Aberrant chromatin regulatory mechanisms in Down syndrome brain

项目总结：迷宫一号 好了！ 唐氏综合症(DS)是世界范围内导致智力残疾的最常见的遗传原因，原因是 人类21号染色体的三倍体(HSA21)。尽管在理解人类基因组的遗传学方面取得了很大进展 DS，HSA21上及以后编码的基因，直接导致智力残疾，以及它们的 相关的分子机制仍然不完全清楚。BRWD1是一个没有特征的大脑 HSA21上DS关键区2内编码的富含染色质‘阅读器’蛋白。BRWD1被认为是 通过与SWI/SNF染色质重塑复合体的相互作用，发挥转录激活剂的作用； 然而，它在神经发育和DS中的作用还有待研究。与一致 以前对人类DS脑中基因表达的分析，我们观察到Brwd1的表达显著 在三体小鼠和DS受试者的HiPSCs来源的神经元中也是升高的。选择组蛋白 翻译后修饰(PTM)，我们已经证明其中一些与BRWD1直接相互作用， 也会改变它们的表达方式。我们已经证明了Brwd1表达的遗传重新正常化 在Ts65Dn小鼠中显着挽救与海马LTP和 三体动物的认知缺陷。此外，外源Brwd1通过病毒介导过表达 野生型动物中成年背侧海马神经元的转导-也导致上下文关系的缺陷 和空间记忆。我们的假设是DS脑中的BRWD1三体导致异常的相互作用 BRWD1和表观遗传格局之间的关系，从而促进全球基因表达 异常和认知缺陷。我们试图在以下独立的、但 协同作用的目标：1)研究Brwd1三体与DS样染色质功能障碍的关系 大脑，主要强调)无偏见地评估组蛋白PTM在DS样中的表达 使用与组蛋白PTM质量偶联的细胞分选方法的神经元和神经胶质细胞(+/-Brwd1救援) 光谱，以及b)描述BRWD1和改变的组蛋白PTMS之间的直接相互作用 新的“设计者”核小体分析和活体验证。2)调查Brwd1的影响 DS样脑中基因表达的三倍化。我们的目标是进行公正的、表观基因组范围的分析 Ts65Dn大脑中Brwd1的丰富-以及相关的组蛋白PTM-随后进行了广泛的比较 利用细胞类型特异性RNA-seq获得基因表达谱。基于CRISPR的操作将 随后执行以直接评估Brwd1靶基因对观察到的行为的贡献 异常现象。3)探讨Brwd1三倍体对特定SWI/SNF染色质募集/活性的影响 用生化和全基因组评估Ts65Dn小鼠脑中的重塑复合体。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.