Understanding cellular and transcriptional regulatory changes in human aging.

摘要 在这个项目中，我们建议添加 Marliette Rodriguez Matos 女士作为我们项目的核心成员来研究 CD4 T 淋巴细胞的老化。 母体 R01 代表了一个雄心勃勃的项目，旨在研究一种初级、纯化的人类细胞类型，该细胞类型已被 与年龄相关的疾病有关，并已被证明表现出 DNA 的系统变化 随着年龄的增长，多种组织类型和物种中发生甲基化。我们的目标是了解有多少 随着年龄的增长，分子基因组特性的变化范围是由于细胞亚型的变化造成的，其中有多少是 细胞自主，这些如何反映细胞信号传导对转录因子生物学的影响，以及个体间如何 DNA 序列变异与这些分子和细胞表型相互作用。 为了实现这些目标，我们正在进行基因分型、T 细胞受体扩增子测序和染色质分析 使用 ATAC-seq 对样本进行可及性研究。我们最初的计划是添加批量 RNA 测序和 DNA 甲基化研究，但我们现在正在探索具有伪批量的单细胞 RNA-seq 方法，以及 担心 DNA 甲基化研究将无法提供信息，因为最近的数据表明这些 变化代表组织中多能干细胞比例随着年龄的增长而减少，这可以解释为什么 DNA 甲基化时钟尚未被证明可以在纯化的细胞中发挥作用。我们已经纯化了我们的细胞 CD4 表面标记，可耗尽所有前体细胞。我们由 Katherine Crocker 博士领导的持续工作是 旨在阐明这个问题，以便我们明智地进行实验投资。 罗德里格斯·马托斯 (Rodriguez Matos) 女士将带头对队列进行选定的一组分子基因组分析 400 个样本。她带着丰富的湿板凳经验来到我们团队，并证明了自己 基因组检测技术卓越。鉴于 COVID-19 造成的广泛项目延误 罗德里格斯·马托斯女士补充说，这一流行病是基于实际考虑，这将使我们能够实现 项目最后分析阶段所需的基因组数据。 我们将培养罗德里格斯·马托斯女士参与该项目，作为结构化培训经验的一部分。我们 在此提案中描述她的项目活动将如何涉及旨在促进可转移的活动 技能。这些技能不仅与老龄化领域有关，还与项目管理、沟通、 领导力、监督技能以及道德和诚信培训。她的分析能力将通过 与纽约基因组中心的联合 PI Tuuli Lappalainen 博士团队合作，完善了她的技能。 罗德里格斯·马托斯女士将她的职业目标明确定义为未来的独立调查员。我们的目标是 R01 项目旨在为她提供技能和出版物，为实现这一成果奠定基础。 我们欢迎有机会培训一名西班牙裔美国女性作为人类基因组学的未来领导者 特别是在衰老研究领域。

ABSTRACT In this project, we propose to add Ms. Marliette Rodriguez Matos as a central member in our project to study the aging of CD4+ T lymphocytes. The parent R01 represents an ambitious project to study a primary, purified human cell type that has been associated with age-related diseases and has been shown to manifest the systematic changes in DNA methylation that occur with age in multiple tissue types and species. Our goal is to understand how much of the spectrum of changes of molecular genomic properties with age are due to cell subtype changes, how many are cell-autonomous, how these reflect cell signalling effects on transcription factor biology, and how inter-individual DNA sequence variation interacts with these molecular and cellular phenotypes. Towards these goals we are performing genotyping, T cell receptor amplicon sequencing, and chromatin accessibility studies using ATAC-seq on the samples. Our original plan was to add bulk RNA-seq and DNA methylation studies, but we are now exploring instead a single cell RNA-seq approach with pseudobulking, and are concerned that the DNA methylation studies will be uninformative given more recent data that indicates these changes represent a decrease in multipotent stem cell proportions in tissues with age, which may explain why the DNA methylation clock has not been demonstrated to work in purified cells. We have purified our cells with the CD4+ surface marker, which depletes all precursor cells. Our ongoing work led by Dr. Katherine Crocker is designed to shed light on this issue so that we make our experimental investments wisely. Ms. Rodriguez Matos will take the lead in performing the chosen set of molecular genomic assays on the cohort of 400 samples. She comes to our group with strong wet bench experience, and has proven herself to be exceptional technically with genomic assays. Given the extensive project delays enforced by the COVID-19 pandemic, adding Ms. Rodriguez Matos is based on the practical consideration that this will allow us to deliver the genomic data needed for the final, analytical stage of the project. We will develop Ms. Rodriguez Matos’ participation in this project as part of a structured training experience. We describe in this proposal how her project activities will involve activities that are designed to foster transferrable skills. These skills will not only be related to the field of aging but also project management, communication, leadership, and supervisory skills, and training in ethics and integrity. Her analytical skills will be fostered by working with the group of co-PI Dr. Tuuli Lappalainen at the New York Genome Center, rounding out her skills. Ms. Rodriguez Matos’ has defined her career goal explicitly as a future independent investigator. Our goal during the R01 project is to give her the skills and publications that will give the foundation needed for that outcome. We would welcome the opportunity to train a Hispanic-American woman as a future leader in human genomics and in the field of aging research in particular.