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.
Germinal Centers (GC) are sites in the body where long-lived immune responses occur that help prevent new infections after vaccination or recovery from an initial infection. GCs continue for weeks to months executing a Darwinian selection process to select the very best responses to be stored for the future. Long noncoding RNAs (lncRNAs) are a new category of genetic information that plays many biological functions but does not make protein. There are tens of thousands of them in the human genome but most of their functiosn are unknown. In this grant we propose to discover new lncRNAs that are expressed in GCs and use genetic means to determine how they control the function and efficiency of the GC process.
