Aberrant chromatin regulatory mechanisms in Down syndrome brain

项目概要：迷宫I ! 唐氏综合征（DS）是世界范围内智力残疾的最常见遗传原因， 人类21号染色体（HSA 21）的三倍。尽管在了解遗传学方面取得了很大进展， DS是HSA 21上编码的基因，直接导致智力残疾， 相关的分子机制，仍然不完全理解。BRWD 1是一个没有特征的大脑， 在HSA 21上的DS关键区2内编码的富集的染色质“阅读器”蛋白。BRWD 1被认为是 通过与SWI/SNF染色质重塑复合物的相互作用作为转录激活因子; 然而，其在神经发育和DS中的作用还有待研究。符合 以前在人类DS脑中的基因表达分析中，我们观察到Brwd 1表达显著增加， 在三体小鼠中，以及在源自DS受试者的hiPSC的神经元中升高。选择组蛋白 翻译后修饰（PTM），其中一些我们已经证明与BRWD 1直接相互作用， 也改变了它们的表达。我们已经证明，Brwd 1表达的遗传再正常化 在Ts 65 Dn小鼠中显著挽救了与海马LTP相关的转录可塑性异常， 三体动物的认知缺陷。此外，外源性Brwd 1过表达-通过病毒介导 野生型动物中成年背海马神经元的转导-也导致两种背景的缺陷， 空间记忆。我们的假设是，在DS脑中BRWD 1三体导致异常的相互作用 BRWD 1和表观遗传景观之间的联系，从而有助于全球基因表达 异常和认知缺陷。我们试图在下面的独立测试中检验这一假设，然而， 协同作用的目的：1）检查Brwd 1三体与DS样细胞中染色质功能障碍之间的关系， 脑，总体重点是a）在DS样细胞中组蛋白PTM表达的无偏评估， 神经元和神经胶质细胞（+/-Brwd 1拯救），使用细胞分选方法结合组蛋白PTM质量 B）使用高光谱描绘BRWD 1和改变的组蛋白PTM之间的直接相互作用 新的基于“设计者”核小体的测定和体内验证。2)调查Brwd 1的影响 三倍于DS样脑的基因表达。我们的目标是进行无偏见的，表观基因组范围的分析， Ts 65 Dn脑中Brwd 1富集-以及相关的组蛋白PTM，随后进行了广泛的比较 使用细胞类型特异性RNA-seq获得的基因表达谱。基于CRISPR的操作将 以直接评估Brwd 1靶基因对观察到的行为 异常3)探索Brwd 1三倍体对特异性SWI/SNF染色质募集/活性的影响 使用生物化学和全基因组评估在Ts 65 Dn小鼠脑中的重构复合物。这些 实验将使我们能够剖析染色质调节的异常模式的功能后果 这将有助于我们理解与大脑可塑性相关的神经表观遗传过程。

Project Summary: Maze I ! Down syndrome (DS) is the most common genetic cause of intellectual disability worldwide resulting from triplication of chromosome 21 (HSA21) in humans. Despite much progress in understanding the genetics of DS, the genes encoded on HSA21–and beyond–that directly contribute to intellectual disability, as well as their associated molecular mechanisms, remain incompletely understood. BRWD1 is an uncharacterized, brain enriched chromatin `reader' protein encoded within the DS critical region 2 on HSA21. BRWD1 is believed to act as a transcriptional activator via its proposed interactions with the SWI/SNF chromatin remodeling complex; however, its roles in the contexts of neurodevelopment and in DS have yet to be studied. Consistent with previous gene expression analyses in human DS brain, we observe that Brwd1 expression is significantly elevated in trisomic mice, as well as in neurons derived from hiPSCs from DS subjects. Select histone posttranslational modifications (PTMs), some of which we have already shown to directly interact with BRWD1, are also altered in their expression. We have demonstrated that genetic re-normalization of Brwd1 expression in Ts65Dn mice significantly rescues transcriptional dysplasticity associated with hippocampal LTP and cognitive deficits in trisomic animals. Furthermore, exogenous Brwd1 overexpression–via viral-mediated transduction of adult dorsal hippocampal neurons in wildtype animals–also leads to deficits in both contextual and spatial memory. It is our hypothesis that BRWD1 trisomy in DS brain results in aberrant interactions between BRWD1 and the epigenetic landscape, thereby contributing to global gene expression abnormalities and cognitive deficits. We seek to test this hypothesis in the following independent, yet synergistic Aims: 1) Examine the relationship between Brwd1 trisomy and chromatin dysfunction in DS-like brain, with an overarching emphasis on a) unbiased assessments of histone PTM expression in DS-like neurons and glia (+/– Brwd1 rescue) using cell sorting methodologies coupled to histone PTM mass spectrometry, and b) delineation of direct interactions between BRWD1 and altered histone PTMs using highly novel `designer' nucleosome-based assays and in vivo validations. 2) Investigate the impact of Brwd1 triplication on gene expression in DS-like brain. We aim to perform unbiased, epigenome-wide analyses of Brwd1 enrichment–along with associated histone PTMs–in Ts65Dn brain, followed by extensive comparisons with gene expression profiles obtained using cell-type specific RNA-seq. CRISPR-based manipulations will subsequently be performed to directly assess Brwd1 target gene contributions to observed behavioral abnormalities. 3) Explore the impact of Brwd1 triplication on recruitment/activity of specific SWI/SNF chromatin remodeling complexes in Ts65Dn mouse brain using biochemical and genome-wide assessments. These experiments will allow us to dissect the functional consequences of abnormal patterns of chromatin regulation in DS brain and will aid in our understanding of the neuroepigenetic processes associated with brain plasticity.