CAREER: Elucidating spatial and epigenetic regulation of gene expression during human development using photopatterning and single-cell multiomics

职业：利用光模式和单细胞多组学阐明人类发育过程中基因表达的空间和表观遗传调控

• 批准号: 2339849
• 负责人: Siddharth Dey
• 金额: $ 106.41万
• 依托单位: University of California-Santa Barbara
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2024
• 资助国家: 美国
• 起止时间: 2024-02-01 至 2029-01-31
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2339849&HistoricalAwards=false
• 关键词: CAREER; Elucidating; spatial; epigenetic; regulation

Complex biological organisms arise from a single cell. Yet our understanding of how this single cell divides and differentiates into all of the specialized cells that make up different tissues within an organism is quite limited. The goal of this proposal is to understand how signaling cues arising from the spatial location of a cell within a tissue and its epigenetic state regulate gene expression during early human development. The function of a cell is tightly controlled by the cells location within a tissue and its intrinsic epigenetic landscape. While single-cell sequencing has revolutionized our understanding of mammalian systems by measuring the transcriptome and epigenome landscape within single cells, spatial information is lost using current methods. To better understand how spatial organization regulates gene expression in individual cells, the PI proposes to use photo-sensitive oligonucleotides to optically stamp the spatial positions of cells during development. This spatial information will be combined with single-cell mRNA sequencing and single-cell epigenetic features. Collectively, these techniques will map how spatial and epigenetic determinants control the specification of human primordial germ cells during early development. The data generated by the research aims will be used for a multi-layered and integrated educational program that includes a computational genomics bootcamp for high school students, a summer wet lab internship for undergraduates, and a mobile app game that introduces gene regulation concepts and targets high school students.To comprehensively study how spatial organization regulates gene expression of individual cells, the PI will design cholesterol-tagged photo-sensitive oligonucleotides (PSO) that incorporate into cell membranes, enabling an optical ‘stamp’ of the position of each cell prior to tissue dissociation and single-cell mRNA sequencing. Quantifying both mRNA and the relative degradation of the PSOs in individual cells will enable mapping spatial single-cell transcriptomes at high-resolution. Further, the PI will develop spatially-resolved single-cell multiomics technologies to simultaneously sequence the transcriptome together with different epigenetic features from the same cell to directly relate how DNA methylation, DNA accessibility and histone marks tune gene expression in varied spatial contexts. Furthermore, by employing iterative rounds of optical labeling, they will capture cell migration with end-point single-cell multiomics sequencing to gain insights into how tissue morphogenesis impacts cellular phenotypes. These transformative methods will be used to specifically study how morphogen gradients, cell-cell interactions, and epigenetic reprogramming play a role in the specification of primordial germ cells (PGC), the precursors to egg or sperm, during human gastrulation. While the emergence of PGCs has been studied in detail in mice, lack of access to human embryos makes similar studies impossible. Therefore, 2D/3D in vitro gastruloid models of human development will be used to address the following: What is the identity of the progenitors that give rise to PGCs, what combination of morphogen gradients and epigenetic remodeling drive PGC specification, and the role of defined niches in giving rise to PGCs. Overall, the development of these spatially-resolved single-cell methods will provide key opportunities in the future to modulate cell identity for varied applications. This project is supported by the Systems and Synthetic Biology Cluster of the Division of Molecular and Cellular Biosciences.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

复杂的生物有机体由单个细胞产生，但我们对这个单个细胞如何分裂和分化成构成有机体内不同组织的所有特化细胞的理解相当有限。该提案的目标是了解信号线索如何产生。细胞在组织内的空间位置及其表观遗传状态在人类早期发育过程中调节基因表达，而单细胞测序已经发生了革命性的变化。我们通过测量转录组来了解哺乳动物系统为了更好地了解空间组织如何调节单个细胞中的基因表达，PI建议使用光敏寡核苷酸在发育过程中以光学方式标记细胞的空间位置。这些信息将与单细胞 mRNA 测序和单细胞表观遗传特征相结合，共同绘制出空间和表观遗传决定因素如何在早期发育过程中控制人类原始生殖细胞的规格。对于一个多层次、一体化的教育项目，包括针对高中生的计算基因组学训练营、针对本科生的暑期湿实验室实习以及针对高中生的介绍基因调控概念的移动应用游戏。全面研究空间组织如何调控基因为了实现单个细胞的表达，PI 将设计掺入细胞膜的胆固醇标记光敏寡核苷酸 (PSO)，从而在组织解离和单细胞 mRNA 定量之前对每个细胞的位置进行光学“标记”。 mRNA 和单个细胞中 PSO 的相对降解将能够以高分辨率绘制空间单细胞转录组图谱。此外，PI 将开发空间分辨单细胞多组学技术，以同时对转录组和不同表观遗传特征进行测序。同一细胞直接关联 DNA 甲基化、DNA 可及性和组蛋白标记如何在不同环境下调节基因表达。此外，通过采用迭代的光学标记，他们将通过终点单细胞多组学捕获细胞迁移。测序以深入了解组织形态发生如何影响细胞表型，这些变革性方法将用于专门研究形态发生素梯度、细胞间相互作用和表观遗传重编程如何在原始生殖细胞（PGC）（原始生殖细胞的前体）的规范中发挥作用。虽然人类原肠胚形成过程中的 PGC 的出现已在小鼠身上进行了详细研究，但由于缺乏人类胚胎，因此无法进行 2D/3D 体外原肠胚模型。人类发育的研究将用于解决以下问题：产生 PGC 的祖细胞的身份是什么，形态发生素梯度和表观遗传重塑的组合如何驱动 PGC 规范，以及确定的生态位在产生 PGC 中的作用。这些空间分辨单细胞方法的发展将为未来调节细胞身份的各种应用提供重要机会。该项目得到了分子与合成生物学部系统与合成生物学集群的支持。细胞生物科学。该奖项反映了 NSF 的法定使命，并通过使用基金会的智力价值和更广泛的影响审查标准进行评估，被认为值得支持。