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

项目摘要 表观遗传机制与许多人类疾病有关，最明显的是癌症。 然而，近年来，越来越多的人类疾病被发现可能存在表观遗传损伤。在 在这方面，我们在费城儿童医院的合作者通过外显子组测序， 发现了H3 FA和H3 FB基因的生殖系突变，这些突变发生在患有类似 神经系统疾病和颅面畸形。这两个基因编码组蛋白变体H3.3， 因此代表了人类组蛋白H3上发现的第一个种系突变（神经组蛋白H3.3突变）。 组蛋白是一种小的碱性蛋白质，它与DNA结合形成染色质结构。沿着DNA 甲基化和长的非编码RNA，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是一种特殊的组蛋白变体 与活性基因有关，在不同的脑癌中发现了这种组蛋白的体细胞突变 只在N-末端尾部。我们的初步数据发现，这些神经组蛋白H3.3突变是 从N端到C端分布在整个蛋白质中。因此，我们假设这些突变， 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做到这一点。在 我们的目标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， 在细胞模型中使用新型质谱（MS）方法研究野生型或突变型组蛋白变体。接下来， 我们希望确定这些神经组蛋白突变体是否会影响染色质的结构或构象， H3.3变体在基因组中的错误掺入。最后，我们将在小鼠中表征蛋白质组表达 使用一种新的子宫内稳定同位素标记方法的突变模型。我们的目标是确定 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， 神经发育和维持，导致这些儿科患者的神经系统疾病。

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.