Understanding cellular and transcriptional regulatory changes in human aging.

摘要 在这个项目中，我们建议增加Marliette Rodriguez Matos女士作为我们项目的主要成员，以研究 CD4T淋巴细胞的老化。 亲本R01代表了一个雄心勃勃的项目，研究一种原代的、纯化的人类细胞类型，这种细胞类型已经 与年龄相关的疾病有关，并已被证明表现出DNA的系统性变化 随着年龄的增长，在多种组织类型和物种中发生的甲基化。我们的目标是了解有多少 分子基因组性质随年龄变化的谱是由于细胞亚型的变化，有多少是 细胞自主，它们如何反映细胞信号对转录因子生物学的影响，以及个体之间如何 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 使用atac-seq对样本进行可及性研究。我们最初的计划是添加大量的RNA-seq和DNA 甲基化研究，但我们现在正在探索一种带有假阻塞的单细胞RNA-SEQ方法，以及 担心DNA甲基化研究将不会提供任何信息，因为最近的数据表明 这些变化表明组织中多能干细胞的比例随着年龄的增长而减少，这可能解释了为什么 DNA甲基化时钟还没有被证明在纯化的细胞中起作用。我们已经用我们的细胞净化了 CD4表面标志物，它耗尽所有的前体细胞。凯瑟琳·克罗克博士领导的我们正在进行的工作是 旨在阐明这个问题，以便我们明智地进行试验性投资。 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个样本中。她带着丰富的湿板凳经验来到我们团队，并证明了自己是 在基因组分析技术上出类拔萃。鉴于新冠肺炎造成的项目大范围延误 罗德里格斯·马托斯女士补充说，这是基于实际考虑，这将使我们能够提供 该项目最后的分析阶段所需的基因组数据。 我们将推动罗德里格斯·马托斯女士参与这一项目，将其作为系统培训经验的一部分。我们 在这份建议书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、 领导力和监督技能，以及道德和诚信方面的培训。她的分析能力将通过 与纽约基因组中心的合作者Tuuli Lappalainen博士一起工作，完善她的技能。 罗德里格斯·马托斯明确将自己的职业目标定位为未来的独立调查员。我们的目标是 R01项目是为了给她提供技能和出版物，为实现这一结果奠定必要的基础。 我们很高兴有机会把一位拉美裔美国女性培养成人类基因组学未来的领导者。 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.