Novel targets of the Roberts Syndrome acetyltransferase Esco2/Eco1

项目摘要 罗伯特综合征（RBS）是一种以短肢畸形为特征的严重遗传性疾病 （鳍状附肢），小头畸形，腭裂，并指畸形，智力残疾， 癫痫，以及心脏和泌尿系统、生殖系统和消化道组织的异常。 重要的是，ESCO 2的突变是引发这种多基因突变的唯一必要条件。 组织和系统表型套件。因此，阐明机制，通过 Esco 2的功能将大大推进我们对途径整合的理解， 人类发展所必需的-从身体到认知。 Esco 2（酵母Eco 1）是一种乙酰转移酶，激活DNA连接复合物- 黏连蛋白Esco 2/Eco 1和cohesin都是染色体正确分离所必需的。在 反过来，RBS（ESCO 2突变）的流行模型是出生缺陷源于有丝分裂失败 （大量非整倍体）和随后的损失（凋亡）的增殖干细胞和发展 组织中以前，我们假设了一个替代模型，其中RBS来自转录 失调一项合作研究确实发现，Esco 2和cohesin共同调节了 骨生长关键基因的转录。因此，测试Esco 2的新模型和替代模型 功能将大大推进我们对人类发展的理解。 无论是基于有丝分裂失败/凋亡还是转录失调，所有 RBS的模型集中于粘附素亚基Smc 3的Esco 2乙酰化。多方面 然而，RBS的性质表明，还有其他信号通路在起作用。这里我们 报告了新的证据表明Eco 1（Esco 2的酵母同系物）可能靶向底物， 内聚素表征这些新型Esco 2/Eco 1底物，并测试它们的作用 特别是在发展中，提供了第一个新的方法来理解RBS， 近年来出现了许多其他的发展性疾病。 本提案的具体目标是相互依存的，并包含概念上不同的 遗传方法，从中确定Eco 1乙酰化反应的新底物。的 这两种筛选的技术方面（无偏的全基因组粘附素旁路筛选和 全基因组条件剂量致死筛选）完成-与遗传相互作用， 手在每个目标中，我们验证候选基因，评估基因产物的乙酰化状态， 和对Eco 1的依赖性，并测试底物乙酰化在酵母中的重要性。然后我们 将研究结果转化为斑马鱼胚胎，我们可以直接测试它们在 开发以及RBS细胞，以测试靶向底物修饰的变化。

Project Summary Robert syndrome (RBS) is a severe genetic disorder characterized by phocomelia (flipper-like appendages), microcephaly, cleft palate, syndactyly, intellectual disabilities, seizures, and abnormalities in the heart and urinary, genital, and alimentary tract tissues. Importantly, mutation of ESCO2 is the single and only requirement necessary to elicit this multi- tissue and systemic suite of phenotypes. It follows that elucidating the mechanisms through which Esco2 functions will significantly advance our understanding of pathway integrations required for human development - from the physical to the cognitive. Esco2 (yeast Eco1) is an acetyltransferase that activates the DNA tethering complex - cohesin. Esco2/Eco1 and cohesin are both essential for proper chromosome segregation. In turn, the prevailing model of RBS (ESCO2 mutated) is that birth defects arise from mitotic failure (massive aneuploidy) and subsequent loss (apoptosis) of proliferative stem cells and developing tissues. Previously, we posited an alternate model in which RBS arises from transcription dysregulation. A collaborative effort indeed discovered that Esco2 and cohesin co-regulate the transcription of genes critical for bone growth. Thus, testing new and alternate models of Esco2 function will significantly advance our understanding of human development. Whether predicated on mitotic failure/apoptosis or transcriptional dysregulation, all models of RBS focus on Esco2 acetylation of the cohesin subunit Smc3. The multi-faceted nature of RBS, however, suggests that additional signaling pathways are at play. Here, we report new evidence that Eco1 (yeast homolog of Esco2) may target substrates beyond cohesins. Characterization of these novel Esco2/Eco1 substrates, and testing their roles specifically in development, provides the first novel approach to understanding RBS, and numerous other developmental maladies, to emerge in recent years. The Specific Aims of this proposal are interdependent and contain conceptually distinct genetic approaches from which to identify novel substrates of Eco1 acetylation reactions. The technical aspects of the two screens (an unbiased genome-wide cohesin-bypass screen and a genome-wide conditional dosage lethality screen) are complete – with genetic interactors in hand. In each aim, we validate the candidates, assess the acetylation state of the gene products and dependency on Eco1, and test the importance of substrate acetylation in yeast. We then translate findings into zebrafish embryos from which we can directly test for roles in development and also RBS cells to test for changes in targeted substrate modifications.