Establishing foundational tools and datasets for investigation of NSD1 gene function in neural development

摘要 Sotos综合征，特征是童年过度生长，全球发育迟缓，智力残疾和 核受体结合集结构域蛋白1(NSD1)单倍体缺陷引起的行为缺陷 它编码组蛋白H3赖氨酸36(H3K36)甲基转移酶。NSD1特异性催化H3K36 二甲基化(H3K36me2)，在DNA甲基化(DNAME)和转录中起重要作用。 以前用小鼠或癌细胞株进行的研究表明，H3K36me2对维持 通过DNA甲基转移酶3A(DNMT3A)的招募，NSD1的缺失导致H3K36me2的减少 和获得拮抗标记组蛋白H3赖氨酸27三甲基化(H3K27me3)，并且NSD1可能 通过促进RNAPII向伸长能力状态的转变来调节基因的表达。蜂窝 表型研究进一步确定了H3K36me2在细胞可塑性和癌症中的重要作用 转移。这些机制如何与Sotos综合征患者的神经发育和功能障碍有关， 然而，这一点仍有待阐明。事实上，NSD1突变和H3K36me失调的影响是 据报道高度依赖于上下文，导致对基本参数(如单元格)的相反影响 跨不同细胞类型的增殖，对NSD1在人脑中的功能和功能障碍知之甚少 与Sotos综合征相关的细胞类型。我们的实验室最近发现了一种特殊而显著的下降 H3K36me2跨越唐氏综合征(由21三体驱动)患者细胞系与整倍体对照的比较 我们一直在通过探索H3K36me2/H3K27me3染色质结合的关系进行研究， DNAME及其转录产物在唐氏综合征中的表达这些数据让我们思考了一种有趣的可能性 唐氏综合征和Sotos综合征可能共享共同的分子扰动，从而导致 常见的神经发育表型。然而，缺乏研究NSD1功能的工具和数据集 在与Sotos综合征(即人类神经发育)相关的背景下，这是一个重大障碍 为进步干杯。因此，在目标I中，我们建议建立一套新的NSD1等基因人类IPSC模型 单倍体功能不全。NSD1上H3K36me2/H3K27me3染色质结合模式的失调 然后，将通过试点切割和标签数据集来评估人类神经元的单倍体不足。严谨 这项提议的执行将为研究新的可持续发展1创造新的灵活和可持续的资源 并测试NSD1单倍体功能不全在生理学上的分子影响 人脑细胞类型。这一建议适用于R03机制，因为结果将开发出新的 基于人体细胞的模型，并生成离散的试点数据，以支持未来的R01提交。

SUMMARY Sotos syndrome, characterized by childhood overgrowth, global developmental delay, intellectual disability and behavioral deficits is driven by haploinsufficiency of Nuclear Receptor Binding SET Domain Protein 1 (NSD1) which encodes a histone H3 lysine 36 (H3K36) methyltransferase. NSD1 specifically catalyzes H3K36 dimethylation (H3K36me2), which has important roles in DNA methylation (DNAme) and transcription. Previous studies using mouse or cancer cell lines have shown that H3K36me2 is essential for maintenance of DNAme via recruitment of DNA Methyltransferase 3A (DNMT3A), that NSD1 loss leads to reduced H3K36me2 and gain of the antagonistic mark histone H3 lysine 27 trimethylation (H3K27me3), and that NSD1 may regulate gene expression by facilitating the transition of RNAPII to an elongation-competent state. Cellular phenotyping studies have further identified important roles for H3K36me2 in cellular plasticity and cancer metastasis. How these mechanisms relate to neural development and dysfunction in Sotos syndrome patients, however, remains to be elucidated. Indeed, the effects of NSD1 mutation and H3K36me dysregulation are reported to be highly context dependent, resulting in opposing impacts on basic parameters such as cell proliferation across different cell types, with little known about NSD1 function and dysfunction in human brain cell types relevant for Sotos syndrome. Our laboratory recently identified a specific and significant decrease in H3K36me2 across Down syndrome (driven by trisomy 21) patient cell lines compared to euploid controls which we have been investigating by probing the relationship between H3K36me2 / H3K27me3 chromatin binding, DNAme and transcript expression in Down syndrome. These data led us to consider the intriguing possibility that Down syndrome and Sotos syndrome could share common molecular perturbations contributing to common neurodevelopmental phenotypes. However, the dearth of tools and datasets to study NSD1 function in a context relevant for Sotos syndrome (i.e., human neural development), has presented a significant barrier to progress. In Aim I, we therefore propose to generate a set of novel isogenic human iPSC models of NSD1 haploinsufficiency. Dysregulation of H3K36me2 / H3K27me3 chromatin binding patterns upon NSD1 haploinsufficiency in human neurons will then be assessed through pilot CUT&Tag datasets. Rigorous execution of this proposal will create new flexible and sustainable resources for studying NSD1 haploinsufficiency and test the molecular impacts of NSD1 haploinsufficiency in a physiologically relevant human brain cell type. This proposal is appropriate for the R03 mechanism, as the results will develop novel human cell-based models and generate discrete pilot data to support a future R01 submission.