LNCFOXP3 CONTROLS CELL IDENTITY AND FUNCTION OF HUMAN REGULATORY T CELLS

Regulatory T cells (Treg) control immunological responses through suppression of effector cells, playing crucial roles both in physiological and pathological contexts. Treg suppressive activity correlates with the tightly regulated expression and stability of their master transcription factor FOXP3. Given the role of lncRNAs in defining cell identity and function, we have previously shown lncRNAs specifically expressed in this lymphocyte subset. Here we present long non-coding FOXP3 (lncFOXP3), a newly identified lncRNA gene located in close proximity to the FOXP3 gene locus. LncFOXP3 defines Treg identity with higher specificity than FOXP3 itself, since its transcription is detectable only in Treg. LncFOXP3 localizes in cytoplasmic and nuclear compartments, whilst it does not associate with chromatin. Downregulation of lncFOXP3 in human Treg decreases FOXP3 protein levels, without significant changes at its transcriptional level. Indeed, lncFOXP3 downmodulation leads to changes in Treg cell protein and gene signature profile associated to their unique molecular blueprints, thus impairing their ability to suppress. To investigate how this lncRNA affects FOXP3 post-transcriptionally, we leveraged a targeted RNA pulldown approach to explore the protein interactors of lncFOXP3 by mass spectrometry. Analysis revealed USP7, a deubiquitinase known to prevent FOXP3 from proteasome-mediated degradation. By exploiting both primary Treg cells and ectopic expression of lncFOXP3 and FOXP3 in a 293T model system, we found that lncFOXP3 contributes specifically to FOXP3 protein stability by promoting its deubiquitination in concert with USP7. Our findings disclosed another layer of FOXP3 regulation and support the role of lncRNAs in the maintenance of cell identity.