PROJECT SUMMARY Background. It is now evident that the majority of the mammalian genome has the potential to express non- coding RNAs (ncRNAs). However, the functionality and mechanism(s) of regulation of these ncRNAs are just beginning to be explored. One challenge that biologists encounter is the detection of these ncRNAs, which often tend to be transcriptionally tightly controlled and rapidly degraded. Using mouse models that allow easy detection of lncRNA, we have recently identified a set of long noncoding RNAs (lncRNA) that are expressed surrounding the immunoglobulin loci genes. These lncRNAs are placed inside topologically associating domains (TADs) that are formed during B cell development. Experiments using mouse models that lack these lncRNAs (published and preliminary data) demonstrate roles in class switch recombination and somatic hypermutation mechanisms. Objectives/Hypothesis. Here we investigate the direct role of lncRNAs in regulating genome local architecture and DNA topology via cis and trans mechanisms. In aim 1: we evaluate the physiological role of lncRNA-expressing loci in regulating IgH recombination via organizing TADIgH; in aim 2, we understand the molecular mechanisms through which lncRNAs are used to control genome architecture; and in aim 3, we focus on understanding the function of lncRNA SµGLT in control of CSR. Study Design: Using mouse models and cell lines that are deficient in specific lncRNAs we aim to investigate their immunologically relevant functions. For evaluating the molecular mechanism of action of the lncRNAs we use biochemical assays to purify lncRNA interacting proteins and perform chromosomal architecture assays such as HiC and 4C-seq. Finally, we perform high-throughput sequencing experiments to evaluate the lncRNA's effect(s) on SHM in the Ig loci genes and elsewhere. Disease Relevance: The proposed studies will lead to a better understanding of the mechanisms in B cell development and function. The lncRNAs being investigated in this application carry polymorphisms in patients with IgA deficiency syndrome and thus our study is relevant for human physiology. Finally, antibody diversification mechanisms are essential for immune system homeostasis but when these mechanisms fail there are increased genomic alterations that are associated with lymphomas. Thus, this study is related with both immunity and lymphomagenesis.