表型，包括头面部缺陷(小头畸形， 眼睛缺陷、听力损失)、肢体尺寸缩小(青光眼)、心脏异常、 胃肠道和呼吸道，以及智力残疾。RBS和RBS的遗传学基础 CDL是已知的-两者都是通过基于粘附素的途径突变而产生的，该途径 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DDB1的转录，DDB1编码的是 库林4环连接酶(CRL4)泛素化复合体。我们假设苏格兰皇家银行 (ESCO2突变)和CDLS(smc3突变)，以及可能的其他发育疾病， 在很大程度上是由于CRL4活性降低所致。为了支持这一假设， 外源表达DDB1可降低发育缺陷的严重程度 否则就会出现在smc3基因敲除的斑马鱼胚胎中。 我们对敲击后的胚胎进行了液质联用。 ESCO2(RBS)、Smc3(CDLS)和DDB1下跌。我们得到了一份按优先顺序排列的清单 我们预测在CRL4下游的候选基因在所有治疗中都很常见 活性，并参与RBS/CDLS表型。在本提案的具体目标1中，我们 验证LC-MS数据，并进一步测试这些目标的击倒程度 同时影响斑马鱼胚胎RBS/CDLS型发育缺陷 ESCO2或Smc3表达降低。在具体目标2中，我们验证了我们的假设 这些候选基因的外源表达是致畸的。总而言之，这些 研究将揭示新的目标，通过这些目标可以降低出生缺陷的严重程度。

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.