Exploring the Role of Long Noncoding RNAs in Germinal Center B cells

探索长非编码 RNA 在生发中心 B 细胞中的作用

• 批准号: 10154493
• 负责人: MARK J SHLOMCHIK
• 金额: $ 18.5万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-12-01 至 2022-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10154493
• 关键词: Affinity; Alleles; Attention; B-Cell Development; B-Lymphocytes; Biological; Biological Models; Biological Process; Biology; CRISPR/Cas technology; Categories; Cell Cycle; Cell Differentiation process; Cell Lineage; Cell Nucleus; Cell physiology; Cells; Cellular biology; Characteristics; Code; Collaborations; Complex; Cytoplasm; DNA cassette; Data; Development; Environment; Epigenetic Process; Exons; Funding; Future; Gene Expression; Genes; Genetic; Genetic Transcription; Genome; Grant; Human; Human Genome; Immune response; Immunize; Immunological Models; Infection; Knock-out; Knockout Mice; Link; Measures; Mediating; Memory; Memory B-Lymphocyte; Messenger RNA; Methods; MicroRNAs; Molecular; Mus; Output; Phenotype; Plasma Cells; Play; Polyadenylation; Principal Investigator; Process; Proteins; RNA; RNA Splicing; Reaction; Recovery; Regulator Genes; Role; Signal Transduction; Site; Specificity; Structure; Structure of germinal center of lymph node; Surface; System; Systems Biology; T-Lymphocyte; Techniques; Time; Tissues; Transcript; Transcription Process; Translations; Untranslated RNA; Vaccination; Work; behavioral response; cell behavior; epigenetic regulation; follow-up; genetic element; genetic information; in vivo; macrophage; mutant; novel; overexpression; prevent; programs; response; scaffold; transcription factor; transcriptome; transcriptome sequencing; vector

The germinal center (GC) response is the basis for effective vaccination, enabling both affinity maturation and memory development. In an iterative process, higher affinity GC B cells (GCBC) are progressively selected. At the same time, a small fraction of GCBC exit cell cycle and differentiate into long-lived progeny—either memory B cells (MBC) or plasma cells (LLPC). These processes are controlled by integrating different signals from the environment (e.g. from Ag, T cell help) that in turn are transmitted via signaling networks to the cytoplasm and nucleus, where cellular behavior and responses are determined. Our lab has studied these processes at multiple levels, including GC surface phenotypes, reprogramming of signaling, and gene expression and epigenetic alterations that are GC-specific. The work of many labs has revealed complex transcription factor (TF) networks that control GC affinity selection and differentiation. While signals that control and induce TFs are clearly important, they are unlikely to be the complete story. RNA-mediated control is one layer that has received considerably less attention. Long non-coding RNAs (lncRNAs)—which share many characteristics of protein- coding mRNAs such as splicing, 5’ cap, and 3’ polyadenylation—have emerged as a broad, complex class of regulatory molecules responsible for modulating key aspects of cell biology. In addition to regulating the processes of transcription and translation, lncRNAs have been implicated in epigenetic regulation of the chromosomal landscape, as well as a host of other diverse cellular processes, including molecular scaffolding and sequestration. The human genome has more lncRNAs than protein-coding RNAs; yet, the function of most of these is unknown. Intriguingly, many lncRNAs are only expressed in one or a few tissues. Recently, a few studies have described lncRNAs expressed in the B cell lineage in human, including in GCBC. However, relative to their abundant representation in the genome, lncRNA encoding genes have been investigated at only a cursory level, and there are no functional data on lncRNA in GCBC. This R21 is a request for funding of our lab’s initial work to investigate this novel arena as part of our efforts to understand GC function and gene regulatory networks. We have used deep RNA-seq and a stringent pipeline to identify putative GC-specific lncRNAs, measured their expression via Q-PCR, then cloned and sequenced them to determine their structure. We focus in this proposal on using genetic and functional approaches to understand the function of three of the most interesting lncRNAs (“GCLnc1, 2 and 3”). For GCLnc1 we have already generated a germline deletion and an overexpression system: both have exciting preliminary phenotypes. Our Aims are: 1) To fully characterize B cell responses of the GCLnc1 KO mouse; 2) Generate transcriptional STOP null alleles of GCLnc1, 2 and 3 and determine initial phenotypes in primary B cell responses; and 3) Overexpress each GCLncRNA in GCBC using a novel cell transfer system developed in our lab and determine the functional effects in vivo. We will do this in collaboration with Dr. Maninjay Atianand, a lncRNA expert who works on lncRNA function in macrophages.

生发中心（GC）应答是有效疫苗接种的基础，使亲和力成熟和免疫应答成为可能。 记忆发展在迭代过程中，逐渐选择较高亲和力的GC B细胞（GCBC）。在 同时，一小部分GCBC退出细胞周期并分化为长寿后代--或者记忆 B细胞（MBC）或浆细胞（LLPC）。这些过程是通过整合不同的信号控制的， 环境（例如，来自Ag，T细胞帮助），其又经由信号传导网络传递到细胞质， 细胞核，决定细胞行为和反应的地方。我们的实验室研究了这些过程， 水平，包括GC表面表型，信号重编程，基因表达和表观遗传 GC特异性的改变。许多实验室的工作揭示了复杂的转录因子（TF）网络 其控制GC亲和选择和分化。虽然控制和诱导TF的信号明显 重要的是，它们不太可能是完整的故事。RNA介导的控制是一个层， 相当少的关注。长链非编码RNA（lncRNA）--它与蛋白质有许多共同的特征-- 编码mRNA如剪接、5'帽和3'多聚腺苷酸化-已经作为一个广泛的、复杂的类别出现， 负责调节细胞生物学关键方面的调节分子。除了调节 在转录和翻译的过程中，lncRNA参与了基因的表观遗传调控。 染色体景观，以及其他不同的细胞过程，包括分子支架 和隔离人类基因组中的lncRNA比编码蛋白质的RNA多;然而，大多数lncRNA的功能 其中的一些是未知的。有趣的是，许多lncRNA仅在一种或几种组织中表达。最近，一些 研究已经描述了在人的B细胞谱系（包括GCBC）中表达的lncRNA。但是相对 由于lncRNA在基因组中的丰富表达，仅在一个特定的时期研究了lncRNA编码基因。 粗略的水平，并且在GCBC中没有关于lncRNA的功能数据。这个R21是我们实验室的资金申请 作为我们理解GC功能和基因调控努力的一部分，我们初步研究了这个新的竞技场 网络.我们已经使用深度RNA-seq和严格的管道来鉴定推定的GC特异性lncRNA， 通过Q-PCR测量它们的表达，然后克隆并测序它们以确定它们的结构。我们专注 在这项关于使用遗传和功能方法来了解三个最重要的功能的建议中， 感兴趣的lncRNA（“GCLnc 1、2和3”）。对于GCLnc 1，我们已经产生了一个种系缺失和一个种系缺失。 过表达系统：两者都具有令人兴奋的初步表型。我们的目标是：1）充分表征B细胞 2）产生GCLnc 1、2和3的转录终止无效等位基因， 确定原代B细胞应答中的初始表型;和3）使用 本实验室开发了一种新型的细胞转移系统，并测定了体内功能效应。我们将在 与Maninjay Atianand博士合作，他是一位lncRNA专家，研究巨噬细胞中的lncRNA功能。