Investigating epigenetic mechanisms in Down syndrome using human cellular models

摘要 唐氏综合征(DS)由21号染色体的一个额外拷贝(HSA21)驱动，与广泛的 神经性和非神经性表型的谱系，深刻的个体间差异和显著 全基因组基因表达和DNA甲基化模式的变化，尽管潜在的机制 仍然没有完全解决。表观遗传重排是实现基因组类型的一个有希望的候选者- 在DS中观察到的广泛变化，以及促成疾病表型的个体间差异。 然而，这个领域的研究，特别是在染色质状态的水平上，仍然几乎完全是 在DS中未被开发。利用人类诱导多能干细胞(IPSC)模型，我们最近进行了一项 对80多种不同组蛋白后的相对丰度进行了新颖、公正和全面的调查 用组蛋白质谱仪检测DS与整倍体对照中的翻译修饰(PTM)。这些 结果，再加上额外的验证实验，揭示了一系列对H3K36me2的新干扰， 我们预测的H3K4me1和H3K23ac丰度对DS疾病生物学有贡献。在这项提案中，我们 目的是扩大我们的分析范围，首次全面了解21三体是如何破坏人类健康的 组蛋白PTMS及其下游对分子和细胞功能的影响。重要的是，这项提议 摆脱以HSA21/基因为中心的DS的观点，传统上使用小鼠模型进行研究，以探索 表观遗传的重新连接可能与一组关键的未回答的问题相交，这些问题在该领域使用生理学 相关的人体细胞模型。从概念上讲，我们的实验旨在探索围绕 编码在整倍体染色体上的基因剂量的显著变化，深刻的个体间 DS的变异，21三体如何以发散或收敛的方式影响不同的细胞类型，以及 DS机制如何与其他疾病重叠。具体地说，在目标一中，我们将系统地定义 利用不同供者个体和细胞类型的队列研究DS患者组蛋白PTM丰度表型的范围 分析个体间和细胞类型的差异，然后将这些变化与染色质结合和 转录输出的选择修饰，以阐明基本的机制。在AIM II中，我们将测试 用药理学和CRISPRA方法研究DS组蛋白表型的分子可逆性 确定DS中潜在的临床相关靶点。在目标III中，我们将检验这样一个假设，即 组蛋白PTMS驱动DS的核心神经生物学表型以进一步了解其功能相关性 并有可能将已知的细胞表型映射到新的分子机制。总体而言，严格和 这一变革性研究奖申请中的创新分析将阐明 DS的表观遗传失调，探索该领域一系列关键的未回答的问题，并最终可能 介绍DS患者的治疗策略。

SUMMARY Down syndrome (DS), driven by an extra copy of chromosome 21 (HSA21), is associated with a broad spectrum of neurological and non-neurological phenotypes, profound interindividual variation and significant changes in genome-wide gene expression and DNA methylation patterns, although underlying mechanisms remain incompletely resolved. Epigenetic re-wiring is a promising candidate for achieving the types of genome- wide changes that are observed in DS as well as contributing to interindividual variation in disease phenotypes. However, this area of investigation, particularly at the level of chromatin states, remains almost entirely unexplored in DS. Leveraging human induced pluripotent stem cell (iPSC) models, we recently conducted a novel, unbiased, and comprehensive survey of the relative abundance of over 80 different histone post- translational modifications (PTMs) in DS versus euploid controls using histone mass spectrometry. These results, coupled with additional validation experiments, revealed a set of novel disruptions to H3K36me2, H3K4me1 and H3K23ac abundance which we predict contribute to DS disease biology. In this proposal, we aim to expand our analyses to obtain, for the first time, a comprehensive view of how trisomy 21 disrupts histone PTMs and the downstream impacts on molecular and cellular function. Importantly, this proposal moves away from a HSA21/gene-centric view of DS, traditionally studied using murine models, to explore how epigenetic re-wiring may intersect with a set of key unanswered questions in the field using physiologically relevant human cellular models. Conceptually, our experiments are designed to explore questions around the significant changes in dosage of genes encoded on euploid chromosomes, the profound interindividual variation in DS, how different cell types may be impacted by trisomy 21 in divergent or convergent ways, and how DS mechanisms may overlap with other diseases. Specifically, in Aim I, we will systematically define the scope of histone PTM abundance phenotypes in DS using a cohort of different donor individuals and cell types to analyze interindividual and cell-type variation, and then connect these changes to chromatin binding and transcriptional output for select modifications to elucidate fundamental mechanisms. In Aim II, we will test the molecular reversibility of histone phenotypes in DS using pharmacological and CRISPRa approaches to identify potential clinically relevant targets in DS. In Aim III, we will test the hypothesis that dysregulation of histone PTMs drives core neurobiological phenotypes in DS to further understand their functional relevance and potentially map known cellular phenotypes to novel molecular mechanisms. Collectively, the rigorous and innovative analyses in this Transformative Research Award application will illuminate new mechanisms of epigenetic dysregulation in DS, explore a set of key unanswered questions in the field and may ultimately inform on therapeutic strategies for DS patients.