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.

项目摘要 表观遗传机制与许多人类疾病和疾病有关，最著名的是癌症。 但是，近年来，发现越来越多的人类疾病可能会出现表观遗传侮辱。 这方面，我们在费城儿童医院的合作者通过外表测序 在患有类似的儿科患者中发现了针对H3FA和H3FB基因的种系线突变 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和 因此，代表了人类组蛋白H3上发现的第一个种系突变（神经组酮H3.3突变）。 组蛋白是小的碱性蛋白质，结合DNA以产生我们的染色质结构。与DNA一起 甲基化和长的非编码RNA，对组蛋白的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是一种专门的组蛋白变体 与活性基因相关，与该组蛋白的体细胞突变已在不同的脑癌中发现 仅在N末端尾巴上。我们的初步数据发现这些神经组酮H3.3突变是 从N-到C末端分布在整个蛋白质上。因此，我们假设这些突变同时 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做到这一点。在 这项建议，我们旨在了解这些神经组织酮突变如何导致神经发育问题。 首先，我们旨在确定这些神经组织酮突变是否影响全球或局部Hisstone修改模式 在细胞模型中使用新型质谱法（MS）方法，野生型或突变组蛋白变体。下一个， 我们希望确定这些神经组酮突变体是否影响染色质结构或构象或导致 基因组中H3.3变体的Misin Corporation。最后，我们将表征小鼠中的蛋白质组表达 使用子宫稳定同位素标记方法中的一种突变模型。我们的目标是确定 这些神经组酮H3.3突变如何影响表观遗传和细胞信号传导机制破坏 神经发育和维持导致这些小儿患者的神经系统疾病。