Investigating epigenetic mechanisms in Down syndrome using human cellular models

总结 唐氏综合征（DS）由21号染色体（HSA 21）的额外拷贝驱动，与广泛的 神经学和非神经学表型的谱，深刻的个体间变异和显著的 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。表观遗传重新布线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变异。 然而，这一领域的研究，特别是在染色质状态的水平上，仍然几乎完全 在DS中未被探索。利用人类诱导多能干细胞（iPSC）模型，我们最近进行了一项研究。 新的，公正的，全面调查的相对丰度超过80个不同的组蛋白后， 使用组蛋白质谱法在DS与整倍体对照中进行翻译修饰（PTM）。这些 结果，加上额外的验证实验，揭示了一组新的H3 K36 me 2破坏， 我们预测的H3 K4 me 1和H3 K23 ac丰度有助于DS疾病生物学。在本提案中，我们 我们的目标是扩大我们的分析，首次全面了解21三体如何破坏 组蛋白PTM及其下游对分子和细胞功能的影响。重要的是，这项提案 从以HSA 21/基因为中心的DS观点出发，传统上使用小鼠模型进行研究，以探索如何 表观遗传重新布线可能与一组关键的未回答的问题在该领域使用生理学 相关的人类细胞模型。从概念上讲，我们的实验旨在探索围绕 在整倍体染色体上编码的基因剂量的显著变化， DS的变化，不同的细胞类型如何以发散或收敛的方式受到21三体的影响，以及 DS机制如何与其他疾病重叠。具体而言，在目标I中，我们将系统地定义 使用不同供体个体和细胞类型队列的DS组蛋白PTM丰度表型范围 分析个体间和细胞类型的变化，然后将这些变化与染色质结合联系起来， 转录输出用于选择修饰以阐明基本机制。在Aim II中，我们将测试 使用药理学和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，我们将检验以下假设： 组蛋白PTM驱动DS中的核心神经生物学表型，以进一步了解其功能相关性 并可能将已知的细胞表型映射到新的分子机制。总的来说， 在这个变革性的研究奖申请创新分析将阐明新的机制， 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，并可能最终 告知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.