Quantitative mass spectrometry for comprehending epigenetic mechanisms in a new underlying neurological developmental disorder

定量质谱分析用于理解新的潜在神经发育障碍的表观遗传机制

• 批准号: 10515832
• 负责人: Benjamin A Garcia
• 金额: $ 53.78万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-16 至 2027-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10515832
• 关键词: Affect; Applications Grants; Biochemical; Biological Assay; Brain; Cell Differentiation process; Cell Line; Cell model; Chromatin; Chromatin Structure; Congenital Epilepsy; Corpus Callosum; Craniofacial Abnormalities; DNA Methylation; DNA-Binding Proteins; Data; Defect; Development; Disease; Epigenetic Process; Fetal Development; Fetal Monitoring; Fetus; Gene Expression; Gene Expression Profile; Gene Mutation; Genes; Genetic Transcription; Genome; Genomics; Germ-Line Mutation; Goals; Heart failure; Histone H3; Histones; Human; In Vitro; Intellectual functioning disability; Knock-out; Lead; Length; Link; Maintenance; Malignant Neoplasms; Malignant neoplasm of brain; Mass Spectrum Analysis; Methodology; Methods; Microcephaly; Missense Mutation; Modification; Molecular Conformation; Mus; Muscle hypotonia; Mutation; N-terminal; Nanotechnology; Nerve Degeneration; Neuraxis; Neurodevelopmental Disorder; Neurodevelopmental Problem; Neurologic; Neurons; Nucleosomes; Outcome; Patients; Pattern; Pediatric Hospitals; Phenotype; Philadelphia; Post-Translational Protein Processing; Proteins; Proteome; Proteomics; Research Personnel; Sampling; Seizures; Series; Signal Transduction; Somatic Mutation; Stable Isotope Labeling; Structure; Syndrome; Tail; Thinness; Time; Tissues; Untranslated RNA; Variant; Xenopus laevis; base; combinatorial; congenital anomaly; developmental disease; embryonic stem cell; epigenome; exome sequencing; histone modification; human disease; improved; in utero; mouse model; mutant; mutant mouse model; nervous system disorder; neurodevelopment; new therapeutic target; novel; oculomotor; pediatric patients; tool

Project Summary Epigenetic mechanisms have been linked to many human disorders and diseases, most notably cancer. However, in recent years more and more human diseases have been found with possible epigenetic insults. In this regard, our collaborators at the Children’s Hospital of Philadelphia have through exome sequencing discovered germline-line mutations to the H3FA and H3FB genes in pediatric patients that suffer from similar neurological disorders and craniofacial abnormalities. These two genes encode for the histone variant H3.3, and thus represent the first germ-line mutations found on histone H3 in humans (neurohistone H3.3 mutations). Histones are small basic proteins that bind DNA to give rise to our chromatin structure. Along with DNA methylation and long non-coding RNA, post-translational modifications to histones regulate gene expression patterns (epigenetic mechanisms) and chromatin organization. Histone H3.3 is a specialized histone variant linked to active genes, and somatic mutations to this histone have been found in different brain cancers exclusively on the N-terminal tail. Our preliminary data have found that these neurohistone H3.3 mutations are spread across the entire protein from the N- to C-terminus. Therefore, we hypothesize that these mutations while resulting in similar phenotypes, do this by disrupting different epigenetic mechanisms involving histone H3.3. In this proposal, we aim to understand how these neurohistone mutations lead to neurodevelopmental problems. First, we aim to determine if these neurohistone mutations affect global or local histone modification patterns on wild-type or mutant histone variants using novel mass spectrometry (MS) approaches in cellular models. Next, we look to determine if these neurohistone mutants affect chromatin structure or conformation, or cause misincorporation of the H3.3 variant in the genome. Lastly, we will characterize proteome expression in a mouse model of one of the mutations using a novel in utero stable isotope labeling approach. It is our goal to determine how these neurohistone H3.3 mutations affect epigenetic and cellular signaling mechanisms to disrupt neurodevelopment and maintenance leading to neurological disorders in these pediatric patients.

项目概要 表观遗传机制与许多人类紊乱和疾病有关，尤其是癌症。 然而，近年来，越来越多的人类疾病被发现可能与表观遗传损伤有关。 在这方面，我们费城儿童医院的合作者通过外显子组测序 在患有类似疾病的儿科患者中发现了 H3FA 和 H3FB 基因的种系突变 神经系统疾病和颅面异常 这两个基因编码组蛋白变体 H3.3，以及 因此代表了在人类组蛋白 H3 上发现的第一个种系突变（神经组蛋白 H3.3 突变）。 组蛋白是一种小的碱性蛋白质，可与 DNA 结合，与 DNA 一起形成我们的染色质结构。 甲基化和长非编码RNA、组蛋白翻译后修饰调节基因表达 模式（表观遗传机制）和染色质组织 组蛋白 H3.3 是一种特殊的组蛋白变体。 与活性基因有关，并且在不同的脑癌中发现了该组蛋白的体细胞突变 我们的初步数据发现，这些神经组蛋白 H3.3 突变仅发生在 N 末端尾部。 从 N 端到 C 端扩散到整个蛋白质，因此，我们捕获了这些突变。 导致相似的表型，通过破坏涉及组蛋白 H3.3 的不同表观遗传机制来实现这一点。 这个提议，我们的目的是了解这些神经组蛋白突变如何导致神经发育问题。 首先，我们的目标是确定这些神经组蛋白突变是否影响全局或局部组蛋白修饰模式 接下来，在细胞模型中使用新型质谱 (MS) 方法分析野生型或突变组蛋白变体。 我们希望确定这些神经组蛋白突变体是否影响染色质结构或构象，或导致 H3.3 变体在基因组中的错误整合最后，我们将表征小鼠中的蛋白质组表达。 我们的目标是使用一种新颖的子宫内稳定同位素标记方法来确定其中一个突变的模型。 这些神经组蛋白 H3.3 突变如何影响表观遗传和细胞信号传导机制并破坏 神经发育和维持导致这些儿科患者的神经系统疾病。