Long Non-Coding RNAs in Allergy

The overall goal of this proposal is to identify fundamental mechanisms controlling allergic responses by the long non-coding RNA (lncRNA) Morrbid. Type 2 allergic responses are characterized by the generation of CD4+ T helper type 2 (Th2) cells, and the recently identified IL-13+ T follicular helper (Tfh13) cells, which drive production of high-affinity anaphylactic IgE. Given their central role in allergy, understanding how T cells are programmed to become Th2 and Tfh13 cells could allow manipulation of T cell responses to mitigate allergy. Recent work has revealed a critical function for lncRNAs in immunity, opening up an exciting area of research that may uncover new targets and pathways for therapeutic intervention. lncRNAs do not encode proteins; rather, many produce functional RNA transcripts that are powerful regulators of cellular identity, function and survival. Our preliminary data show that the lncRNA Morrbid controls CD4+ T cell function and is required for type 2 immune responses in vivo. Single-cell RNA-sequencing (scRNA-Seq) of CD4+ T cells revealed Morrbid to be most highly expressed in Il13-expressing Th2 and Tfh13 cells, and Tfh13 cells are reduced during type 2 responses in Morrbid-/- mice. Our hypothesis is that Morrbid is an epigenetic regulator of Th2 and Tfh13 differentiation during allergic responses. In Aim 1 we will identify cell types that require Morrbid to generate type 2 immune responses. Using mice with a conditional Morrbid allele crossed to different Cre-expressing lines, we will test the function of Morrbid in dendritic cells, B cells, T cells, and Tfh cells during type 2 responses in vivo. We will analyze expression of human MORRBID in T cells from donors with or without allergies using scRNA-Seq, to determine whether Th2 and Tfh cells overexpress MORRBID in allergy. Finally, using CRISPR/Cas9-based epigenome editing in primary human T cells, we will determine the impact of MORRBID silencing and overexpression on CD4+ T helper cell polarization in vitro. In Aim 2 we will dissect molecular mechanisms by which the Morrbid locus regulates gene expression in T cells. To this end we have developed a novel genetic targeting strategy based on pre-tRNA processing to ablate lncRNA transcripts without blocking transcription in vivo. In Aim 3 we will map the Morrbid interactome to determine how Morrbid controls T cell function. To identify genes directly bound by Morrbid, we will employ a novel method that allows simultaneous mapping of both lncRNA-chromatin interactions and lncRNA-associated chromatin loops genome-wide, called RNA ChIA-PET (RNA-Chromatin Interaction Analysis by Paired-End Tag sequencing). To identify regulatory proteins interacting with Morrbid, we will use RAP-MS (RNA Antisense Purification coupled with Mass Spectrometry). Through completion of these Aims, we will elucidate new regulatory pathways controlling type 2 immunity that could potentially be exploited to treat allergy. In addition, we will have established and validated two novel tools of significant benefit to the research community which suffers from a dearth of standard methodology for analyzing lncRNA function; a lncRNA-specific genetargeting approach, and a method to map genome-wide lncRNA-associated chromosomal interactions.